table of contents table of contents6 i nno v ation lászló vÍgh deputy director general for...

1

TABL

E O

F CO

NTE

NTS

InformatIon ...........................................................2IntroductIon .........................................................3dIrectors ..................................................................4InnovatIon ...............................................................6

InstItute of BIophysIcs .............................. 7Pál ormos .....................................................................8andrás dÉr .................................................................. 10Géza Groma ...............................................................13Lóránd KeLemen ......................................................15Ilona LacZKÓ ............................................................. 17Zsolt toKaJI ................................................................ 18György vÁrÓ ...............................................................20László ZImÁnYI ......................................................... 21tibor PÁLI.....................................................................23alajos BÉrcZI ..............................................................25Balázs sZaLontaI ....................................................26csaba BaGYInKa ......................................................28László sIKLÓs ..............................................................30Árpád PÁrducZ ........................................................32mária deLI ...................................................................34István KrIZBaI ...........................................................36Kornél L. KovÁcs.......................................................38Kornél L. KovÁcs and Gábor rÁKHeLY ..............39Gábor rÁKHeLY and Balázs LeItGeB ..................41selected publications ...................................................42

InstItute of BIochemIstry ...................... 47antal KIss .....................................................................48György PÓsfaI ............................................................50csaba PÁL ..................................................................... 52Balázs PaPP ..................................................................54sándor BenYHe and anna BorsodI ...................56mária sZŰcs ................................................................58csaba tÖmBÖLY and Géza tÓtH..........................60miklós sÁntHa .........................................................62Imre m. Boros ...........................................................64Péter deÁK ...................................................................65andor udvardY .......................................................67Ibolya KIss ....................................................................69ernő duda ..................................................................71csaba vIZLer ..............................................................72László vÍGH .................................................................73ervin WeLKer ............................................................ 76selected publications ...................................................78

InstItute of enzymology ........................85László BudaY ..............................................................86Péter frIedrIcH .......................................................87Károly LILIom .............................................................88Judit ovÁdI ..................................................................90László PattHY ........................................................... 91

László PoLGÁr ...........................................................93István sImon...............................................................95Gergely sZaKÁcs .......................................................97Péter tomPa ...............................................................99mária vas ...................................................................100andrás vÁradI ........................................................102Beáta G. vÉrtessY ..................................................105Péter ZÁvodsZKY ...................................................107Péter ZÁvodsZKY and Péter GÁL ........................109selected publications ................................................. 111

InstItute of genetIcs ............................. 117Henrik GYurKovIcs ............................................. 118László sIPos ...............................................................120József mIHÁLY ..........................................................122Géza ÁdÁm ...............................................................124miklós erdÉLYI .......................................................126István KIss ..................................................................129István andÓ .............................................................130Ilona dusHa ..............................................................132Gabriella endre .......................................................134Lajos HaracsKa .....................................................136Ildikó unK .................................................................138Éva monostorI .....................................................140István rasKÓ ............................................................142Gyula HadLacZKY ................................................144Zsolt PÉnZes .............................................................146selected publications .................................................148

InstItute of plant BIology ................... 153ferenc naGY ..............................................................154Imre vass ...................................................................157Győző GaraB ...........................................................159Zoltán GomBos ....................................................... 162Gábor v. HorvÁtH .................................................164Zoltán maGYar ....................................................... 167attila feHÉr .............................................................. 169László sZaBados .................................................... 171selected publications ................................................. 173

laBoratorIes of core facIlItIes .......... 177scIentIfIc secretarIat .................................. 178anikó PÁY ................................................................... 179sándor BottKa .......................................................180László G. PusKÁs ..................................................... 182Katalin f. medZIHradsZKY ...............................184sándor PonGor ....................................................... 185ferhan aYaYdIn ......................................................188selected publications .................................................190

ImPrInt ....................................................................192

Table of ConTenTs

2

INFO

RMAT

ION

BIOLOGICAL RESEARCH CENTER,HUNGARIAN ACADEMY OF SCIENCESCENTER OF EXCELLENCE OF THE EUROPEAN UNION

H-6726 szeged, temesvári krt. 62.H-6701 szeged, P.o. Box 521, Hungary

Phone: +36-62-599-600fax: +36-62-432-576http://www.szbk.u-szeged.huor http://www.brc.hu

3

INTR

OD

UCT

ION

In 1962, the Hungarian academy of sciences decided to concentrate a substantial part of its means on supporting research in the field of modern biology. Professor Bruno straub f. initiated the foundation of the Biological research center. szeged was chosen as the site for a number of reasons. It is an university town with numerous excellent traditions. Work began in the Brc in 1971, but the official opening ceremony was held in 1973, when the construction of the whole building was finished.

Based on the concept of interdisciplinarity, scien-tific work in the Brc is carried out in 5 institutes: Biophysics, Biochemistry, Enzymology, Genetics and Plant Biology. In 2009, the Brc employs 479 persons in the four institutes located in szeged (and 90 persons in the Institute of enzymology located in Budapest), 257 (68) of whom belong to the scientific staff in addition to 66 (16) students in various Phd programs and 9 gradu-ate students of the International training course.

In 1998, the European Molecular Biology Organization (EMBO) and in 2001, the International scientific advisory Board performed a detailed evaluation of research groups in the Brc institutes. Both committees concluded that in certain fields such as developmental genetics, enzymology and plant biology, the research performance meets the highest international standards. In 2000, the Brc was selected as a Center of Excellence of the European Union. scientists from Brc have published 250–300 papers with 1000–1100 impact factors yearly. The Brc institutes in szeged have been supported by the Hungarian academy of sciences with eur 5,75 million every year as basic funding, and a similar amount has been generated from governmental and european grants. The research priorities as listed in this book are changing continuously according to the international trends in biological sciences. structural biology, system biology, functional genomics, environmental adaptation, neurosciences and lipidomics can all be found among the new initiatives of the Brc projects.

competitive research in various fields of biology requires constant improvement of the instrumentation. Therefore the Brc has organized several laboratories of core facilities for functional genomics, proteomics, bioinformatics, cellular imaging, dna synthesis, dna sequencing and flow cytometry.

scientists in the Brc are encouraged to participate actively in the educational programs of the university of szeged. In addition to the International training course, the Biology and Medical PhD Schools are also

important components of the educational activities of the Biological research center. Phd students are active contributors to the research projects and, after receiving their Phd degrees from the university, they continue their training in laboratories abroad. several successful postdoctoral scientists return to the center and play key roles in the initiation of new competitive projects. The intellectual and infrastructural capacity of the Brc provides a unique situation to serve as a regional center both in Hungary and in the central european region.

a few years ago the Brc initiated the organization of the Biopolisz Szeged—Life Science Consortium that integrates activities in the field of biology, medicine, biotechnology and plant breeding in the town. functional genomics and bioinformatics serve as a common platform to improve teaching and research performance. furthermore, it is essential to organize an efficient technology transfer procedure presently carried out by the Biopolisz Szeged Innovation Services Ltd. (www.biopolisz.hu). during the last years 14 spin-off companies have been founded by scientists from Brc. as a response to the grant possibilities reflecting the governmental r&d policy, applied research activities have been increased in the center. This stimulated the organization of various consortia, primarily between partners from the szeged university (Knowledge Center of Neurobiology) and from the cereal research non-Profit company (Wheat Consortia). There is a consensus that the industrial development in szeged city can be primarily based on bioindustry and biotechnology. to reach this goal, the Brc proposed the establishment of a bioincubator (Genomic Innovation Center and Szeged Bioinnovation Park). The collaborations of Brc scientists with pharmaceutical companies (Gedeon richter) can be an essential element in the realization of the Szeged-Biopolisz concept.

each autumn the Brc organises a three-day conference, which provides a forum for the scientists to present their new research achievements. We dedicate these scientific meetings to Professor Bruno straub f., the first director General of the Biological research center.

Szeged, 2009.

Prof. dénes dudits Director General

bIoloGICal ReseaRCH CenTeR,Hungarian academy of sciencesCenter of excellence of the european Union

4

DIR

ECTO

RS

Péter ZávODSZKYDirector (2007– ), Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences

H-1113 Budapest, Karolina út 29.H-1518 Budapest, P.o.Box 7, H-1518, Hungary

Phone: 36-1-209-3535fax: 36-1-466-5465e-mail: [email protected]

secretary: Ágnes szikraPhone: 36-1-279-3113financial officer: mária WagnerPhone: 36-1-209-3536

DIReCToRs

György PóSFAIDirector (2004– ), Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences



secretary: olga miklós and mónika BaliPhone: 36-62-599-654financial officer: mónika KordásPhone: 36-62-599-656

Pál ORMOSDirector General (2010– ), Biological Research Center, Hungarian Academy of Sciences


Phone: 36-62-599-613fax: 36-62-433-133e-mail: [email protected] [email protected]

Director (1994– ), Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences

secretary: andrea GémesPhone: 36-62-599-614

Dénes DUDITSDirector General (1997–2009), Biological Research Center, Hungarian Academy of Sciences



secretary: Zsuzsa KeczánPhone: 36-62-599-769office manager: Judit szabadPhone: 36-62-599-761; e-mail: [email protected]

financial officer: anikó HrkPhone: 36-62-599-609

5

DIR

ECTO

RS

DIReCToRs

János BáTHORIFinancial Director (2004– ), Biological Research Center, Hungarian Academy of Sciences


Phone: 36-62-599-736 fax: 36-62-433-494e-mail: [email protected]

secretary: anna miletinPhone: 36-62-599-735

Miklós ERDÉLYIDirector (2010– ), Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences



secretaries: csilla soltész and marinetta HerczegPhone: 36-62-599-657financial officer: tünde abonyiPhone: 36-62-599-656

István RASKóDirector (1994–2009), Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences



secretary: csilla soltész and marinetta HerczegPhone: 36-62-599-657financial officer: tünde abonyiPhone: 36-62-599-656

Imre vASSDirector (2000– ), Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences



secretary: mariann KárolyiPhone: 36-62-599-714financial officer: Judit KanalasPhone: 36-62-599-713

6

INN

OvA

TIO

N

László vÍGHDeputy Director General for Innovation Biological Research Center, Hungarian Academy of Sciences


Phone/fax: 36-62-432-048e-mail: [email protected]

secretary: mónika BaliPhone: 36-62-599-654

Spin-off CompanIes lInkeD To bRC

Company Contact persons

acheuron Hungary Ltd. tamás Letoha

avIcor Ltd. László Puskás

avIdIn Ltd. László Puskás

Biofotonika Ltd. Győző Garab

curamach Ltd. Gyula Hadlaczky

creative Labor Ltd. vilmos tubak

delta Bio 2000 Ltd. Lajos Haracska

JsW Hungary Ltd. miklós sántha

Lipidart Ltd. Zsolt török, László vígh

Pharmacoidea Ltd. tamás Letoha

Planta cosmetix Ltd. Gábor v. Horváth, dénes dudits

Therbiogen Ltd. vilmos tubak, róbert Katona

transmentIX Ltd. László Puskás

t-sejt Ltd. vilmos tubak, Éva monostori

InnovaTIon

7

BIO

PHYS

ICS

Institute of BiophysicsH-6726 Szeged, Temesvári krt. 62.H-6701 Szeged, P.O. Box 521, Hungary

Brc

8

BIO

PHYS

ICS

Light carries momentum, i.e. it exerts force upon objects. In everyday life this force is negligible. In the microworld, however, it is different: if a micrometer-sized particle is illuminated by light of moderate in-tensity (10 mW), the light pressure may be significant. If a particle with an index of refraction higher than that of its surroundings is placed in a focussed light beam, it will be trapped in the focus. micrometer-sized particles can be trapped this way. typical forces fall in the pn range: this is just the range to manipu-late biological objects (cells, cell organelles) in water and also of forces exterted by biological machines. consequently, this is a procedure with immense po-tential in biology.

In the basic case the position of spherical objects is controlled by optical tweezers. It would offer an addi-tional degree of control if the orientation could also be controlled, thereby extending the manipulation pos-sibilities. In our laboratory we investigate the interac-tion of optical tweezers with objects of special shape.

We can produce micrometer-sized particles of arbi-trary shape by two-photon excited photopolymeriza-tion of light-hardening photoresits, and we use such objects to study new phenomena of trapping. In addi-tion, based on the trapping of such objects we develop novel tools of micromanipulation. We present two typical methods.

Particles of helical, propeller shape will rotate in optical tweezers. objects to which such rotors are at-tached can be rotated, and micromechanical machines can be driven by them. They can be used for the gen-eration of complex machines for use in biology.

If the optical tweezers is generated by linearly po-larized light, flat objects will be trapped such that they will orient along the plane of polarization. With this method objects can be oriented. If we attach a mole-cule to such a particle, we can exert or measure torque on this molecule. In this way we can twist molecules, and their torsional properties can be determined. This is very important in biology, since there are numerous processes involving rotation. for example, to access the information stored in dna it has to be untwisted, consequently information about the torsional prop-erties of dna is fundamental for understanding the encoded information. With our method we can de-termine the torsional elasticity of dna, an important parameter for the function. numerous dna/protein interactions and protein motions involving rotation can be studied by this method.

Figure 1. Twisting a macromolecule by the optical tweezers. By rotat-ing the plane of polarisation, we rotate the flat object at the end of the DNA

opTICal mICRomanIpUlaTIon

Pál ORMOS / research Professor, Group Leader

László OROSZI / staff scientist Sándor vALKAI / staff scientist András BÚZáS / Phd studentMária KISSNÉ DOMONKOS / technician

9

BIO

PHYS

ICS

Optical control in microfluidics

In modern biochemical and medical diagnostical research there is a need for devices that handle small amounts of material but are able to process large numbers of samples in a short time. These requirements can be met by decreasing size, and microfluidics (lab-on-a-chip) is the development seeking solution in this direction. In the size range of these devices the physics of processes is quite different from that of the macroscopic world, consequently the solution is not merely a scaling down of known cases: development is progressing in different directions.

We believe that optical control can be very useful in microfluidics and we perform research in this area studying the processes on which future devices can be based.

using the photopolymerisation technique, we build microfluidics systems where channels and optical waveguides are integrated into a single system. Here light is used to investigate material (cells, molecules) in the channel; in addition, light is also used to manipu-late objects. for example, the fluorescence of cells can be measured using the integrated optical waveguides and selected ones are separated by the pressure of light also carried in integrated waveguides. disposable all-optical microfluidic cell sorters were built this way.

to extend optical control, we developed the con-cept of light-controlled electroosmosis. In a liquid-filled channel the surface charge of the wall is neutral-ized by opposite charges collecting at the wall that can be moved by an electric field parallel to the wall. In microchannels the total volume can be agitated this way, and this is electroosmosis. We cover the chan-

nel wall with photosensitive material, and in this way electroosmosis can be modulated by light.

We have developed different flow control schemes: in a single channel we can turn the flow on and off. We have built a light-controlled flow switch: in a bifurcating channel, the direction of flow is selected by light. In microfluidics flow is always laminar, therefore mixing, a key process in biochemical reactions is always a problem. Light-controlled electroosmosis again offers a solution: by illuminating the light-sensitive channel wall with light of appropriate pattern, the flow pattern can be controlled within a single microchannel.

The elaborated methods are useful for studying the flow properties of microchannels, at the same time they have great potential offering methods for controlling mi-crofluidics systems by light. our aim is the development of completely light-controlled microfluidics systems.

Figure 2. Simulation of a light-controlled flow switch. The distribution of electric field and flow pattern are determined by the finite element method

Contact: [email protected]

Pál ORMOS OPTICAL MICROMANIPULATION

10

BIO

PHYS

ICS

Bioelectronics has a double meaning in scientific lit-erature. on the one hand, as a branch of basic biophysi-cal sciences, it deals with electric phenomena appear-ing on any organization level of living systems (a). on the other hand, as a recently developed discipline of in-formation technological science, it explores the poten-tial of biological materials for application in molecular electronics (B). These two areas of research are in close interaction not only with each other, but also with oth-er disciplines of basic and applied sciences. our main goal is to develop novel methods on integrated micro- and nanotechnological platforms for the investigation of light-induced processes in biological membranes, and utilize them in both branches of bioelectronic sci-ence. Besides its impact on basic biophysical science, this research is expected to have applications in various branches of molecular electronics.

A) Electric signals associated with membrane transport processes

electric phenomena, ubiquitous in living systems, carry a lot of information about basic physiological processes (see, e.g., ecG or eeG) that are inaccessible to other techniques. They can all be traced back to the cellular level, namely to membrane-coupled signal- and energy transduction processes. The importance of methodological developments aiming at the detection of the associated electric signals is underpinned, e.g., by the nobel Prize given for the patch clamp technique (neher and sackmann, 1991).

However, application of the most commonly used microelectrode methods to the investigation of trans-membrane ionic currents often fails due to technical limitations, whereas alternative optical techniques still suffer from fundamental sensitivity and time resolution problems. active pump currents, therefore, are still measured on suspensions of cell organelles or cells by macroelectrode methods, in whose elabora-tion our institute in szeged played a determining role. The generalization of one of our techniques allowed the detection of intramolecular electric signals in all the three spatial dimensions.

bIoeleCTRonICs of Ion-TRanspoRTInG membRane pRoTeIns

András DÉR / Principal Investigator

Lajos KESZTHELYI / Professor emeritusRudolf TóTH-BOCONáDI / staff scientistLászló FáBIáN / Phd studentAnna MATHESZ / Phd student

11

BIO

PHYS

ICS

from the application of the technology and its com-bination with molecular dynamics simulations, we expect fundamental information concerning changes in the electric field distribution inside the molecule during its function. We demonstrated the ability of our technique on the simplest and best-characterized active ion transport protein, the light-driven proton pump, bacteriorhodopsin (br), where the description of the molecular function is close to atomic-level pre-cision.

recent developments in nanotechnology also offer the opportunity of a natural extension of our tech-niques to the investigation of bioelectric phenomena of other objects such as single cells.

B) Protein-based integrated optical switching

since the start of integrated electronics, the expan-sion of development has been described by “moore’s law”: the density (performance) of integrated elec-tronic circuits doubles about every 1.8 years. While this “law” has remained valid for a remarkable peri-od of 30 years, there is a general perception that the evolutionary development has reached a limit. It is agreed that future development needs revolutionary new principles. Presently, all possible candidates are explored in the search for new routes. molecular elec-tronics combined with optical data processing is re-garded as being among the most promising emerging alternative technologies.

coupling of optical data-processing devices with microelectronics, as well as sensory functions, is one of the biggest challenges in molecular electronics. suit-able nonlinear optical (nLo) materials with high sta-bility and sensitivity are being intensively researched. In addition to organic and inorganic crystals, biologi-cal molecules have also been considered for use in op-toelectronics, among which br has generated the most interest.

our recent results demonstrated the applicability of this protein as an active, programmable nonlin-ear optical material in all-optical integrated circuits. Based on these findings, a usa patent [Light-driven integrated optical device (us 6,956,984 B2)] has re-cently been registered. In addition, preliminary results suggest that all-optical switching utilizing the Br to K transition is also possible. taking into account the fast kinetics of the K intermediate (picosecond rise time), its application in integrated optics or telecommunica-tion could be especially advantageous. our long-term

András DÉR BIOELECTRONICS OF ION-TRANSPORTING MEMBRANE PROTEINS

12

BIO

PHYS

ICS

goal is to elaborate br-based films supporting applica-tions of optical and optoelectronical components and devices.

Hofmeister effects

Water is the third most abundant molecule in the universe (after H and co), and the most abundant on earth. The major part of living organisms is made up of water (on each level of organization). If water is ex-tracted, proteins do not function. “Water is a matrix providing stability and flexibility of proteins at the same time.” (Philip Ball)

some unique physical-chemical properties: high electric dipole moment, network of H-bonds, fast proton exchange. according to molecular dynamics modelling, such cluster-formations are more frequent at lower than at higher temperatures:

The reason is the change of H-bond strength versus temperature. What consequences could this have on proteins? temperature change is the most straightfor-ward tool to change the strength of H-bonds; however, this has an impact on the Brownian motion of protein molecules as well. addition of salts which do not af-fect pH, and do not specifically interact with proteins might help this problem. The non-specific effects of neutral salts on protein aggregation and conformation have been known for a long time, and are called Hof-meister effects after their first investigator. according

to the investigations, the effects are dominated by ani-ons rather than cations. In 1888, Hofmeister ordered the anions according to their ability of precipitating globular proteins from water:

SO4-- > F- > CH3COO- > Cl- > Br- > I- > ClO4-, SCN- (1)

cl- has the least effect (in the middle of the row), while those on the left-hand side of (1) are called ko-smotrops (increase in aggregation: “salting out”), and the right-hand-side ones are called chaotrops (increase in solubility: “salting in”). Interestingly, the same series was found for protein conformation and activity, too: normally, kosmotrops stabilize conformation and in-crease activity, whereas chaotrops destabilize confor-mation and decrease activity. disturbingly, however, the tendency is just the opposite in some cases. such exceptions make the elaboration of a coherent theory of Hofmeister effects rather difficult. The main goal of our research is to develop and apply a comprehen-sive theory of Hofmeister effects. The starting point is that both in aggregation and conformational changes, there is a change in water-exposed protein surface area. according to our hypothesis already supported by a growing line of experimental evidence, the salt-dependence of protein-water interfacial tension holds the explanation of Hofmeister effects.

We are going to provide a microscopic interpre-tation of the effects by the investigation of protein conformational fluctuations. another goal is to use Hofmeister effects as an experimental tool to pinpoint large conformational changes during protein func-tion.


András DÉR BIOELECTRONICS OF ION-TRANSPORTING MEMBRANE PROTEINS

13

BIO

PHYS

ICS

Géza GROMA / Principal Investigator

Zsuzsanna HEINER / staff scientistFerenc SARLóS / staff scientistAndrás MAKAI / Programmer

femtobiology is a novel branch of science, focusing on ultrafast processes in biological systems, occurring on a femtosecond (10-15 s) timescale. although com-mon biological reactions are much slower, elemen-tary molecular events, such as chemical bonding and unbonding, as well as vibrational and rotational mo-tions take place in this time range. In this context all chemistry is femtochemistry and all biology is femto-biology. By the methods of classical spectroscopy the above processes could be investigated only indirectly in the frequency domain. The advent of ultrafast lasers made possible studying these events directly in time domain, yielding detailed, previously unobtainable information. (The spectrum can be calculated from the time-evolution, but not vice versa.)

The ultrafast charge redistribution in the active center of bacteriorho-dopsin induces electromagnetic radiation.

our recent femtobiological research – carried out in national and international collaborations – mainly focused on the light-induced primary charge separa-tion processes taking place in bacteriorhodopsin (br), a protein utilizing light energy. our calculations indi-

cated that these ultrafast charge motions – as sources of the familiar Hertz dipole radiation – could emit electromagnetic radiation at an experimentally detect-able level. Based on this finding, we started spectro-scopic experiments with femtosecond time-resolution to study the components of this radiation falling to the far infrared and terahertz regime. The main results of this study are the following:

Terahertz radiation of bacteriorhodopsin and its modelling by electron and proton translocation.

By coherent infrared emission of 10 fs resolution we demonstrated for the first time the phenomenon of optical rectification on a biological sample, originating from the excited-state electron polarisation in the retinal chromophore of br. for exact interpretation of the effect we elaborated the theory of resonant optical rectification, derived from the fundamentals of quantum electrodynamics. following the excitation we also observed the existence of time-domain synchronized (coherent) vibrations. It was found that these vibrations partially originate from modes characteristic of the excited state, reporting among

femTobIoloGICal sTUDIes on baCTeRIoRHoDopsIn

14

BIO

PHYS

ICS

others about the isomerisation of retinal, a functionally important process in the energy conversion.

also for the first time we detected coherent terahertz radiation from a biological sample by a measuring apparatus built directly for this purpose and ensuring the investigation of somewhat slower (200 fs – 5 ps) charge motions. This system made feasible to follow the complete process of the excited-state charge redistribution in retinal. In addition, in the emission signal we observed an additional component of several ps life-time, attributable to the appearance of the primary functional proton motions.

for the analysis of the above and further experi-ments, we also studied the ultrafast absorption ki-netics of native and chemically modified br samples. furthermore, we experimentally determined the opti-cal refractive index of br in a wide spectral range, and

calculated the corresponding sellmeier coefficients, describing the dispersion properties of the protein.

Further research plans, possible utilization of the results

In our laboratory the construction of an ultrafast optical pump-probe unit is in progress, with the capa-bility of measuring absorption kinetics as well as fluo-rescence up-conversion in the time range of 100 fs – 1 ns. our aim is to operate this laboratory as a national service for solving appropriate problems in the area of both research and development.


Géza GROMA FEMTOBIOLOGICAL STUDIES ON BACTERIORHODOPSIN

15

BIO

PHYS

ICS

Lóránd KELEMEN / Principal Investigator

Badri PRASHAD / Phd student

recent technological developments enable the re-placement of large, complicated and expensive instru-mentation with cheaper and smaller ones. among these are the widely studied lab-on-a-chip systems ap-plying microfluidic methods. In these systems chan-nels, reactors and reservoirs are built on a microscope slide within a few mm2 area in order to carry, mix, react and detect minute volumes of sample. These microscopic labs require the application of mechani-cal devices of a few micrometer dimension.

In our lab we are developing micromachinery for microfluidic applications. The structures with a typi-cal size of less than ten micrometer and sub-micro-meter resolution are made of polymer that hardens in a pre-defined 3d shape upon illumination with fo-cused laser light. The beam consisting of femtosecond pulses initiates two-photon absorption in the polymer exclusively in the vicinity of the focal spot.

Electron microscopic image of a surface-integrated, light-driven micro-motor and light-guide

This two-photon polymerization method enables the production of structures with features down to 200 nanometers.

Polymerization of light-driven microstructures

The first group of polymerized structures is actu-ated by radiation pressure when the impulse of light is transferred to the objects via reflection, thereby ini-tiating their movement. a typical example for these structures is a surface-attached micro-motor of 10µm diameter. The motor rotates on an axis when illumi-nated by a beam emerging from a light guide, also polymerized to the surface by a focused laser beam. similar wheels are intended to serve as power sources for complex structures performing various microflu-idic tasks. We are developing gear shift systems for this kind of machinery, similar to those of the mac-roscopic world. Besides the successful polymerization of various gears, we have polymerized other mechani-cal parts, such as spiral springs and tested their per-formance.

Polymerization of micromanipulators for optical tweezers applications

our new research area is the polymerization of mi-crotools for optical trapping systems. optical tweez-ers are based on the trapping force of a focused laser beam exerted on transparent micrometer-sized ob-jects with refractive indexes higher than that of the surrounding medium. When the task is to interact with biological objects, most trapping systems use

DevelopInG aRTIfICIal mICRoTools foR bIoloGICal applICaTIons

16

BIO

PHYS

ICS

Lóránd KELEMEN DEVELOPING ARTIFICIAL MICROTOOLS FOR BIOLOGICAL APPLICATIONS

chemically functionalized microbeads. The beads en-able only translational manipulative motion. With the tools we polymerize, the optical trap will be ca-pable of manipulation with more complicated forms of motion. due to the variability of the tools’ shape, non-trivial movements such as the twisting of the sample will become possible. We tested the manipula-tion capabilities of the microtool-optical trap system in an experiment where a complex 3d superstructure was assembled, an operation which was only possi-ble with high-precision translational and rotational movements.

Electron microscopic images of the basic units polymerized for the 3D assembly experiment, and optical microscopic image of the process of the assembly

Polymerization with modified laser beams

The most important reason for the investigation of the application of modified laser beams is the accelera-tion and simplification of the polymerization process. Here the initial single laser beam is altered by a spatial Light modulator (sLm) such that several identical la-ser beams or extended illuminated patterns are pro-jected into the sample and perform polymerization. When we create several distinct but identical focal spots, the result of this parallel-type polymerization is the same number of identical objects (a couple of tens of them). When a complex and continuous light pat-tern is focused into the sample, the polymer hardens in the same shape, without the need of scanning of the beam.

Surface activation of the polymerized microstructures

The su8 polymer used for polymerization is an epoxy-based resin which is chemically quite inert, therefore it practically does not interact with biologi-cal objects without further treatment. In many cases this is a requirement, but for the micromanipulators designed for optical trapping applications, the inter-action is a must. for this the surface of the polymer has to be activated through one of the processes de-scribed in the literature. We are adopting the surface treatment protocols to the microstructures, equipping them with small functional groups, macromolecules, proteins, as well as metal colloids. our initial results show the coating of microrods’ and manipulators’ sur-face with the protein streptavidin in high density.

Electron and fluorescent optical microscopic image of polymerized heli-cal rods. The optical image shows the fluorescence of the labeled protein streptavidin applied on the rods’ surface

Future plans, applications

The polymerized microstructures can be used in microchannel environment to pump or mix liquids of picoliter volumes. The manipulators made for optical trapping systems, depending on their artificially cre-ated surface quality, will enable researchers for various localized measurements on cell surfaces.


17

BIO

PHYS

ICS

Ilona LACZKó / Principal Investigator

The main field of chiroptical spectroscopy is the investigation of the steric structure of biopolymers such as proteins and nucleic acids. circular dichroism (cd) is a phenomenon that results when chromo-phores in an asymmetrical environment interact with polarized light. In proteins the major optically active groups are the amide bonds of the peptide backbone. The far-uv cd is generally reflective of the backbone conformation and different secondary structures in polypeptides and proteins give characteristic far-uv cd spectra. cd is a simple and quick method, its time-scale is below the femtosecond region. due to the low time-scale, any cd spectrum can be resolved into the component spectra of the main conformer types (α-helix, β-sheet, random, etc.). This method can also be successfully combined with other vibra-tional spectroscopic methods of similar time-scale [fourier transform infrared (ftIr), raman, vibra-tional circular dichroism].

recently several studies have been performed in national and international cooperations, the most im-portant are the following:

– Aggregation of Aβ(1-42) amyloid peptide in the presence of short peptides. one of the most character-istic pathological markers of alzheimer’s disease is the accumulation of the neurotoxic amyloid polypeptide consisting of 42 amino acids. The aggregation of this peptide – which induces neuronal apoptosis - is pre-ceded by changes in the secondary and tertiary struc-tures, which can be followed by cd spectroscopy. With the aid of combined cd and ftIr spectroscopies the effect of different small peptides on the aggregation profile of aβ(1-42) is studied.

– Complex-formation between antisense oligonu-cleotides (AON) and cell-penetrating peptides (CPP). The application of aons modulating gene expression is a promising approach in medicinal therapeutics. The method is based on the delivery of aon into the cells by peptides capable of translocation through the cell membrane. The major limitation of the technique is the low efficiency of membrane translocation and targeting. Hence, the main purpose of our work in this field is screening for cPPs which are highly effective in membrane translocation and targeting. complex for-mation between cPP and aon can be followed by cd, ftIr spectroscopies and atomic force microscopy and the cPP/aon molar ratio optimal for cell penetration also can be estimated.

– Secondary and tertiary structures of different hy-drophilic enzymes. There is growing interest in the use of enzymes in aqueous organic solutions. as compared with other catalysts, enzymes are stereoselective. non-aqueous enzymology is of particular relevance when the desired reactants are poorly soluble in aqueous so-lution and when the reversal of a hydrolytic reaction is desired. The enzymes are able to catalyze reactions in organic solvents at low water content; however, they are less stable than in water. There is a direct relation-ship between the stability and the conformation of en-zymes. Therefore we investigate the effect of organic solvents and stabilizers on the secondary and tertiary structures of various enzymes such as trypsin, chymo-trypsin, papain, pepsin, etc.


applIeD CHIRopTICal speCTRosCopY

18

BIO

PHYS

ICS

movement is a fundamental feature of living or-ganisms and systems, and the same is true for man, the most developed living system. The importance of the information content of these movements (fre-quently representing deeper, e.g. molecular or even in-tramolecular processes as well) have long been known intuitively. (Think about e.g. the effects of ethanol con-tained in alcoholic drinks). However, methods that are really suitable for the quantitative study of these mo-tions have either been developed only recently, or are now in the process of being developed.

Presently the two main (methodological) focuses of our research activities are to study human body move-ments also affected voluntarily by actigraphy, and an internal movement that is practically uncontrollable voluntarily, the continuous changes in pupil size by video-pupillography.

The actigraph usually is a wrist-wearable watch-sized device that is capable of recording movement activi-ties for a relatively long period of time (for several days or weeks), and the collected time-activity data can be transferred into a computer for the purpose of analysis. actograms carry a lot of information about the biologi-cal clock, daily routine, jet lag and various psychiatric disorders. some of their characteristics are already rou-tinely used in medical therapy, but the complex struc-ture of actograms is still mostly unexplored. The reason for this paradox is that while fourier analysis, the main tool used for the evaluation of actograms, is effective in revealing periodic components, it fails to analyse sto-chastic events—resulting in fluctuations—that seem to be prevalent on the ultradian scale (fig. 1a ).

Figure 1: A) Activity recording of a typical day. B) Integral function of the curve in A)

It is a key question in life sciences whether the stochastic dynamics of physiological rhythms are an essential feature for their function, or they are either simple consequences of environmental fluctuations or associated with certain malfunctions. our aim is to find and characterize some new, general features in human physical activity patterns that are expected to shed more light on the nature of complex physiologi-cal processes underlying activity signals.

In the case of video-pupillography, the movements of the subject’s pupil(s) are recorded for a relatively short or long period of time (usually for 1–15 minutes) by a video-camera system, and analyzed by computer programs.

HUman bIopHYsICs anD movemenT analYTICs

Zsolt TOKAJI / Principal Investigator

András DÉR / Principal InvestigatorKrisztina SZABó / Phd student

19

BIO

PHYS

ICS

Figure 2: Probabilities of the appearance of different pupil diameters as a function of time during an 11-minute pupillographic sleepiness test in the case of an alert and a sleepy subject.

Both of these main methods were able to find and show differences between healthy state and the one accompanying affective disorder (depression). Both of these methods are extremely suitable for the meas-urement of alertness, which is presently hardly meas-urable by other objective methods. In these alertness measurements, the strength of video-pupillography is the ability to measure the actual/short term level of alertness as well, whereas actigraphy is excellent for measuring average alertness and its periodicity.

using actigraphy, we have proved and characterized the appearance of a higher order structure of human daily activity. figure 3 shows the result of a statistical analysis of the spikes in fig. 1a.

Figure 3: Probability density functions (PDFs) of activity spikes recorded under conditions described in Fig.1. The time scale of the daily recordings was divided into equal intervals (“boxes”), and activity values were averaged within each box. PDFs were calculated from these averaged values. Data are shown for some characteristic box lengths between 1 and 720 minutes distinguished by different colours. Curves in (A) and (B) are obtained from the original recordings and the scrambled control, respectively.

note that an explicit depression is built up in the middle of the curve by increasing box size, persisting for box lengths of several hours. The meaning of this feature becomes obvious when we take a look at the integral function of the daytime activity trace of an

average day (fig.1b). It is apparent that there are two characteristic slopes of this function, corresponding to the two peaks in the Pdf. during the active periods average activities are similar, and centered to a higher value (right peak in fig. 2a, or the bigger slope in fig. 1b), while it is easy to distinguish resting periods with activities close to zero (left peak in fig. 2a, or plateaus in fig. 1b). In other words, on this time scale the hu-man physical activity is quasi-binary: it is distributed such that well-defined bursts are followed by passive periods. This novel finding, generally characteristic of all cases studied, is expected to inspire new math-ematical models of human physical activity.

although video-pupillography is a relatively rap-idly performable experimental method, we have used it successfully in darkness for the detection of changes of alertness due to natural reasons (e.g. in the case of children with attention-deficit hyperactivity disorder) or drug effects (e.g. nicotine). under room light con-ditions video-pupillography can provide information about changes in the equilibrium of the sympathetic/parasympathetic nervous activities originating either in natural causes, or resulting from drug use (e.g. methyl-phenidate) or a treatment (e.g. bright light exposure).

In our laboratories, unconventional methods (e.g., distribution function or wavelet analysis) for the evalu-ation of actigraphic and video-pupillographic data are also applied. several new methods are under develop-ment or are being currently introduced into practice.


our methods are suitable for achieving a better un-derstanding and characterization of human behaviour, with respect to its temporal organization and patho-logical alterations. The effects of drugs on the brain can be studied, their transport into and elimination from the brain can be monitored by these methods. By using drug/chemical interactions, conclusions can also be drawn even for intramolecular alterations con-cerning e.g. receptor-mediated processes.


Zsolt TOKAJI HUMAN BIOPHYSICS AND MOVEMENT ANALYTICS

20

BIO

PHYS

ICS

In the last years wonderful results have been pub-lished in the field of single molecule visualization and manipulation techniques. The atomic force microscope is increasingly used in biological research. The instru-ment was developed in the 20th century. a small tip at the end of a µm-size cantilever scans the studied sur-face. during the scan the deflection of the cantilever is proportional to the force acting between the tip and the surface. The spatial resolution of the instrument is de-termined by the sharpness of the tip. on the highest res-olution images even atoms can be distinguished. com-pared with the electron microscope a great advantage of the instrument is that biological samples can be studied in fluids, in their natural environment. This makes pos-sible to observe proteins or cells during function.

With the aid of atomic force microscope results impossible to attain by other techniques have been achieved in biological systems.

Here we present several results from our atomic force microscopy research:

– By studying oligonucleotides we observed that short sequences of nucleic acids self-assemble on mica

surface in a long chain-like formation. This self-assem-bly could have a role in the origin of life, by forming dna or rna, the information carrying molecules.

– on protein level we studied the interaction of the photosynthetic reaction center with carbon nano-tubes. The complex formed is a promising material for biotechnological applications.

– studying the proton pumping protein bacteri-orhodopsin, we measured the size change during its function, which was direct detection of the conforma-tional change of the protein.

– during the study of endothelial cells we observed that mannitol treatment influences their volume and elasticity. upon prolonged calcium treatment a change in cell shape could be detected.

– We demonstrated a difference in shape and elas-ticity between wild-type and mutant bacteria.

Further developments

The accumulated knowledge helps develop new nanotechnological materials and contributes to un-derstanding the action mechanism of drugs during medical treatments.


bIoloGICal applICaTIon of THe aTomIC foRCe mICRosCope

György váRó / Principal Investigator

Zsolt SZEGLETES / staff scientistAttila Gergely vÉGH / Phd studentKrisztina NAGY / Phd student

21

BIO

PHYS

ICS

László ZIMáNYI / Principal Investigator

Katalin TENGER / staff scientistPetro KHOROSHYY / staff scientist

cytochromes are heme-containing proteins, which carry out diverse physiological functions, such as electron transfer, an important process in the energy metabolism of living cells. c type cyto-chromes are distinguished from other cytochromes by the covalent attachment of the heme group to the protein. The advantages of this covalent binding are so far unclear. The maturation of mitochondrial cy-tochrome c, i.e. the covalent binding of the heme co-factor is catalysed by the enzyme cytochrome c heme lyase. despite the importance and the widespread oc-currence of these proteins from yeast to human, we know little about the catalytic process or about the interaction of the two proteins and the heme. recent studies have also shown that the mitochondrial cyto-chrome c has another fundamental role besides elec-tron transfer in triggering programmed cell death (apoptosis).

We express cytochrome c heme lyase in a bacte-rium and purify the protein. We study the interac-tion of the purified heme lyase, the heme and the apocytochrome (cytochrome c without heme) in or-der to clarify the details of cytochrome c maturation on a molecular level. according to our hypothesis the structure of both the heme lyase and the cyto-chrome may alter as a result of their interaction with each other and with the heme. Indirect information about this interaction is supplied by our observation of spontaneous cytochrome c maturation (covalent heme attachment in the absence of the cytochrome

c heme lyase) at a much lower efficiency than in the presence of heme lyase. However, the spontaneously matured cytochrome c has slightly different physico-chemical parameters as compared to the native pro-tein. This indicates that the function of heme lyase is not only the catalysis (acceleration) of covalent heme attachment, but also to facilitate the formation of the final, native conformation (structure) of cytochrome c. Without heme lyase the first process can take place (albeit slowly, with low efficiency), but the second one cannot.

cytochrome c is but one component of a complex energy transducing and storing apparatus consist-ing of various proteins and membranes. metabo-lism is based on the migration of electrons through well-organized protein chains towards lower energy, analogously to the current of electrons in the elec-tric circuit from one pole through a load to the other

maTURaTIon, sTRUCTURe, anD eleCTRon TRansfeR pRopeRTIes of CYToCHRome C

22

BIO

PHYS

ICS

László ZIMáNYI MATURATION, STRUCTURE, AND ELECTRON TRANSFER PROPERTIES OF CYTOCHROME C

pole. There is, however, a basic difference between the mechanism of electronic conductance in a metal wire and in a protein. our goal is to better understand the mechanism of electronic conductance in proteins, and to study whether or not nature has optimized (and how) the conductance in certain important proteins. our experiments are mainly performed on cytochrome c. We label the surface of the cyto-chrome with a molecule which becomes an electron source after its irradiation with a short laser pulse, and which donates its electron to the heme group within the protein, and recovers the electron after-wards. We can measure the rate of electron transfer and thereby compare various regions on the protein surface as well as various prospective routes (direc-tions) within the protein in terms of electronic con-ductance. We attempt to explain the link between the efficiency of electronic conductance and the structure of the protein with model calculations. In the figure we coloured the surface of cytochrome c according to the calculated efficiency of electron transfer from the heme (in the middle) to the various regions of the surface as green, red, and blue for average, good, and poor conductance, respectively.


We plan to investigate the electron transfer routes, the efficiency of electron (and charge) transfer and its energetic utilization in the more complex but simi-larly important physiological oxidizing partner of cytochrome c, the cytochrome c oxidase protein. cy-tochrome c itself is a promising candidate as a com-ponent of biomolecule-based sensors, bioelectronic designs, therefore the understanding of its electric conductance is a high priority. Within the framework of an international collaboration we also plan to incor-porate cytochrome c into hybrid biophotonic archi-tectures – photonic crystals based on porous silicon. Thereby we expect to tune the optical properties of the semiconductor-based photonic crystals using the col-oured cytochrome protein. conversely, we intend to study the effects of the interesting nonlinear optical phenomena characteristic of the photonic crystals on the optical properties of the protein.


23

BIO

PHYS

ICS

Tibor PáLI / Principal Investigator, Group Leader

Balázs SZALONTAI / senior scientistZoltán KóTA / staff scientistCsilla FERENCZ / Phd studentErika KóNYA / technician

Proton pumping by a membranous molecular motor, the vacuolar proton-ATPase (V-ATPase)

The v-atPase is a membrane-bound molecular ro-tary engine, which converts the chemical energy from atP hydrolysis to the rotation of the rotor domain via a torque between specific subunits. This leads to trans-membrane proton pumping in the interface between the stator and rotor domains. The v-atPase plays an important role in diseases like osteoporosis and in the metastasis of tumours. Therefore, specific inhi-bition of certain sub-classes of the v-atPase family has direct medical and pharmaceutical relevance. to date there is no atomic resolution structure of the v-atPase known and its mechanism of function is not known either.

The proton-translocating trans-membrane machinery of the vacuolar proton-ATPase: the subunit c ring (rotor) and subunit a (stator).

In extensive international collaborations we have shown earlier that the c subunit of the Vo domain is a representative of the ductins family of highly homolo-gous and structurally similar membrane proteins. We have determined the assembly of the subunit c ring in native membranes as a hexamer of four-helix bundles

and found that highly specific v-atPase inhibitors perturb the lipid-protein interface of the Vo domain. We localized a unique cysteine and the glutamic acid residue, essential for proton transport, which were found to face lipids.The primary long-term objective is the better un-derstanding of structure-function relationship and the identification of functionally relevant structural changes in the engine. This project is aimed at the study of the proton pumping and atP hydrolyzing functions of the Vo and V1 domains, respectively, their connection and interaction; the arrangement and interactions, also with lipids, of the Vo subunits; the rotation of the rotor domain; structural stability and the effect of structural and functional agents, e.g. spe-cific inhibitors, on all these features in intact vacuoles, vacuolar vesicles and in reconstituted v-atPase-lipid vesicles. We aim to develop the structural models of the intra-membranous a and c subunits based on our structural data, whereas the function will be inter-preted in physical models.

Protein insertion, folding and assembly in membranes and on membrane surfaces

Protein folding in general and membrane protein folding in particular are most challenging problems in biophysics today, because membrane lipids and proteins are coupled structurally, dynamically and functionally. The protein-lipid interface takes several different forms, all of which are crucial to biology.

speCTRosCopY-baseD sTRUCTURal bIoloGY of bIomembRanes anD membRane pRoTeIns

24

BIO

PHYS

ICS

Tibor PáLI SPECTROSCOPY-BASED STRUCTURAL BIOLOGY OF BIOMEMBRANES AND MEMBRANE PROTEINS

for biological membranes the protein-lipid interface may be either polar in the case of surface-bound or absorbed peripheral proteins, or apolar in the case of integral trans-membrane proteins. focusing on the protein-lipid interface requires studies on the struc-ture, the dynamics and the function of both mem-brane proteins and lipids. We are also interested in this problem, because the work on membrane proteins requires they be inserted and assembled properly in the bilayer to achieve functional reconstitution. our objective is to obtain experimental data on factors guiding insertion, folding and assembly of proteins and polypeptides in membranes and on membrane surfaces. These data are used to guide and constrain molecular and physical models (as shown in the fig-ures taken from our publications). We pay increas-ing attention to theoretical approaches to membrane protein folding too. at present, we focus on three groups of proteins: trans-membrane helix (v-atPase subunits and polypeptides), beta-barrel (E. coli outer membrane proteins) and soluble proteins interact-ing with bio- and model membranes (lysozyme).

Structure prediction for a 4 trans-membrane helix electron transport membrane protein unit based on homology and structural constraints.

Bovine rhodopsin (PDB ID: 1L9H) surrounded by a single (partly hidden) shell of energy-minimised bilayer lipids.

Approaches and techniques

The working strategy for the above membrane-pro-tein systems is that structural, dynamic and thermo-dynamic data on native and reconstituted membranes are measured, during permanent control of the bio-logical function, wherever possible, using a range of biophysical techniques, which are then consistently interpreted in detailed molecular models and related to the biological function. data are obtained with a variety of techniques and their combinations. These include fourier-transform infrared (ftIr), site-spe-cific spin-labelling and spin-trapping electron para-magnetic resonance (ePr), polarized attenuated total internal reflection ftIr, uv, visible and fluorescence spectroscopy; high-sensitivity differential scanning calorimetry (dsc); theory and computation (physical models, spectrum simulations and molecular model-ling). This approach can be termed as function-con-trolled spectroscopy-based structural biology.


25

BIO

PHYS

ICS

Alajos BÉRCZI / Principal Investigator

In 1971 scandinavian researchers discovered a b-type cytochrome in bovine chromaffin granule membranes that (1) participated in trans-membrane electron transport but was not a member of the mito-chondrial electron transport chain, (2) was essential for the synthesis of neurotransmitters, and (3) needed the presence of ascorbate (vitamin c) for its biological activity. The newly identified protein was named cyto-chrome b561 (cyt-b561) protein.

It has recently become obvious that proteins simi-lar to the bovine chromaffin granule cyt-b561 protein are common in most, if not all, eukaryotic organisms, including plants, animals and humans too. moreover, these organisms, in general, contain more than one protein with physical and chemical properties very similar to those of bovine cyt-b561 protein. such pro-teins accept electron (e—) from ascorbate (asc) on one side of a biomembrane, transfer the electron—across their „protein body”—from one side to the other side of the biomembrane, and donate the electron to some electron acceptor molecule (aox) on the other side of the biomembrane. on the basis of their physical and chemical properties, such proteins form the cyt-b561

protein family. The electron acceptor is ascorbate free radical (aox≡asc*) for the chromaffin granule cyt-b561; however, the electron acceptors of the other cyt-b561 proteins are not known. at present 5 members of the cyt-b561 protein family are known and charac-terized in some detail. one major aim of the present research is to identify the electron acceptors for and the physical-chemical properties of the new members of cyt-b561 protein family, since this information is essential and needed for understanding the biological function of the newly discovered cyt-b561 proteins.

It is already evident that members of the cyt-b561 protein family take part in vitamin c metabolism. Preliminary laboratory experiments suggest that (1) some cyt-b561 proteins also play a role in fe-metab-olism, and (2) one member of the cyt-b561 protein family is expressed with other tumour suppressor proteins in cancerous cells and thus participates in their elimination.

Future possibilities

since the newly discovered members of the cyt-b561 protein family are integral trans-membrane, electron-transporting proteins definitely participating in vitamin c metabolism and probably participating in fe-metabolism, detailed knowledge of their structure, function, and regulation would open new perspectives in plant nutrition, health, medical, and pharmaceuti-cal research.


THe CYToCHRome B561 pRoTeIns

26

BIO

PHYS

ICS

Without membranes, there are no living organ-isms. membranes separate the cells from the outside world, but at the same time membranes embed those proteins, which sense the signals of the outside world and perform material transport through them. In the interiors of the cells, the generation of energy takes place in membranes, including the primary energy source for all forms of life on earth, photosynthesis. after sequencing the complete genome of several or-ganisms, it seems that about 30% of their sequences encode membrane proteins. In contrast, from the sev-eral tens of thousands of proteins whose 3d structure is known in atomic resolution, only a few dozens are membrane proteins. The reason for this discrepancy between abundance and knowledge is that membranes are very complex assemblies, where proteins and lip-ids together form functional units. The lipids supply the insulation capacity of the membranes, and their hydrophobic double layer provides the working con-ditions for membrane proteins. Isolated membrane proteins are therefore very difficult, and frequently practically impossible to study, and there is always the intriguing question: to what extent the obtained struc-tures, features correlate with those existing under in-membrane conditions.

for the resolution of this problem, two interrelated lines of research can be pursued. on the one hand, whole, possibly intact biological membranes can be studied as they are, by applying methods selectively sensitive for different membrane properties. on the other hand, model systems can be developed which, by mimicking natural conditions as close as possible, permit the study of individual components or particu-lar processes. Then, by compiling the data obtained by

the two approaches, one may hope to obtain a com-prehensive picture of the lipid-protein interactions in biological membranes.

according to this strategy, we apply and further de-velop the perfectly non-invasive fourier transform in-frared (ftIr) spectroscopy in biological membranes. This method has the advantage of having the lipid- and protein-related spectral regions well separated. Thus, both lipids and proteins can be studied individ-ually and, furthermore, via the correlations between changes in the lipid- and protein-related regions the lipid-protein interaction in the membranes can also be addressed.

Figure 1: Schematic view of the attenuated total reflection (ATR) mode of the Fourier transform infrared (FTIR) measurement. PEI – a positively charged polyelectrolyte, which adheres very well to surfac-es. PGA – Poly(glutamic acid) – a negatively charged poly amino acid; PLL – Poly(lysine) – a positively charged poly amino acid.

In the other direction, developing better model sys-tems, we use a nano-technological approach. We build protein-like surfaces layer-by-layer from oppositely charged polyelectrolytes. Thus, we can obtain surfac-es, which mimic the cytoskeleton of the cells. Then, biological and model membranes can be adsorbed

lIpID-pRoTeIn InTeRaCTIons In bIoloGICal anD moDel sYsTems

Balázs SZALONTAI / Principal Investigator

27

BIO

PHYS

ICS

onto these surfaces. In this way, transport processes and lipid-protein interactions can be studied on a stable support on both fully natural membranes and “hybrid” systems built from natural and synthetic components. Infrared spectroscopy also plays an im-portant role in these studies, since ftIr spectroscopy when used in attenuated total reflection mode (atr) (which means that we measure the adsorbed sample on the surface of an internal total reflection element) can follow the structural changes associated step-by-step with the buildup of the polyelectrolyte film and the adsorption of any further component.

concerning the complexity of biological membranes and the need for developing new techniques for their study, at the beginning our research is purely basic re-search, and we are in this phase now. We keep in mind, however, that detailed knowledge and effective experi-mental techniques can have important bio-medical applications. Therefore, when selecting the systems for study, we consider biologically, medically important ob-jects (selected lipid species, ion-channel proteins, etc.).


Balázs SZALONTAI LIPID-PROTEIN INTERACTIONS IN BIOLOGICAL AND MODEL SYSTEMS

28

BIO

PHYS

ICS

1. Structure and function of redox proteins

redox proteins play an important role in the bio-energetic processes of living organisms. energy pro-ducing, utilizing, transforming processes are present in all living systems; the research of redox proteins can hardly be overestimated. most of the redox proteins contain some kind of metal (iron, copper, etc.).

our group isolates proteins from the purple, sul-phur, photosynthetic organism Thiocapsa roseoper-sicina. several metal-containing and redox active proteins have already been purified. The long-time favourite and most widely investigated protein is Hyn hydrogenase (see next section). We have also identified and characterized a number of other metal-containing proteins, including a flavocytochrome, a cytochrome c4 (yet unknown from photosynthetic organism) and a blue copper protein. We have determined their char-acteristics (redox potential, molecular mass, etc.).

despite the fact that the organism does not survive temperatures above 30 °c, the cytochrome c4 is stable and functional at high temperatures (up to 60 °c). The protein undergoes conformational changes upon in-creases in temperature. These conformational changes are reversible under anaerobic conditions and irre-versible if oxygen is present.

Development and exploitation of the results

The investigation of these proteins helps to under-stand the temperature tolerance of the proteins. on the other hand the techniques developed in the course of research are also important and can be used for other research. The most important technique is a new pro-

tein-sequencing methodology, which was developed in cooperation with an american ms group in texas. The protein sequence is determined with the help of a mass spectrometer. usually the protein sequence is determined by determining the nucleic acid sequence and translating it to protein sequence. It is a quite time-consuming, complicated and not always success-ful method for genetically unmapped organisms (and most organisms belong to this group). furthermore the sequence does not necessarily fit the sequence of the working protein. our new method is competitive with sequence determination from the gene, and we hope that with some improvement it will be possible to decrease the required amount of protein to the level of two-dimensional gel spots. The drawback of this method is that it needs expensive mass spectrometers.

2. Investigation of autocatalytic and oscillating enzyme reactions

autocatalytic reactions are quite common in living systems; they are very easy to observe macroscopi-cally. Proliferation and reproduction are typical auto-catalytic processes. In order to produce posteriors, in addition to food one or two parents are necessary.

autocatalytic processes are easily recognized by their characteristic spatial patterns. In the absence of other disturbing effects the reaction fronts are spher-ical (or, in the case of flat reaction arrangements, circular). The radii of the objects are continuously increasing in time (see the figure). The spread of hu-mankind on earth has a very similar pattern.

although autocatalytic reactions are common in living systems, they are quite rare in elementary re-

sTRUCTURe anD fUnCTIon of ReDox meTallopRoTeIns

Csaba BAGYINKA / Principal Investigator

Gabriella PANKOTAI-BODó / staff scientistRui Miguel MAMEDE BRANCA / staff scientistJudit ŐSZ / staff scientist

29

BIO

PHYS

ICS

actions. We investigate such elementary reactions on bio-molecules and enzymes.

The main object of our study is the hydrogenase enzyme. It can be found mostly in prokaryotes and ar-chae, but some eukaryotes might contain it as well.

Hydrogenase catalyzes a very simple reaction: it splits hydrogen gas into protons and electrons and, as all catalysts, it works in the reverse direction as well, i.e. it produces hydrogen gas from electrons and protons. The enzyme contains metal cofactors, iron, and some hydrogenases also contain nickel. We have found that during the enzyme reaction there is at least one autocatalytic step involved.

The autocatalytic reaction of hydrogenase in thin layer.


autocatalytic reactions are also present in other biological processes. the development of “prion” diseases (creutzfeldt-Jakob disease, scrapie, bovine spongiform encephalopathy, etc.) is also described as an autocatalytic process. In the case of prion proteins, however, the investigation of the kinet-ics of the autocatalytic reaction (how the reaction proceeds, what happens during the reaction) is very difficult. since the reaction of hydrogenase is easy to follow and the concentration of different com-ponents can easily be changed, there is hope that the information and the techniques developed in research on this reaction can be applied to the case of prion reactions.

the autocatalytic reaction of hydrogenase has biotechnological importance. We do not think that with the help of hydrogenase bio-hydrogen would be produced in industrial quantity by splitting the water. But we do think that hydrogenase can be uti-lized as a key constituent of fuel cells. It might sub-stitute the expensive platinum, because it is cheaper, and it doesn’t have to be mined, because it can be produced utilizing solar energy. for its utilization, however, it is essential to precisely know the reac-tion kinetics.


Csaba BAGYINKA STRUCTURE AND FUNCTION OF REDOx METALLOPROTEINS

30

BIO

PHYS

ICS

one paradoxical consequence of the increase of the life expectancy of mankind is the increase of the incidence of those intractable diseases whose risk fac-tor increases with age. The degenerative alterations of the nervous system, associated with such diseases, are at the top of this list. a disease of the motor system, amyotrophic lateral sclerosis (aLs), which currently does not have a cure, deserves special attention com-pared to the other neurodegenerative disorders. first, since it is relatively easy to design predictive clinical trials in this case, this disease is preferred for drug testing by the pharmaceutical companies compared to the other diseases in which the putative drugs are ex-pected to be distributed in a larger market (e.g. among alzheimer’s disease patients).

a reason for this policy is based on the document-ed similarity in the majority of destructive processes observed in the degenerative diseases of the nervous system, thus the results can be generalized. secondly, since the disease also affects the motor axon terminals

located in the skeletal muscle, these parts of the nerv-ous system can be sampled without any complications in the patients, and the results of these tests are easily comparable to those obtained from the animal experi-ments. on the other hand, since neither the cause, nor the exact pathomechanism of the disease is known, explorative basic research is still necessary.

The research of the mechanisms of aLs is built around the anatomy of the voluntary motor system: the pyramidal cells project to the lower motor neurons, which are located in distinct anatomical regions of the brainstem and the ventral horn of the spinal cord. The innervating projections of the lower motor neurons, called peripheral nerves are terminating at the skeletal muscles of the body. during the disease, the progres-sive destruction of the motor neurons causes the loss of innervation of the muscles and leads to the death of the patients if vital muscles are affected. although the complete machinery leading to the destruction of the motor neurons is still unknown, some of the details are already disclosed. such mechanisms are: exhaus-

neURonal DeGeneRaTIon

László SIKLóS / Principal Investigator, Group Leader

Ngo Thi Kue DUNG / staff scientistMelinda PAIZS / staff scientistErika MARáCZINÉ RáCZ / technicianSzabolcs áBRAHáM / technician

31

BIO

PHYS

ICS

tion of the motor neurons due to over-excitation, dam-age of protein and membranous constituents of motor neurons due to the insufficient antioxidant defense, development of deformed cellular structure due to in-adequate assembly of the cytoskeleton, immunologi-cal and auto-immune reactions, and self- amplifying toxic processes due to the impairment of the calcium homeostasis. according to most recent research data, we are aware that during all these pathological proc-esses not only the motor neurons are involved, but the neighbouring astrocytic and microglial cells also take an active role.

our recent studies are aimed at unravelling the role of the stability of calcium homeostasis of the motor neurons during degeneration, and to examine if the neighbouring astrocytic and microglial cells could

modify their resistance. methodically, our experi-ments are based on in vivo, i.e. animal experiments: either production of suitable transgenic animals, or pharmacological treatment or surgery of wild-type and/or transgenic animals would help to answer our specific questions. detection will be mainly by elec-tron microscopic techniques. This technique, in ad-dition to ultra-structural resolution, could provide information on the composition of the sample under investigation on the micro-analytical scale.

In our light microscopic studies we intend to in-vestigate—in a similar experimental paradigm—the structural and molecular changes in the neighbour-ing cells. as an example: after characterizing the mor-phology and distribution of the resting microglial cells in the spinal cord (left panel), motor neurons are experimentally injured and start to express a distress signal (red colour, middle panel), then the change in the microglial population could be determined (mid-dle panel green signal; right panel).


László SIKLóS NEURONAL DEGENERATION

32

BIO

PHYS

ICS

our understanding of the role of gonadal steroids is actually undergoing a major reassessment. experi-mental data indicate that apart from being neuroen-docrine regulators, these hormones exert a variety of morphogenetic and organizing effects in the nervous system. They influence the development of certain brain regions and also affect the differentiation of spe-cific neuronal and glial populations and the establish-ment of synaptic connectivity. moreover, it has been discovered that these hormones can be produced in the central nervous system as well and they act locally, resulting in well-defined structural and functional changes. These data have led to the birth of two cat-egories: neurosteroids, i. e. brain-derived steroids and neuroactive steroids, i.e. steroids acting on the brain but produced in the gonads.

The research interest of the Group is focused on the role of gonadal steroids in neuro-glial plasticity, more specifically on the mechanism of neurodegeneration and neuroprotection. our aim is to perform basic research to study the cellular and molecular mecha-nisms responsible for the neuroprotective effects of certain neurosteroids, namely estrogen and one of its precursors, dehydroepiandrosterone (dHea). at this phase of research the aim is not the develop-ment of new drugs, but on the long run we focus on the possible therapeutic use of neuroactive steroids and neurosteroids. according to our opinion the de-tailed knowledge of hormonal action may provide new pieces of evidence for the understanding of the regen-erative capacity of the central nervous system. on the basis of our results we may work out a model system

to study the neuroprotective effect of new synthetic steroids. any knowledge in this field will be especially useful for the elder generation, because at the time of approaching senescence there is a significant decrease in the plasma concentrations of dHea, IGf-I and es-tradiol (and its precursor testosterone) both in women and men, consequently the neuroprotective effect of these molecules is also diminished.

classical studies supported the idea of extensive neuronal loss even in normal ageing, and neuronal degeneration in the hippocampus and cerebral cor-tex was thought to contribute directly to age-related cognitive deficits. It is now recognised that biological

neURonal plasTICITY anD neURopRoTeCTIon: Role of HoRmones anD neURosTeRoIDs

árpád PáRDUCZ / Principal Investigator

Zsófia HOYK / staff scientistTibor HAJSZáN / staff scientistEszter CSáKváRI / staff scientistAndrea GYENES / staff scientist

33

BIO

PHYS

ICS

changes in the healthy ageing brain are more subtle than it was previously thought and mainly involve shrinkage of neuronal cell bodies and a decrease in the density of neurones and synapses. This is encour-aging news, because it means that treating normal age-related dysfunctions of the nervous system such as memory loss will be possible.

research carried out in different laboratories has demonstrated that the ageing nervous system retains its capacity for regeneration and that age-dependent biological changes such as memory impairment and myelin abnormalities can be reversed by the admin-istration of estrogen and other neurosteroids. Their

findings also show that the reduction in neurosteroid levels during ageing renders the nervous system more susceptible to degeneration.

our working hypothesis is that the beneficial effect of these compounds is not limited to the protection of neurons, but may affect preserving and/or remodelling synaptic connections as well. such hormonally driven synaptic plasticity can explain memory improvement and the functional recovery observed after neuronal injury.


árpád PáRDUCZ NEURONAL PLASTICITY AND NEUROPROTECTION: ROLE OF HORMONES AND NEUROSTEROIDS

34

BIO

PHYS

ICS

The capillaries of the central nervous system form-ing the blood-brain barrier (BBB) have specific proper-ties and functions. They regulate the brain microenvi-ronment by restricting ionic, fluid and cell movements between the blood and the brain, by supplying brain cells with essential nutrients, and by protecting them from toxic molecules in the blood.

Prof. ferenc Joó, the first head of the Laboratory of molecular neurobiology and his co-workers created the first in vitro model of the BBB by the successful isolation of viable brain microvessels more than 35 years ago. ex-periments on isolated brain microvessels provided im-portant data on cerebral endothelial receptors, transport-ers and signalling mechanisms. continuing their work we have developed new BBB models by the co-culture of brain endothelial cells, glial cells and pericytes to study cell–cell interaction in the neurovascular unit, modula-tion of BBB permeability in physiological, pathological, and pharmacological conditions and to screen drug can-didate molecules to predict brain penetration (fig. 1).

brain endothelial cell pericyte astroglia

Figure 1: Co-culture model of the BBB (Nakagawa, Deli et al., 2007; Nakagawa, Deli et al., 2009)

BBB functions under physiological and pathological conditions

the protective functions of the blood-brain bar-rier is injured in several diseases, like bacterial and viral infections of the nervous system and neu-rodegenerative diseases. In these pathologies the amount of nutrients actively transported by brain endothelial cells is reduced. In parallel, the perme-ability of the blood-brain barrier is increased for potentially harmful substances from blood, like albumin, which contribute to neuronal death and the progression of the disease. even relatively small changes in the functions of the blood-brain barrier may lead to serious and chronic neuronal dysfunc-tions.

We study the effects of key pathogenic factors in nervous system diseases on the morphology and functions of the blood-brain barrier, and how the blood-brain barrier can be protected from the dam-aging effects. We demonstrated that amyloid pep-tides, participating in the pathogenesis of alzheimer's and prion diseases, can directly exert a toxic effect on brain endothelial cells and inhibit their func-tions. Pentosan treatment protected the cells against the toxic effect. Pentosan, an active ingredient from a plant with structural similarities to heparin, was also effective against bacterial lipopolysaccharide-mediated endothelial cell damage.

blooD-bRaIn baRRIeR paTHoloGY anD pHaRmaColoGY

Mária DELI / Principal Investigator

Szilvia vESZELKA / staff scientistLevente KÜRTI / Phd studentAndrea TóTH / Phd studentBoglárka CSISZáR / Phd studentLóránd KISS / Phd student

35

BIO

PHYS

ICS

Drug targeting to brain

In collaboration with the department of Pharma-ceutical technology of the university of szeged, we search for new methods to target drugs to the central nervous system. We focus on (i) the nasal pathway as an alternative gate to the brain (fig. 2) as well as to the systemic circulation and (ii) nanoparticles, namely ni-osomes, bilayer vesicles made up of non-ionic surface active agents (fig. 3) to cross the blood-brain barrier by targeting transport systems.

Figure 2: Pathways of nasal drug delivery

Fig. 3A: Vesicular structure of a niosome

Fig. 3B: Niosomes seen by atomic force microscopy

AFM: Dept. of Optics and Quantum Electonics, University of Szeged


The protection of brain endothelial cells and the im-provement of BBB functions in pathological conditions, the exploration of new approaches for drug transport/targeting to brain may have a therapeutic potential in the treatment of central nervous system diseases.


Mária DELI BLOOD-BRAIN BARRIER PATHOLOGY AND PHARMACOLOGY

36

BIO

PHYS

ICS

the blood-brain barrier (BBB) plays a key role in the maintenance of the homeostasis of the cen-tral nervous system (cns). the morphological basis of the BBB is formed by cerebral endothelial cells lining the capillaries and microvessels of the brain. cerebral endothelial cells are interconnected by a continuous line of tight and adherens junctions and come into close contact with pericytes and astro-cytes.

due to its complex functions the BBB plays an important role in clinical practice. first, there is increasing evidence that the BBB is involved in the pathomechanism of a large number of cns diseases like stroke, brain trauma and tumours as well as neu-rodegenerative disorders. second, due to the relative impermeability of the barrier, many drugs are unable to reach the cns in therapeutically relevant concen-tration, making the BBB one of the major impedi-ments in the treatment of cns disorders.

By using an in vitro model of the BBB, our research is focused on the elucidation of molecular mecha-nisms regulating endothelial function under physio-logical and pathological conditions. recently we have shown that in different cns disorders like cerebral ischemia or hemorrhagic shock, complex signalling events are initiated accompanied by changes in the integrity of the junctional complex. Presently we are investigating the signalling mechanisms involved in the regulation of BBB permeability and transmigra-tion of tumour cells through the BBB.

Model system

In order to be able to directly investigate molecu-lar mechanisms regulating BBB function, we use an in vitro model based on the culture of cerebral endothe-lial cells.

The model can be used for basic research and ap-plied research for the study of the interaction of drugs with the BBB and the transport of different drugs through the BBB.

our methods of investigation include Western-blots, phosphorylation analysis, proteomics, real-time Pcr, rna silencing, zymography, immunofluorescence etc.

Figure 1: Triple co-culture model of the BBB

Signalling in brain endothelial cells under physiological and pathological conditions

The permeability of the BBB is under complex con-trol. This control is mediated by a large number of signalling molecules, many of which are localized to

bRaIn enDoTHelIal ReseaRCH

István KRIZBAI / Principal Investigator

Attila FARKAS / staff scientistImola WILHELM / staff scientistCsilla FAZAKAS / Phd studentPéter NAGYŐSZI / Phd studentJános HASKó / Phd student

37

BIO

PHYS

ICS

the junctional complex. our research is focused on the signal transduction pathways in cerebral endothelial cells activated by pathological conditions like oxida-tive stress, hyperosmosis or the presence of inflam-matory mediators. recently we were able to identify src kinase and the receptor tyrosine kinase axl as key elements of endothelial signalling.

Figure 2: Axl signalling in brain endothelial cells

Interaction of metastatic tumour cells with the BBB

Brain metastases are clinically diagnosed in the majority of patients having secondary localizations of melanoma. The prognosis for these patients is very

poor and treatment can be difficult because of rapid progression of the disease. since the brain does not have lymphatic vessels, melanoma cells have to trans-migrate through the blood-brain barrier in order to reach the brain parenchyma. during this process the tumour cells either must disrupt intercellular junc-tions or use the transcellular route. We are currently investigating the molecular mechanisms of these phe-nomena.

Figure 3: A2058 melanoma cells (green) in contact with brain endothe-lial cells (red: ZO-1 staining).


István KRIZBAI BRAIN ENDOTHELIAL RESEARCH

38

BIO

PHYS

ICS

Biogas

Photosynthesis and photosynthetic organisms are widespread on earth, hence an oxidizing atmosphere had developed long time ago. as a consequence, most compounds found in the environment as waste or contamination are in oxidized state. Their conversion and utilization in many cases is only possible through reduction. There is no better reductant for biotech-nological elimination of these compounds than the hydrogen produced by certain bacteria on site. This principle is used for the intensification of biogas pro-duction, a biotechnological process, which decomposes organic materials yielding biogas. Biogas is formed by a consortium of microbes in an oxygen-free environ-ment. The microbial community consumes the com-plex molecules of organic waste or biomass, because they exploit the chemical energy stored in the carbon-carbon bonds of these substrates. since none of the in-dividual microbes can break all carbon-carbon bonds, a well-organized, efficient microbiological food-chain develops in order to liberate all of the chemical energy that is used to fuel their own metabolism and growth. The organic molecules are split to smaller pieces, un-til there is no more carbon-carbon bond left and the leftover is released as biogas, i.e. a mixture of meth-ane and carbon dioxide. The biological activity of the

biogas-producing natural consortia is not optimized for maximum production of the gas, which is useless for the bacteria. We have demonstrated that the com-plex biochemical and microbiological reaction chain can run faster and more efficiently if one properly in-terferes with some of the individual steps. The strat-egy was assessed both in laboratory experiments and field tests using animal slurry and manure, waste wa-ter sludge, various energy plants, industrial waste and solid household waste. The biotechnological manage-ment of the microbial activity leads to more economi-cal and cost-effective biogas production technology.


mICRobIal Gas meTabolIsm

Kornél L. KOváCS / Principal Investigator, Group Leader

Gábor RáKHELY / senior scientistAndrás TóTH / staff scientistGergely MARóTI /staff scientistZoltán BAGI / staff scientistErika MOLNáR / technicianRózsa vEREBÉLY / technician

39

BIO

PHYS

ICS

Biohydrogen

at the dawn of the 21st century a global environ-mental and energy crisis threatens the sustainable lifestyle of mankind. as long as there is plenty of fos-sil energy carrier available, every aspect of our life is supported by the use of these resources. It is, however, an undeniable fact, supported by several lines of in-dependent evidence that the uncontrolled consump-tion of fossil energy damages the environment to a degree that threatens the survival of the human race and many other plant and animal species. Hydrogen is the simplest molecule and is considered to become the renewable energy carrier of the future, which is capable of replacing the carbon-based fossil fuels in a sustainable manner. If hydrogen is produced at an industrial scale ultimately from solar energy, it can alleviate the environmental disasters foreseen due to global warming and eliminate political tensions among various geographical regions.

Hydrogen, just like electricity, is an energy carrier that cannot be exploited using conventional mining

technologies. currently it is produced from fossil en-ergy sources, mainly from natural gas. This approach does not solve any of the environmental and geopo-litical concerns. Hydrogen could be generated from renewable primary energy sources with an acceptable efficacy; these biotechnological methods evolve and improve rapidly and offer promise for a future hydro-gen-based economy.

The key enzyme of hydrogen production and uti-lization is hydrogenase, a protein containing redox active metallocentre(s). The main aim of our research program is to understand the molecular details of this enzyme function, the regulation of its biosynthesis and, in general, to study the metal-protein interaction in redox metalloenzymes. The results are not ready yet for large-scale practical application, but we could already create modified bacteria, which display out-standing hydrogen productivity. most of these minia-ture hydrogen factories derive from an easy-to-handle phototrophic bacterium.

Bioremediation

many different microbial strains contain enzymes and metabolic pathways that can be exploited to de-grade hazardous, poisonous chemicals, e.g. the recal-citrant chlorinated hydrocarbons. The bioremediation methods identify the best microorganisms for the elimination of the target compound and improve its performance by traditional training or via molecular biological tools. We have developed strains that de-grade hydrocarbons, their halogenated derivatives, lipids, chemicals in the waste streams of the pharma-ceutical, pesticide, and food processing industries. These studies also took us to the field of biosurfactants,

Kornél L. KOváCS / Principal InvestigatorGábor RáKHELY / Principal Investigator

40

BIO

PHYS

ICS

i.e. biodegradable detergents and to the production of industrial enzymes.

an industrial fermentation facility, operated by the spin-off corax-Bioner Inc. produces about 20,000 m3/year of microbial cultures for bioremediation and organic farming. our team provides the scientific background for the product development of the com-pany on the basis of a long-term collaboration agree-ment.

Contact: [email protected]; [email protected]

Kornél L. KOváCS, Gábor RáKHELY BIOHYDROGEN, BIOREMEDIATION

41

BIO

PHYS

ICS

Bioactive peptides are a group of diverse intercel-lular signalling molecules. almost half a century of research on this topic has resulted in an enormous amount of data. Bacteria, fungi or higher eukaryotic cells and tissues express a variety of biologically active peptides and these cell lines are also able to respond to many different bioactive peptides, indicating that multiple receptors and signal transduction pathways are present in a single cell. Bioactive peptides might have very diverse effects: they might be antimicrobials, or serve as signalling molecules which are involved in development, cellular responses or control processes.

numerous neurodegenerative diseases are related to conformational changes and/or aggregation of small proteins or peptides, such as polyalanine or poly-glutamine. The molecular mechanisms of their action might also vary: they can aspecifically interact with the membrane, but more typically, they are bound by spe-cific receptors. usually, the interaction of these peptides with their receptors requires special conformation of the partners. The bioactive conformation of a peptide can be studied by few experimental methods, but cheap and powerful computer-based approaches are also

available to explore the conformational space of a given molecule, to identify the intramolecular interactions stabilizing the structure and to describe the molecular events in time. The peptide – receptor interaction can be further characterized by in silico docking, provided the 3d structure of the receptor is available.

The prediction of a bioactive conformation allows us to get a deeper insight into the molecular basis of biological processes and to design new peptidomimet-ic compounds/drugs for therapeutic purposes.

In the last few years we studied bioactive peptides either related to neurodegenerative disorders or hav-ing antimicrobial effects. We disclosed the secondary structures of homopolyamino acids, especially poly-alanine and polyglutamine, whose aggregation seems to be related to neurodegenerative disorders such as alzheimer’s disease. molecular dynamics calculations were performed to simulate the temporal folding proc-esses. additionally, in silico structural analyses were performed for cyclic nonapeptides, conopressins hav-ing neuromodulator effects. among the antimicrobial peptides we explored the conformational space of in-dolicidin and other molecules of palindromic struc-ture. These studies might form the basis to design new antimicrobial drugs, which can be generally used for protecting human health.


sTRUCTURal bIoInfoRmaTICs of bIoaCTIve pepTIDes/moleCUles

Gábor RáKHELY / Principal Investigator

Gábor JANZSó / Phd studentLiza HUDOBA / Phd student

Balázs LEITGEB / senior scientist

42

BIO

PHYS

ICS

Pál OrmosGalajda, P. and ormos, P. (2001). complex micromachines pro-duced and driven by light. Applied Physics Letters 78: 249-251.

Galajda, P. and ormos, P. (2003). orientation of flat particles in optical tweezers by linearly polarized light. Opt. Express 11: 446-451.

oroszi, L., Galajda, P., Kirei, H., Bottka, s. and ormos, P. (2006). direct measurement of torque in an optical trap and its application to double-strand dna. Phys. Rev. Lett. 97: art. nr. 058301.

oroszi, L., dér, a., Kirei, H., rakovics, v. and ormos, P. (2006). control of electro-osmostic flow by light. Appl. Phys. Lett. 89: art. nr. 263508.

oroszi, L., dér, a., Kirei, H., rakovics, v. and ormos, P. (2009). manipulation of microfluidic flow pattern by opti-cally controlled electroosmosis. Microfluidics and Nanofluid-ics 6: 565-569.

András Dérdér, a., oroszi, L., Kulcsár, Á., Zimányi, L., tóth-Boconádi, r., Keszthelyi, L., stoeckenius, W. and ormos, P. (1999). Inter-pretation of spatial charge displacements in bacteriorhodopsin in terms of structural changes during the photocycle. Proc. Natl. Acad. Sci. U.S.A. 96: 2776-2781.

dér, a. and Keszthelyi, L. (2001). charge motion during the photocycle of bacteriorhodopsin. (review) Biochemistry 66: 1234-1248.

dér, a. and Keszthelyi, L. (eds.) (2001). Bioelectronic appli-cations of Photochromic Pigments, Ios Press nato science series, vol. 335.

ormos, P., fábián L., oroszi L., ramsden, J.J., Wolff, e.K. and dér, a. (2002). Protein-based integrated optical switching and modulation. Appl. Phys. Lett. 80: 4060-4062.

tóth-Boconádi, r., dér, a., taneva, s.G. and Keszthelyi, L. (2006). excitation of the L intermediate of bacteriorhodopsin: electric responses to test X-ray structures. Biophys. J. 90: 2651-2655.

dér, a., valkai, s., fábián, L., ormos, P., ramsden, J.J. and Wolff, e.K. (2007). Integrated optical switching Based on The Protein Bacteriorhodopsin. Photochem. Photobiol. 83: 393-396.

dér, a. and ramsden, J.J. (1998). evidence for loosening of a protein mechanism. Naturwissenschaften 85: 353-355.

neagu, a., neagu, m. and dér, a. (2001). fluctuations and the Hofmeister effect. Biophys. J. 81: 1285-1294.

dér, a., neagu, a. and neagu, m. (2001). active transport modulated by barrier fluctuations. In: dér, a., Keszthelyi, L. (ed.) Bioelectronic Applications of Photochromic Pigments, Ios Press, nato science series vol. 335. pp. 225-243.

dér, a., Kelemen, L., fábián, L., taneva, s.G., fodor, e., Páli, t., cupane, a., cacace, m.G. and ramsden, J.J. (2007). Inter-facial Water structure controls Protein conformation. J. Phys. Chem. B. 111: 5344-5350.

Géza GromaGroma, G.I., colonna, a., Lambry, J.c., Petrich, J.W., váró, G., Joffre, m., vos, m.H. and martin, J.L. (2004). resonant optical rectification in bacteriorhodopsin. Proc. Natl. Acad. Sci. U.S.A. 101: 7971-7975.

colonna, a., Groma, G.I. and vos, m.H. (2005). retinal isomer-ization dynamics in dry bacteriorhodopsin films. Chem. Phys. Lett. 415: 69-73.

colonna, a., Groma, G.I., martin, J.L., Joffre, m. and vos, m.H. (2007). Quantification of sudden light-induced polari-zation in bacteriorhodopsin by optical rectification. J. Phys. Chem. B. 111: 2702-2710.

Groma, G.I., Hebling, J., Kozma, I.Z., váró, G., Hauer, J., Kuhl, J. and riedle, e. (2008). tHz radiation from bacteriorhodopsin reveals correlated primary electron and proton transfer proc-esses. Proc. Natl. Acad. Sci. U.S.A. 105: 6888-6893.

Heiner, Z. and osvay, K. (2009). refractive index of dark-adapt-ed bacteriorhodopsin and tris(hydroxymethyl)aminomethane buffer between 390 and 880 nm. Appl. Opt. 48: 4610-4615.

Lóránd KelemenKelemen, L., valkai, s. and ormos, P. (2006). Integrated opti-cal motor. Applied Optics 45: 2777-2780.

Jákl, P., Šery, m., Ježek, J., Zemánek, P., ormos, P., valkai, s. and Kelemen, L. (2007). opto-fluidic micromanipulation sys-tem based on integrated polymer waveguides. J. Optoelectr. Adv. Mater. 9: 2148–2151.

rodrigo, P.J., Kelemen, L., alonzo, c.a., Perch-nielsen, I.r., dam, J.s., ormos, P. and Glückstad, J. (2007). 2d optical manipulation and assembly of shape complementary planar microstructures. Opt. Expr. 15: 9009-9014.

Kelemen, L., valkai, s. and ormos, P. (2007). Parallel pho-topolymerisation with complex light patterns generated by diffractive optical elements. Opt. Expr. 15: 14488-14497.

rodrigo P.J., Kelemen, L., Palima, d., alonzo, c.a., ormos, P. and Glükstad, J. (2009). optical microassembly platform for constructing reconfigurable microenvironments for biomedi-cal studies. Opt. Expr. 17:6578-6583.

Ilona Laczkósimon, L.m., Kotormán, m., Garab, Gy. and Laczkó, I. (2002). effects of polyhydroxy compounds on the structure and activ-ity of chymotrypsin. Biochem. Biophys. Res. Commun. 293: 416-420.

InsTITUTe of bIopHYsICsseleCTeD pUblICaTIons:

43

BIO

PHYS

ICS

Laczkó, I., Bottka, s., tóth, G.K., malvy, c., Bertrand, J.r. and Hollósi, m. (2004). Interaction of fusogenic peptides with an antisense oligonucleotide in solution and in the presence of micelles: conformational studies. Biochem. Biophys. Res. Com-mun. 313: 356-361.

szegedi, v., Juhász, G., rózsa, e., Juhász-vedres, G., datki Zs., fülöp, L., Bozsó Zs., Lakatos, a., Laczkó, I., farkas, t., Kis, Zs., tóth, G., soós, K., Zarándi, m., Budai, d., toldi, J. and Penke, B. (2006). endomorphin-2, an endogenous tetrapeptide protects against aβ(1-42) in vitro and in vivo. FASEB J. 20(8): 324-333.

Laczkó, I., váró, G., Bottka, s., Bálint, Z., Illyés e., vass, e., Bertrand, J.r., malvy, c. and Hollósi, m. (2006). n-terminal acylation of the sv40 nuclear localization signal peptide en-hances its oligonucleotide binding and membrane transloca-tion. Arch. Biochem. Biophys. 454: 146-154.

Laczkó, I., vass, e., soós, K., fülöp, L., Zarándi, m. and Penke, B. (2008). aggregation of aβ(1-42) in the presence of short peptides. J. Peptide Sci. 14: 731-741.

Bertrand, J.r., malvy, c., auguste, t., tóth, G.K., Kiss-Ivánko-vits, o., Illyés, e., Hollósi, m., Bottka, s. and Laczkó, I. (2009). synthesis and studies on the cell-penetrating peptides. Biocon-jugate Chemistry 20: 1307-1314.

Zsolt Tokajiszabó, Z., tokaji, Z., Kálmán, J., oroszi, L., Pestenácz, a. and Janka, Z. (2004). The effect of bright light exposure on pupillary fluctuations in healthy subjects. J. Affect. Disord. 78: 153-156.

szabó, Z., antal, a., tokaji, Z., Kálmán, J., Kéri, s., Benedek, G. and Janka, Z. (2004). Light therapy increases visual contrast sen-sitivity in seasonal affective disorder. Psychiatry Res. 126: 15-21.

dancshazy, Z., der, a., Groma, G.I., Janka, Z., Jardanhazy, t., makai, a., szentistvanyi, I. and vasadi, a. (2004). Phase-syn-chronization of daily motor activities can reveal differential circadian patterns. Chronobiol. Int. 21: 309-314.

tokaji, Z., szabó, K., nagyova, K. and vetró, Á. (2006). dark-ness-induced alertness-increase in children with attention def-icit hyperactivity disorder and its diminution by methylpheni-date. Eur. Neuropsychopharmacol. 16: s532-s532.

tokaji, Z., szabó, Z., Kovács, Z. and nagyova, K. (2007). Lack of the large aftereffect of bright light on pupillary fluctuations in patients with non-seasonal depression. Eur. Neuropsychop-harmacol. 17: s322-s322.

nagyova, K., szabó, K., vetró, Á. and tokaji, Z. (2007). methyl-phenidate-induced stabilization of the sympathetic/parasym-pathetic tone in children with adHd. Eur. Neuropsychophar-macol. 17: s574-s575.

György VáróBálint, Z., Krizbai, I.a., Wilhelm, I., farkas, a.e., Párducz, Á., szegletes, Z. and váró, G. (2007). changes induced by hyper-osmotic mannitol in cerebral endothelial cells: an atomic force microscopic study. Eur. Biophys. J. 36: 113-120.

Bálint, Z., végh, G.a., Popescu, a., dima, m., Ganea, c. and váró, G. (2007). direct observation of the protein motion dur-ing the photochemical reaction cycle of the bacteriorhodopsin. Langmuir 23: 7225-7228.

Wilhelm, I., farkas, a.e., nagyőszi, P., váró, G., Bálint, Z., végh, G.a., couraud, P.o., romero, I.a., Weksler, B. and Krizbai, I.a. (2007). regulation of cerebral endothelial cell morphology by extracellular calcium. Phys. Med. Biol. 52: 6261-6274.

Bálint, Z., nagy, K., Laczkó, I., Bottka, s., végh, G.a., sze-gletes, Z. and váró, G. (2007). adsorption and self-assembly of oligodeoxynucleotides onto a mica surface. J. Phys. Chem. C. 111: 17032-17037.

László ZimányiKotlyar, a.B., Borovok, n., Khoroshyy, P., tenger, K. and Zimányi. L. (2004). redox photochemistry of thiouredopyren-etrisulfonate. Photochem. Photobiol. 79(6): 489-493.

tenger, K., Khoroshyy, P., Leitgeb, B., rákhely, G., Borovok, n., Kotlyar, a., dolgikh, d.a. and Zimányi, L. (2005). complex kinetics of the electron transfer between the photoactive redox label tuPs and the heme of cytochrome c. J. Chem. Inf. Mod. 45(6): 1520-1526.

tenger, K., Khoroshyy, P., Kovács, K.L., Zimányi, L. and rákhely, G. (2007). Improved system for heterologous expres-sion of cytochrome c mutants in Escherichia coli. Acta Biol. Hung. 58: 23-35.

martin, m., Palestino, G., cloitre, t., agarwal, v., Zimányi, L. and Gergely, cs. (2009). Three dimensional spatial resolution of the nonlinear photoemission from biofunctionalized po-rous silicon microcavity. Appl. Phys. Lett. 94: art. nr: 223313.

Tibor PáliHolzenburg, a., Jones, P.c., franklin, t., Páli, t., Heimburg, t., marsh, d., findlay, J.B.c. and finbow, m.e. (1993). evi-dence for a common structure for a class of membrane chan-nels. European Journal of Biochemistry 213(1): 21-30.

Páli, t., finbow, m.e., Holzenburg, a., findlay, J.B.c. and marsh, d. (1995). Lipid-protein interactions and assembly of the 16-kda channel polypeptide from Nephrops norvegicus. studies with spin-label electron spin resonance spectroscopy and electron microscopy. Biochemistry 34(28): 9211-9218.

Páli, t., finbow, e.m. and marsh, d. (1999). membrane as-sembly of the 16-kda proteolipid channel from nephrops nor-vegicus studied by relaxation enhancements in spin-label esr. Biochemistry 38(43): 14311-14319.

Kostrzewa, a., Páli, t., froncisz, W. and marsh, d. (2000). mem-brane location of spin-labeled cytochrome c determined by par-amagnetic relaxation agents. Biochemistry 39(20): 6066-6074.

Bashtovyy, d., marsh, d., Hemminga, H.m. and Páli, t. (2001). constrained modelling of spin-labelled major coat protein mutants from m13 bacteriophage in a phospholipid bilayer. Protein Science 10(5): 979-987.


44

BIO

PHYS

ICS

Páli, t. and marsh, d. (2001). tilt, twist and coiling in beta-barrel membrane proteins: relation to infrared dichroism. Bio-physical Journal 80(6): 2789-2797.

Kóta, Z., Horváth, L.I., droppa, m., Horváth, G., farkas, t. and Páli, t. (2002). Protein assembly and heat stability in develop-ing thylakoid membranes during greening. Proc. Natl. Acad. Sci. U.S.A. 99(19): 12149-12154.

Bashtovyy, d., Bérczi, a., asard, H. and Páli, t. (2003). struc-ture prediction for the di-heme cytochrome b561 protein fam-ily. Protoplasma 221: 31-40.

Páli, t., Garab, G., Horváth, L.I. and Kóta, Z. (2003) function-al significance of the lipid-protein interface in photosynthetic membranes. Cellular Molecular Life Sciences 60(8): 1591-1606.

marsh, d. and Páli, t. (2004). The protein-lipid interface: per-spectives from magnetic resonance and crystal structures. Bio-chimica et Biophysica Acta - Biomembranes 1666(1-2): 118-141.

Páli, t., dixon, n., Kee, t.P. and marsh, d. (2004). Incor-poration of the v-atPase inhibitors concanamycin and in-dole pentadiene in lipid membranes. spin-label ePr studies. Biochimica et Biophyisica Acta – Biomembranes 1663(1-2): 14-18.

dixon, n., Páli, t., Kee, t.P. and marsh, d. (2004). spin-labelled vacuolar-atPase inhibitors in lipid membranes. Biochimica et Biophyisica Acta - Biomembranes 1665(1-2): 177-183.

marsh, d. and Páli, t. (2006). Lipid conformation in crystal-line bilayers and in crystals of transmembrane proteins. Chem-istry and Physics of Lipids 141: 48-65.

Páli, t., Bashtovyy, d. and marsh, d. (2006). stoichiometry of lipid interactions with transmembrane proteins - deduced from the 3-d structures. Protein Science 15: 1153-1161.

dixon, n., Páli, t., Kee, t.P., Ball, s., Harrison, m.a., findlay, J.B.c., nyman, J., vaananen, K., finbow, m.e. and marsh, d. (2008). Interaction of spin-labelled inhibitors of the vacuolar H+-atPase with the transmembrane vo-sector. Biophysical Journal 94(2): 506-514.

fodor, e., fedosova, n., ferencz, c., marsh, d., Páli, t. and esmann, m. (2008). stabilization of na,K–atPase by ionic interactions. Biochimica at Biophysica Acta – Biomembranes 1778(4): 835-843.

Kóta, Z., Páli, t., dixon, n., Kee, t.P., Harrison, m.a., find-lay, J.B.c., finbow, m.e. and marsh, d. (2008). Incorporation of transmembrane peptides from the vacuolar H+-atPase in phospholipid membranes: spin-label electron paramagnetic resonance and polarized infrared spectroscopy. Biochemistry 47(12): 3937–3949.

Alajos BércziBérczi, a., su, d., Lakshminarasimhan, m., vargas, a.s. and asard, H. (2005). Heterologous expression and site-directed mutagenesis of an ascorbate-reducible cytochrome b561. Arch. Biochem. Biophys. 443: 82-92.

Bérczi, a. and asard, H. (2006). characterization of an ascor-bate-reducible cytochrome b561 by site-directed mutagenesis. Acta Biol. Szeged. 50: 55-59.

Bérczi, a., su, d. and asard, H. (2007). an arabidopsis cyto-chrome b561 with trans-membrane ferrireductase capability. FEBS Lett. 581: 1505-1508.

Bérczi, a. and asard, H. (2008). expression and purification of the recombinant mouse tumor suppressor cytochrome b561 protein. Acta Biol. Szeged. 52: 257-265.

Balázs Szalontaischwinte, P., voegel, J.c., Picart, c., Haikel, Y., schaaf, P. and szalontai, B. (2001). stabilizing effects of various polyelectro-lyte multilayer films on the structure of adsorbed/embedded fibrinogen molecules: an atr-ftIr study. J. Phys. Chem. B. 47: 11906-11916.

szalontai, B., Kóta, Z., nonaka, H. and murata, n. (2003). structural consequences of Genetically engineered satura-tion of the fatty acids of Phosphatidylglycerol in tobacco Thylakoid membranes. an ftIr study. Biochemistry 42: 4292-4299.

Pilbat, a.m., Ball, v., schaaf, P., voegel, J.c. and szalontai, B. (2006). Partial Poly(glutamic acid) t Poly(aspartic acid) ex-change in Layer-by-Layer Polyelectrolyte films. structural alterations in the Three-component architectures. Langmuir 22: 5753-5759.

Pilbat, a.m., szegletes, Zs., Kóta, Z., Ball, v., schaaf, P., voegel, J.c. and szalontai, (2007). Phospholipid bilayers as biomem-brane-like barriers in layer-by-layer polyelectrolyte films. Langmuir 23: 8236-8242.

Gergely, c., szalontai, B., moradian-oldak, J. and cuisinier, f.J.G. (2007). Polyelectrolyte-mediated adsorption of amelo-genin monomers and nanospheres forming mono- or multi-layers. Biomacromolecules 8: 2228-2236.

szalontai, B. (2009). membrane protein dynamics: Limited lip-id control. Pmc Biophysics 2: 1-17. doi:10.1186/1757-5036-2-1

Csaba BagyinkaBagyinka, cs., Ősz, J. and száraz, s. (2003). autocatalytic oscillations in the early phase of the photoreduced methyl viologen-initiated fast kinetic reaction of hydrogenase. J. Biol. Chem. 278(23): 20624-20627.

Ősz, J. and Bagyinka, cs. (2005). an autocatalytic step in the reaction cycle of hydrogenase from Thiocapsa roseopersicina can explain the special characteristics of the enzyme reaction. Biophysical Journal 89: 1984-1989.

Ősz, J., Bodó, G., Branca, r.m.m. and Bagyinka, cs. (2005). Theoretical calculations on hydrogenase kinetics: explanation of the lag phase and the enzyme concentration dependence of the activity of hydrogenase uptake. Biophysical Journal 89: 1957-1964.


45

BIO

PHYS

ICS

tomcová, I., Branca, r.m.m., Bodó, G., Bagyinka, cs. and Kutá smatanová, I. (2006). cross-crystallization method used for the crystallization and preliminary diffraction analysis of a novel di-haem cytochrome c4. Acta Cryst. f62: 820-824.

Branca, r.m.m., Bodó, G., várkonyi, Zs., debreczeny, m., Ősz, J. and Bagyinka cs. (2007). oxygen and temperature-de-pendent structural and redox changes in a novel cytochrome c4 from the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina. Archives Biochem. Biophys. 467: 174-184.

Branca, r.m.m., Bodó, G., Bagyinka, cs. and Prókai, L. (2007). de novo sequencing of a 21-kda cytochrome c4 from Thiocap-sa roseopersicina by nanoelectrospray ionization ion-trap and fourier-transform ion-cyclotron resonance mass spectrom-etry. J. Mass Spectrometry 42: 1569-1582.

Pankotai-Bodó, G., Branca, r.m.m., Bagyinka, cs., tóth, a. and Horváth, d. (2008). autocatalytic reaction of hydroge-nase from Thiocapsa roseopersicina. FEBS Journal 275: suppl. 1. 204-204.

Bodó, G., Branca, r.m.m., tóth, a., Horváth, d. and Bagyin-ka, cs. (2009). concentration-dependent front velocity of the autocatalytic hydrogenase reaction. Biophysical Journal 96: 4976-4983.

László Siklóssiklós, L., engelhardt, J.I., Harati, Y., smith, r.G., Joó, f. and appel, s.H: (1996). ultrastructural evidence for altered calci-um in motor nerve terminals in amyotrophic lateral sclerosis. Annals of Neurology 39: 203-216.

siklós, L., engelhardt, J.I., alexianu, m.e., Gurney, m.e., sid-dique, t. and appel, s.H. (1998). Intracellular calcium parallels motoneuron degeneration in sod-1 mutant mice. Journal of Neuropathology and Experimental Neurology 57: 571-587.

siklós, L., engelhardt, J.I., adalbert, r. and appel, s.H. (1999). calcium-containing endosomes at oculomotor terminals in animal models of aLs. NeuroReport 10: 2539-2545.

siklós, L., engelhardt, J.I., reaume, a.G., scott, r.W., adalbert, r., obál, I. and appel, s.H. (2000). altered ca-homeostasis in spinal motoneuorons but not in oculomotor neurons of sod-1 knock-out mice. Acta Neuropathologica 99: 517-524.

adalbert, r., engelhardt, J.I. and siklós, L. (2002). dL-homo-cysteic acid application disrupts calcium homeostasis and in-duces degeneration of spinal motor neurons in vivo. Acta Neu-ropathologica 103: 428-436.

engelhardt, J.I., soós, J., obál, I., vigh, L. and siklós, L. (2005). subcellular localization of IgG from the sera of aLs patients in the nervous system. Acta Neurologica Scandinavi-ca 112: 126-133.

vigh, L., smith, r.G., soós, J., engelhardt, J.I., appel, s.H. and siklós, L. (2005). sublethal dose of 4-hydroxynonenal reduces intracellular calcium in surviving motor neurons in vivo. Acta Neuropathologica 109: 567-575.

siklós, L. and appel, s.H. (2005). calcium binding proteins in selective vulnerability of motor neurons. In: Neurodegen-erative Diseases: Neurobiology, pathogenesis and therapeutics. (eds. Beal m.f., Lang a.e., Ludolph a.c.) cambridge univer-sity Press, pp. 65-79.

obál, I., engelhardt, J.I. and siklós, L. (2006). axotomy induces contrasting changes in calcium and calcium-binding proteins in oculomotor and hypoglossal nuclei of Balb/c mice. Journal of Comparative Neurology 499: 17-32.

Paizs, m., engelhardt, J.I. and siklós, L. (2009). Quantitative assessment of relative changes of immunohistochemical stain-ing by light microscopy in specified anatomical regions. Jour-nal of Microscopy (Oxford) 234: 103-112.

Árpád Párduczschumacher, m., Weill engerer, s., Liere, P., robert, f., fran-klin, rjm., Garcia segura, L.m., Lambert, J.J., mayo, W., mel-cangi, r,c., Parducz, a., suter, u., carelli, c., Baulieu, e.e. and akwa, Y. (2003). steroid hormones and neurosteroids in nor-mal and pathological aging of the nervous system. Progress in Neurobiology 71: 3-29.

Hoyk, Z. Parducz, a. and Garcia segura, L.m. (2004). dehy-droepiandrosterone regulates astroglia reaction to denerva-tion of olfactory glomeruli. Glia 48: 207-216.

Juhasz vedres, G., rozsa, e., rakos, G., dobszay, m.B., Kis, Z., Wolfling, J., toldi, J., Parducz, a. and farkas, t. (2006). dehy-droepiandrosterone sulfate is neuroprotective when adminis-tered either before or after injury in a focal cortical cold lesion model. Endocrinology 147: 683-686.

csakvari, e., Hoyk, Z., Gyenes, a., Garcia ovejero, d., Garcia segura, L.m. and Parducz, a. (2007). fluctuation of synapse density in the arcuate nucleus during the estrous cycle. Neuro-science 144(4): 1288-1292.

Parducz, a., Hajszan, t., macLusky, n.J., Hoyk, Z., csakvari, e., Kurunczi, a., Prange Kiel, J. and Leranth, c. (2006). syn-aptic remodeling induced by gonadal hormones: neuronal plasticity as a mediator of neuroendocrine and behavioral re-sponses to steroids. Neuroscience 138: 977-985. review

naftolin, f., Garcia-segura, L.m., Horvath, t.L., Zsarnovszky, a., demir, n., fadiel, a., Leranth, c., vondracek-Klepper, s., Lewis, c., chang, a. and Parducz, a. (2007). estrogen-induced hypothalamic synaptic plasticity and pituitary sensitization in the control of the estrogen-induced gonadotrophin surge. Re-prod. Sci. 14(2): 101-116. review

csákvári, e., Kurunczi, a., Hoyk, Z., Gyenes, a., naftolin, f. and Párducz, Á. (2008). estradiol-induced synaptic remod-eling of tyrosine hydroxylase immunopositive neurons in the rat arcuate nucleus. Endocrinology 149: 4137-4141.

Hajszán, t., dow, a., Warner-schmidt, J.L., szigeti-Buck, K., sallam, n.L., Párducz, Á., Léránth, c. and duman, r.s. (2009). remodeling of hippocampal spine synapses in the rat learned helplessness model of depression. Biol. Psychiatry 65: 392-400.


46

BIO

PHYS

ICS

Mária Delideli, m.a., Ábrahám, cs., Kataoka, Y. and niwa, m. (2005). Permeability studies on in vitro blood-brain barrier models: physiology, pathology and pharmacology. Cell. Mol. Neuro-biol. 25: 59-127.

andras, I., deli, m.a., veszelka, sz., Hayashi, K., Hennig, B. (2007). toborek, m. The nmda and amPa/Ka receptors are involved in glutamate-induced alterations of occludin expres-sion and phosphorylation in brain endothelial cells. J. Cerebr. Blood Flow Metab. 27: 1431-1443.

nakagawa, s., deli, m.a., Kawaguchi, H., shimizudani, t., shimono, t., Kittel, a., tanaka, K. and niwa, m. (2009). a new blood-brain barrier model using brain endothelial cells, peri-cytes and astrocytes. Neurochem. Int. 54: 253-263.

Horvát, s., fehér, a., Wolburg, H., veszelka, s., tóth, a., Kis, L., Kurunczi, a., Balogh, G., sipos, P., Kürti, L., erős, I., sza-bó-révész, P. and deli, m.a. (2009). sodium hyaluronate as a mucoadhesive component in nasal formulation enhances de-livery of molecules to brain tissue. Eur. J. Pharm. Biopharm. 72: 252-259.

deli, m.a. (2009). Potential use of tight junction modulators to reversibly open membranous barriers and improve drug deliv-ery. Biochim. Biophys. Acta, 1788: 892-910.

István Krizbaitraweger, a., fuchs, r., Krizbai, I.a., Weiger, t.m., Bauer, H.c., Bauer, H. (2003). The tight junction protein Zo-2 local-izes to the nucleus and interacts with the hnrnP protein saf-B. J. Biol. Chem. 278: 2692-2700.

Krizbai, I.a., Bauer, H., Bresgen, n., eckl, P.m., farkas, a., szatmári, e., traweger, a., Wejksza, K. and Hans-christian, Bauer. (2005). effect of oxidative stress on the junctional pro-teins of cultured cerebral endothelial cells. Cell. Mol. Neuro-biol. 25: 129-139.

farkas, a., szatmari, e., orbok, a., Wilhelm, I., Wejksza, K., Hutamekalin, P., Bauer, H., Bauer, Hc., traweger, a. and Kriz-bai, I.a. (2005). Hyperosmotic mannitol Induces src-Kinase-dependent Phosphorylation of Beta-catenin In cerebral en-dothelial cells. J. Neurosci. Res. 80: 855-861.

Wilhelm, I., farkas, a.e., nagyőszi, P., váró, Gy., Bálint, Z., végh, G.a., couraud, P.-o., romero, I.-a., Weksler, B. and Krizbai, I.-a. (2007). regulation of cerebral endothelial cell morphology by extracellular calcium. Phys. Med. Biol. 52: 6261-6274.

Wilhelm, I., nagyőszi, P., farkas, a.e., couraud, P.o., romero, I.a., Weksler, B., fazakas, cs., dung, n.t.K., Bauer, H., Bauer, H.c. and Krizbai, I.a. (2008). Hyperosmotic stress induces axl activation and cleavage in cerebral endothelial cells. J Neuro-chem. 107: 116-126.

Kornél L. Kovács and Gábor RákhelyBálint, B., Bagi, Z., tóth, a., rákhely, G., Perei, K. and Kovács, K.L. (2005). utilization of keratin-containing biowaste to pro-duce biohydrogen. Appl. Microbiol. Biotechnol. 69: 404-410.

Kovács, K.L., maróti, G. and rákhely, G. (2006). a novel ap-proach for biohydrogen production. Int. J. Hydrogen Energy 31: 1460-1468.

Bagi, Z., Ács, n., Bálint, B., Horváth, L., dobó, K., Perei, K.r., rákhely, G. and Kovács, K.L. (2007). Biotechnological intensi-fication of biogas production. Appl. Microbiol. Biotechnol. 76: 473-482.

Kars, G., Gündüz, u., rákhely, G., Yücel, m., eroglu, I. and Kovács, K.L. (2008). Improved hydrogen production by uptake hydrogenase deficient mutant strain of Rhodobacter sphaer-oides o.u.001. Int. J. Hydrogen Energy 33: 3056-3060.

Ivanova, G., rákhely, G. and Kovács, K.L. (2008). Hydrogen production from biopolymers by Caldicellulosiruptor saccha-rolyticus and stabilization of the system by immobilization. Int. J. Hydrogen Energy 33: 6953-6961.

Palágyi-mészáros, L., Balogh, J., Latinovics, d., Balogh, t., Klement, É., medzihradszky, K., rákhely, G. and Kovács, K.L. (2009). electron-transfer subunits of the nife hydrogenases in Thiocapsa roseopersicina. BBs. FEBS J. 276: 164-174.

Ivanova, G., rákhely, G. and Kovács, K.L. (2009). Thermophil-ic biohydrogen production from energy plants by Caldicellulo-syruptor saccharolyticus and comparison with related studies. Int. J. Hydrogen Energy 34: 3659-3670.

maróti, G., tong, Y., Yooseph, s., Baden-tillson, H., smith, H.o., Kovács, K.L., frazier, m., craig venter, J. and Xu, Q. (2009). discovery of a [nife]-hydrogenase in metagenomic sargasso sea dna: cloning and functional analysis in Thio-capsa roseopersicina. Appl. Environ. Microbiol. 75: 5821-30.

Gábor Rákhely and Balázs LeitgebLeitgeb, B., szekeres, a., manczinger, L., vágvölgyi, c. and Kred-ics, L. (2007). The history of alamethicin: a review of the most extensively studied peptaibol. Chem. Biodiv. 4(6): 1027-1051.

Leitgeb, B., Kerényi, Á., Bogár, f., Paragi, G., Penke, B. and rákhe-ly, G. (2007). studying the structural properties of polyalanine and polyglutamine peptides. J. Mol. Model. 13(11): 1141-1150.

Leitgeb, B. (2007). structural investigation of endomorphins by experimental and theoretical methods: Hunting for the bio-active conformation. Chem. Biodiv. 4(12): 2703-2724.

Janzsó, G., rákhely, G. and Leitgeb, B. (2009). comprehensive structural characterization of the cyclic disulphide-bridged nonapeptides, arg- and Lys-conopressins. J. Mol. Graph. Mod-el. 27(8): 881-888.


47

BIO

CHEM

ISTR

Y

Institute of BiochemistryH-6726 Szeged, Temesvári krt. 62.H-6701 Szeged, P.O. Box 521, Hungary

Brc

48

BIO

CHEM

ISTR

Y

SssI DNA-methyltransferase: enzyme engineering for targeted DNA-methylation

In mammalian genomes a significant fraction of cytosine residues is methylated (5-methylcytosine) and this modified nucleobase is found in 5’-cG sequences (cpG sites). dna methylation is catalyzed by dna (cytosine-5) methyltransferases (c5-mtase), which transfer the activated methyl group from s-adenosyl-L-methionine to the target cytosine. methylation of the promoter region of a substantial fraction of genes leads to transcriptional inactivation (gene silencing). silencing selected genes by exogenous, targeted meth-ylation, besides being a promising research tool for studying dna methylation in vivo, can lead to thera-peutic applications. There are approaches described in the literature that used genetically constructed fusions between sequence-specific dna binding proteins, in most cases zinc finger proteins (ZfP), and c5-mtases to direct methylation to predetermined sites. a key is-sue of targeted dna methylation is specificity, i.e. the difference between levels of exogenous methylation at the targeted site vs. at non-targeted sites. methylation

of non-targeted sites occurs because of the inherent af-finity of dna mtases to their recognition sequences. non-targeted methylation limits the use of mtase fu-sions as a research tool, and would be a serious obsta-cle for therapeutic application.

We use the prokaryotic c5-mtase m.sssI as a model system to study conditions of targeted methylation. We wish to develop m.sssI into a targetable mtase with little off-target methylation. an advantage of m.sssI is that it recognizes the short sequence 5’-cG, thus has the same specificity as mammalian mtases. no X-ray structure is available for m.sssI, and little is known about the de-tails of specific dna recognition. In collaboration with elizaveta Gromova’s group (moscow state university, moscow, russia), and using a computational model of the m.sssI-dna complex as guide, site-directed mu-tants of m.sssI were constructed. The mutant enzymes are characterized in vivo and in vitro. We are mainly interested in m.sssI mutants showing reduced dna-binding affinity, because the binding specificity of ZfP-m.sssI hybrids containing such mtase mutants are ex-pected to be dominated by the ZfP domain, leading to reduced level of background methylation. Interactions

The prokaryotic DNA methyltransferase M.SssI shares the specificity (5’-CG) of mammalian DNA methyltrans-ferases qualifying it as a model system and a research tool for the study of many aspects of DNA methylation in higher eukaryotes. Our goal is to develop M.SssI into a targetable DNA methyltransferase that can selectively methylate predetermined sites in genomic DNA. Mutant variants of M.SssI are genetically fused to zinc finger pro-teins, which act as targeting domains. Specificity of targeted methylation is tested in vivo in E. coli. In a separate project DNA recognition and cleavage mechanism of a monomeric Type II restriction enzyme is studied.

Dna meTHYlTRansfeRases anD ResTRICTon enZYmes: Tools foR Genome manIpUlaTIon(sYnTHeTIC/sYsTems bIoloGY UnIT)

Antal KISS / Principal Investigator, Group Leader

Krystyna ŚLASKA-KISS / staff scientistIldikó STIER / Phd studentIbolya ANTON / technician

49

BIO

CHEM

ISTR

Y

between m.sssI and substrate dna are studied, and the information obtained is used to engineer fusions between the m.sssI variants and ZfPs recognizing 9–18 bp sites (fig. 1). targeted methylation specificity of the chimeric mtases is tested in e. coli cells using a target plasmid containing a single target site and numerous non-target cG sites. methylation specificity is analyzed by restriction digestion and bisulfite sequencing.

In a parallel project, we found that split fragments of m.sssI, which are inactive by themselves, can as-semble to form active enzyme in vivo. The potential application of this phenomenon in increasing specifi-city of targeted methylation is also studied.

Figure 1. Scheme of ZFP-M.SssI chimeric methyltransferase bound to the ZFP binding site adjacent to the target CpG site.

BspRI, a monomeric Type IIP restriction endonuclease

the BsprI restriction endonuclease, originally isolated from Bacillus sphaericus, recognizes the palindromic sequence GG/cc and cleaves it as in-

dicated to produce blunt ends. BsprI is interesting, because it also cuts single-stranded dna and ex-ists as a monomer (304 amino acids). cleavage of double-stranded dna proceeds via a nicked inter-mediate (fig. 2).

Figure 2. Conversion of supercoiled plasmid DNA into linear form by BspRI digestion. Agarose gel electrophoresis.

This mechanism is consistent with the enzyme acting as monomer and cleaving the two strands in two consecutive reactions. substrate recognition and dna cleavage by BsprI is studied by various biochemical methods. to analyze BsprI structure by X-ray crystallography, we collaborate with J. Horton (emory university school of medicine atlanta, Ga, u.s.a.).


Antal KISS Dna meTHYlTRansfeRases anD ResTRICTon enZYmes: Tools foR Genome manIpUlaTIon

50

BIO

CHEM

ISTR

Y

Towards a minimal Escherichia coli cell

E. coli K-12 is a model organism of basic research, the workhorse of molecular biology, and the platform of choice for the production of dna, metabolites and many proteins of therapeutic or commercial interest. In spite of being one of the best understood model or-ganisms, roughly one third of its ~4300 genes have no experimentally verified functions assigned to them. moreover, as E. coli evolved in the intestinal tracts of animals, it has many genes that are not relevant to prac-tical applications and some that may be detrimental. one of the objectives of this research is to eliminate as many of these unnecessary/unknown genes as possible to develop core-genome strains of E. coli K-12 mG1655 with robust metabolic performance, to which genetic modules carrying out specific practical applications can be added. By streamlining the genome, simpler and bet-ter characterized cells could be developed. moreover, due to the removal of unnecessary products, the energy and metabolic resources of the cell could be redirected to useful biomolecule production.

systems biology and comparative genomics meth-ods are applied to identify dispensable genomic re-gions. Genomes of E. coli strains are now recognized to be mosaics in which a backbone of conserved genes

in conserved order is interspersed with strain-specific horizontally transmitted “islands”. The islands con-tain genes supporting niche-specific adaptation as well as transposable elements. using scarless genomic reduction of the sequenced K-12 strain mG1655, we physically realize strains whose genome consists of the backbone elements common to most e. coli strains.

In published studies, we have described the dele-tion of up to 43 genomic segments of mG1655, result-ing in a >15% reduction of the genome (Kolisnychenko et al., 2002; Pósfai et al., 2006). This work progressed to the construction of multideletional strains with >70 deletions (>20% genome reduction). regions deleted include large K-islands, prophages, phage remnants, restriction-modification genes, flagellar and chemo-taxis related genes, genes with unknown functions, and transposable elements.

strain characteristics important for laboratory applications (growth rate, biomass production, elec-troporation efficiency, plasmid yield and quality) were not adversely affected by the deletions. In fact, compared with the parental wild-type strain, some multideletional strains display a number of beneficial changes: increase/improvement in growth rate, physi-ological uniformity, electroporability, plasmid main-tenance and genome stability. currently we are pursu-

Using approaches of synthetic biology, we are focusing on the rational large-scale remodeling of the genome of Escherichia coli K-12. Our goal is to construct an improved, minimal genome E. coli to serve both as a reduced-complexity model organism, and as a programmable cellular chassis for biotechnological applications. In the proc-ess of gradually streamlining the genome, we are investigating the effect of genome architecture and gene content on the adaptation and evolution of the cells.

Genome enGIneeRInG(sYnTHeTIC/sYsTems bIoloGY UnIT)

György PóSFAI / Principal Investigator, Group Leader

Tamás FEHÉR / staff scientistGabriella BALIKó / staff scientistZsuzsanna GYŐRFY / staff scientistIldikó KARCAGI / staff scientistBálint CSÖRGŐ / Phd studentKinga UMENHOFFER / Phd studentágnes SZALKANOvICH / technician

51

BIO

CHEM

ISTR

Y

ing large-scale characterization of the multideletional strains, including the matching of experimental data with metabolic models, as well as the analysis of newly emerging phenotypic traits.

The genome reduction/optimization work is done in collaboration with f.r. Blattner (scarab Genom-ics LLc, madison, usa). systems biology analyses are done in collaboration with B. Papp and c. Pál (Brc).

The role of mobile genetic elements in adaptation and evolution

Insertion sequence (Is) elements in E. coli have tra-ditionally been considered mostly “selfish” dna, i.e.,

evolutionarily neutral. Having a unique tool, a multi-deletional strain stripped of its mobile genetic ele-ments, we are investigating the potential role of Iss in adaptation to stressful conditions. measuring muta-tion rates under various conditions, we find evidence of stress-induced transposition of Iss, resulting in genomic rearrangements and inactivation of protein overexpressing plasmid clones. comparing isogenic strains with or without Is elements, their individual contribution to the adaptation of the cell can be in-vestigated.


György PóSFAI Genome enGIneeRInG

52

BIO

CHEM

ISTR

Y

I. Mechanisms and evolution of gene dispensability

Perhaps one of the most striking discoveries of modern molecular genetics was the extent by which organisms appear to tolerate mutations or even com-plete loss of their genes. systematic single gene dele-tion studies on microbes have revealed that 70–80% of the single mutant strains are viable with no apparent phenotypic deformation (see table 1).

our research concentrates largely on yeast (S. cer-evisiae) and E. coli, and we seek to understand the physiological and evolutionary mechanisms behind this pattern. The following questions sum up our re-search:• Are these seemingly dispensable genes redundant or

do they have important contribution under special environmental conditions not yet tested in the labo-ratory?

• How far can the deleterious impacts of gene dele-tions be mitigated during evolution, and what fac-tors limit the extent of compensatory evolution?

• Is it likely that some of these genes increase the rate of evolutionary adaptation?

to address these issues, we combine evolutionary genomics with systems biology and laboratory experi-mental evolution protocols.

The ability of cellular systems to adapt to genetic and environmental perturbations is a fundamental but poorly understood process both at the molecular and evolutionary level.

There are both physiological and evolutionary reasonings why mutations often have limited impact on cellular growth. First, perturbations that hit one target often have no effect on the overall performance of a complex system (such as metabolic networks), as perturbations can be adjusted by changed regulation and expression of the corre-sponding genes. Second, due to the fast evolvability of microbes, the effect of a perturbation can readily be alleviated by the evolution of compensatory mutations at other sites of the network. Understanding the extent of intrinsic and evolved robustness in cellular systems demands integrated analyses that combine functional genomics and computa-tional systems biology with microbial evolutionary experiments. In collaboration with several leading research teams in the field, we are investigating the following issues. First, we ask how accurately genome-scale metabolic network models can predict the impact of genetic deletions and non-heritable perturbations. Second, we investigate how far epistatic interaction networks are influenced by global regulatory modulators, such as Hsp90. Third, to understand how the impact of genetic and drug perturbations can be mitigated during evolution, we pursue large-scale lab evolu-tionary protocol, and compare results with computational model predictions and bioinformatics analyses.

evolUTIonaRY sYsTems bIoloGY / pal lab(sYnTHeTIC/sYsTems bIoloGY UnIT)

Csaba PáL / Principal Investigator, Group Leader

Béla SZAMECZ / staff scientistKálmán SOMOGYI / staff scientistBalazs BOGOS / staff scienstist Orsolya MÉHI / Phd studentEmma BORKA Andrásné / technician

53

BIO

CHEM

ISTR

Y

II. Evolution of epistatic interaction networks

redundant functions (and the extent of systems ro-bustness) can be uncovered by comparing the fitness of single and double knock-out strains. understanding the relevant genetic and environmental factors that in-fluence epistatic interactions across genes is of central importance for at least two reasons. first, it helps our understanding on the physiological and evolutionary contribution of genes with identified biochemical func-tions. second, understanding the impact of certain genes on the genetic interaction landscape will shed new lights on the cellular mechanisms of buffering. In collaboration with the Papp lab, we integrate machine learning protocols, metabolic network analyses with large-scale mapping of genetic interactions in yeast (S. cerevisiae). We ask (i) how reliably systems biology models can predict genetic interactions, (ii) to what extent genetic interactions depend on the cellular en-vironment investigated, and (iii) how far interactions between mutations are influenced by global regulatory modulators.

In the long run, a more complete picture of the interaction between multiple mutations and envi-ronmental conditions, and of the phenotypic conse-quences of these interactions would be required (i) to understand complex genetic diseases, (ii) to ration-

ally identify novel antimicrobial drug targets, (iii) to comprehend the accumulation of genetic variation in natural populations, (iv) to understand how cel-lular networks evolve and (v) to rationally construct simplified microbial cells by means of genome reduc-tion.

III. Evolution of drug resistance

evolution of antibiotic resistance is a problem that continues to challenge the healthcare sector. drug re-sistance mechanisms are often complex and involve many complementary changes that can affect trans-port processes, target enzymes and may also cause global reorganization of gene expression patterns. also, several case studies indicate the probability of acquired drug resistance when certain drug combina-tions are employed.

first, we ask how global transcriptional regulatory genes affect the potential for de novo evolution of re-sistance in E.coli. second, we are developing a heuris-tic algorithm with the aim to optimize the composi-tion of antimicrobial drug cocktails (for more details, see Papp lab projects).


Csaba PáL evolUTIonaRY sYsTems bIoloGY / pal lab

54

BIO

CHEM

ISTR

Y

systems biology aims at understanding the design principles and multi-level properties of large cellular subsystems arising from numerous molecular interac-tions. While recent technological advancements has enabled the rapid collection of data on the molecular components of cells and their interactions, there is an increasing need for automated methods that can ex-tract useful knowledge from these data and build com-putational models that accurately describe both nor-mal cellular physiology and the phenotypic impact of mutations and environmental perturbations (e.g., drug treatments). The unicellular yeast Saccharomyces cer-evisiae is an ideal candidate for systems biology studies due to the availability of large and diverse sets of post-genomic information and experimental tools. We are developing novel computational methods to analyze functional genomic datasets and to automate scientific discovery in the fields of systems biology and drug dis-covery by focusing on the following research topics:

(i) Understanding genetic interaction networks

Why most single gene deletions do not show a lethal phenotype? How do mutations in different genes inter-

act to enhance or suppress the phenotype? How com-mon are genetic interactions? What is the functional role of genes with an especially large number of genetic interactions? answers to these questions have relevance not only to functional genomics, but also to problems such as which mutational paths are accessible for evolu-tion and how deleterious mutations are eliminated from the population. With the recent availability of systemat-ic genetic interaction maps in yeast, we are in a position to gain new insights into the above issues. In particu-lar, we use data mining methods to integrate informa-tion on genetic interactions with other types of omics data (e.g., gene expression, protein-protein interactions, etc.) to infer cellular pathways and modules. We also investigate the mechanistic cause and evolution of gene dispensability (i.e., the apparent lack of growth defect after gene removal) by analyzing single and double gene deletion phenotypes under different environmental conditions. for example, we demonstrated that a large fraction of gene pairs that can compensate mutations in each other under standard laboratory conditions dis-play non-redundant functions under some other con-ditions. This finding supports the view that functional redundancy among genes is more apparent than real.

Recent advances in the generation of high-throughput functional genomics data has driven the need for sys-tems-level approaches in molecular biology to understand how cellular behavior arises from the interaction of its components. One of the central challenges of systems-level approaches, however, is the automation of various steps of the scientific discovery process, that is, the replacement of error-prone and tedious human operation by high-throughput automated laboratory and computational methods. Our research group aims at (i) developing machine learning methods to extract knowledge from large-scale phenotype datasets and transform this knowledge into predictive mathematical models of cellular operation, (ii) automating experimental design and hypothesis genera-tion in antimicrobial drug discovery, and (iii) deciphering the mechanisms and evolution of genetic interactions.

evolUTIonaRY sYsTems bIoloGY/ papp lab(sYnTHeTIC/sYsTems bIoloGY UnIT)

Balázs PAPP / Principal Investigator, Group Leader

Dániel KÖvESI-NAGY / staff scientistKároly KOváCS / Phd student Balázs SZAPPANOS / Phd student

55

BIO

CHEM

ISTR

Y

A conceptual model to explain conditional synthetic lethal genetic in-teractions in metabolism. A key metabolite (yellow circle) can be synthe-sized via three independent pathways. Metabolic genes A and B show synthetic lethality in Environment I, where starting nutrients of both pathways are present in the medium. However, B is unable to compen-sate deletion of A in Environment II, and the double mutant is rescued by the third pathway in Environment III.

(ii) Automated refinement of metabolic network models

Genome-scale metabolic models give a mechanistic mapping between genotype and phenotype and can be used to understand the behavior of gene networks, to design novel strains for metabolic engineering appli-cations, and to examine the process of genome evolu-tion. The construction and refinement of such mod-els, however, is largely performed manually, which is slow, difficult to reproduce, prone to biases, and can-not make efficient use of all types of high-throughput data. automating model inference from systematic phenotype data is therefore of paramount importance. We are applying machine learning techniques to im-prove the metabolic network model of the yeast Sac-charomyces cerevisiae, based on an unprecedented set of quantitative phenotypic data on millions of mutants (published single gene deletion data and unpublished double gene deletion data provided by our collabora-tor, charles Boone, toronto).

(iii) Automated discovery of optimal antimicrobial drug combinations

evolution of antimicrobial drug resistance is a problem that continues to challenge the healthcare in-dustry. In addition to discover new compounds, there is an increasing need to identify optimal combinations of existing drugs (‘drug cocktails’) that are highly ef-fective against resistant strains. most high-throughput experimental screens aim to test all possible pair-wise combinations in a given drug compound library, which is not feasible for large libraries, or when the goal is to test the synergistic effects between three or more com-pounds. Therefore, robotic protocols coupled with in-telligent experimental selection are needed to explore the vast chemical space of drug compounds in rapid and cost-effective ways. We are developing a heuris-tic algorithm that would optimize the composition of antimicrobial drug cocktails by iteratively performing experiments, using automated laboratory equipments, and automatically evaluating them. The project is run in collaboration with the Pál lab (Brc, szeged).


Group homepage: www.brc.hu/sysbiol/

Balázs PAPP evolUTIonaRY sYsTems bIoloGY/ papp lab

56

BIO

CHEM

ISTR

Y

Phylogenetic diversity of endogenous opioid peptides

Leu- and met-encephalin were the first endogenous opioid peptides identified in various mammalian spe-cies including the human. comparative biochemical and bioinformatic evidence indicates that encepha-lins are not limited to mammals. various prodynor-phin (PdYn) sequences in lower vertebrates revealed the presence of other encephalin fingerprints in these precursor polypeptides. among the novel encephalins Ile-encephalin (tyr-Gly-Gly-Phe-Ile) was primarily observed in the african clawed frog (Xenopus laevis) PdYns, whereas the structure of Phe-encephalin (tyr-Gly-Gly-Phe-Phe) was predicted by analyzing brain cdna sequences encoding a PdYn of the african lung-fish (Protopterus annectens). Ile-encephalin can also be found in the PdYns of other fish species including the eel, bichir, zebrafish and tilapia, but no further occur-rence for the Phe-encephalin motif is available as yet. Based on sequencing data, the biological relevance of Phe- and Ile-encephalin is suggested, because both can arise by regular posttranslational enzymatic processing

of the respective neuropeptide precursors. Phylogenetic variability of the endogenous neuropeptides offers an ‘evolutionary established peptide combinatorial library’ that is expectedly increasing as more and more bioin-formatic data become available.

Emerging role of opioids and nociceptin in epilepsy

The incidence of epilepsy in the human popula-tion is very high, giving a tremendous challenge for

G-protein coupled receptors (GPCRs, 7TM receptors) represent an important superfamily of membrane proteins mainly involved in transmembrane signaling. Opioid and antiopiate receptors play a major role in the process of pain relief and are principally involved in the development of opiate addiction. Natural opioid peptides derived from polypeptide macromolecular precursors serve as endogenous ligands of the opioid receptors. Both endogenous opioid peptides and their receptors display significant multiplicity. This group investigates the ligand-receptor in-teraction and regulation to achieve a better understanding of the molecular basis of heterogeneity. The ultimate goal is to develop new analgesics with reduced side effects.

sTRUCTURal anD fUnCTIonal HeTeRoGeneITY of THe opIoID (moRpHIne) ReCepToR sYsTem(neURobIoloGY UnIT)

Sándor BENYHE / Principal Investigator, Group Leader Anna BORSODI / Principal Investigator

Fanni TóTH / staff scientistEngin BOJNIK / staff scientistFerenc ZáDOR / Phd studentKatalin PAPP / technicianZsuzsanna CANJAvEC / technician

57

BIO

CHEM

ISTR

Y

the successful medication. for better understanding the mechanisms behind the disease, extensive mo-lecular studies are required. We conducted our stud-ies in animal models with compounds acting in pain relief and playing a role in epilepsy as well. nociceptin heptadecapeptide influences the threshold for elicit-ing afterdischarge, generation and spread of seizure activity. The effect is different in partial and general-ized seizures. nociceptin does not modify behavioral changes, spike frequency and duration of discharge. autoradiographic experiments carried out with ra-dioactive compounds showed a different distribution of the receptors in the brain regions studied, with enhancement of receptor numbers for nociceptin as well as for opioid receptors recognizing morphine. It is shown that nociceptin facilitates the development of seizures in partial, but not in generalized seizures.

Bivalent ligands targeting MOP-NOP receptor heterodimers

numerous studies have established the presence of GPcrs as dimers in heterologous cell expression sys-tems and also in vivo. In this context, heterodimers of opioid receptors would constitute new targets of spe-cific interest, each entity possessing original proper-ties in terms of function and pharmacology.

mu opioid receptor (moPr) activation induces an-algesia, while noPr activation would produce hyper-algesia. Thus, in order to fight pain, it would be impor-tant to synthesize one molecule combining the agonist effect for the moP and the antagonist effect for the noPr receptors, in order to reinforce the synergistic effect on analgesia. The objective of this project is to conceive new analgesic molecules by combining two pharmacophores targeting the moPr/noPr het-erodimers. This research should therefore speed up comprehension of the physiological roles of pronoci-ceptin-derived neuropeptides and may provide new targets and tools to develop novel, potent, non-opiate and non-addictive analgesic drugs.


Sándor BENYHE, Anna BORSODI

sTRUCTURal anD fUnCTIonal HeTeRoGeneITY of THe opIoID (moRpHIne) ReCepToR sYsTem

58

BIO

CHEM

ISTR

Y

New directions in receptor research; promiscuity versus specificity, and establishment of a new model of opioid tolerance/dependence

GPcrs are the largest class of cell-surface recep-tors and are encoded by >1,000 genes in the human genome. more than 50% of all current drugs regulate GPcr function, and some 30% of these drugs direct-ly target GPcrs. currently, our laboratory is focus-ing on the opioid and cannabinoid receptor families and their interactions. We have demonstrated cross-antagonism between G-protein signaling of GaBaB and cannabinoid cB1 receptors, which altered the pharmacology of the individual receptors. The in-teraction was tissue-specific and only manifested in membranes of hippocampus, but not in cerebral cortex or spinal cord. The cross-talk of the two re-ceptors is being investigated by confocal microscopy and Bret studies in cell lines co-transfected with GaBaB and cB1 receptors in Hungarian and us col-laborations.

many GPcrs exhibit constitutive activity in the absence of agonists that can be selectively blocked by ligands that is referred to as inverse agonists. The cB1 receptors display a significant level of constitutive ac-tivity. We have shown that the cB1 specific antagonist rimonabant (sr141716) displays cB1 receptor-inde-pendent inverse agonist properties at high concen-trations. It was found to be able to block G-protein signaling of the highly mu-opioid receptor specific ag-onist, damGo in cHo cells expressing recombinant mu-opioid receptors (mor-cHo). co-application of sr141716 with damGo unmasked a PtX-insensitive, novel, mor-mediated G-protein signaling. notably, the novel signaling was desensitized by chronic mor-phine treatment. These results provide new directions to understand drug tolerance/dependence.

The new model of tolerance, to which our previous results has contributed suggests that there is an inverse relationship between the extent of receptor internali-zation and the abuse potential of the ligand. to test this hypothesis, rats are treated acutely or chronically with various ligands, followed by pharmacological,

My laboratory is predominantly interested in two research areas: one of them is to reveal the molecular mecha-nism of receptor functioning from the molecular level analysis of ligand-receptor interactions to in vivo studies; the other is to understand the molecular basis of drug tolerance/dependence. Evidence is accumulating to show that receptors do not always function as monomers, but may also interact with each other either via cross-talk at the signaling level, or by forming homo- and hetero-oligomers. These molecular complexes may have altered binding and signaling properties. Understanding their interactions may provide new targets for drug discovery with less side-effects. In recent years, we have been focusing on two systems, the opioid and cannabinoid receptors, both of which play key roles in analgesia and drug addiction. The ligand-specific nature of the molecular changes underly-ing opioid analgesic tolerance is being explored. These studies contribute to our understanding of drug addiction, thus may be useful in developing potent analgesic drugs with less side-effects.

laboRaToRY of moleCUlaR pHaRmaColoGY(neURobIoloGY UnIT)

Mária SZŰCS / Principal Investigator

Erika BIRKáS / staff scientistResat CINAR / staff scientistIldikó NÉMETH / technician

59

BIO

CHEM

ISTR

Y

receptor binding and signaling as well as autoradiog-raphy measurements. a wide spectrum of ligands is being used to test the correlation between the extent of receptor internalization and the chemical structure, the efficacy and the abuse potential of the ligand. mor-phine, the most widely used opioid analgesic, which has high abuse potential and is a partial agonist in rat brain, did not internalize surface mors, but elic-ited significant intracellular changes and altered sig-naling. all the full agonists tested, namely damGo, endomorphins and etorphine decreased the density of the surface mors. It is concluded that analgesic tolerance/dependence is a complex phenomenon that cannot be described by a single molecular mechanism. The changes observed are ligand- and tissue-specific, and are further investigated.

to develop new opioid ligands with improved prop-erties (higher specificity, selectivity or longer half-life), new peptides, synthesized by the neuropeptide Laboratory of the Brc, are being studied in receptor

binding assays. a new, conformationally constrained analog of the well-known delta-opioid antagonist tIPP was radiolabeled and the resulting ligand, [3H]tyr-tic-(2s,3r)-β-mePhe-Phe-oH characterized in binding assays. The new ligand was 4× more potent delta-antagonist than the parent ligand. Based on structure-activity relationship studies performed with 16 new endomorphin peptidomimetics, two ‘lead’ molecules have been selected. [3H]cisacPc-endomor-phin-2 and [3H]cisacHc-endomorphin-2 [results to be published] have been prepared and characterized in binding and functional assays. They were found to be high affinity and selectivity mor agonists with long half-lives. These two new ligands may be helpful tools in mapping the bioactive conformation of mors. These new ligands will also be evaluated in future in vivo chronic studies.


Mária SZŰCS laboRaToRY of moleCUlaR pHaRmaColoGY

60

BIO

CHEM

ISTR

Y

Peptide and protein synthesis

The therapeutic value of opioid type drugs is high, but their side-effects and their misuse cause serious medical and social problems. The diverse physiological effects of the opiates and opioid pep-tides are mediated by the heterogeneous G-protein coupled opioid receptors. However, the lack of their high-resolution structure restricts the design of opio-ids free of side effects. as an alternative, structure-activity studies of ligand sets with well defined three dimensional structures made it feasible to determine the spatial arrangement of pharmacophore elements, and in an iterative way to find more selective ligands with high affinity. such structure-activity studies were performed on unnatural amino acid contain-ing endomorphin, deltorphin and tIPP analogues. It turned out that the gauche(–) orientation of the Phe4 side chain is important for binding to the μ-opioid receptor. furthermore, bending of the tetrapeptide backbone was achieved by incorporating a spirocyclic benzazepinone into the endomorphin-2 sequence, and this forced secondary structure was preferred by the μ-opioid receptor. The incorporation of alicy-

clic β-amino acids into the endomorphin sequences resulted in another ligand set with variable opioid receptor affinities and selectivities that depended on the absolute configuration of the substituent amino acid.

Because the genome-sequencing projects have re-vealed hundreds of thousands of new proteins without isolating and characterizing them, our very recent re-search efforts target the incorporation of site-specific covalent modifications into proteins to support their physicochemical and functional characterization. chemical synthesis alone or in combination with recombinant techniques is a powerful approach for this purpose, as it allows both the covalent structure to be varied and the incorporation of non-native ele-ments. The model system is the membrane-associated prion protein (PrP), where the chemical synthesis of fluorescent-labeled membrane anchor molecules and the total chemical synthesis of the full length PrP are explored. different anchor mimetics are conjugated to the recombinant PrP, and the resulting semisynthetic proteins are used for in vitro and in vivo studies of PrP misfolding, the basic phenomenon during prion pathogenesis (collaboration with e. Welker, Brc,

Chemistry based research in our group is focused on the understanding of how proteins work and how the structural features of receptor ligands influence protein – small molecule interacting systems. One major area of our activity is to develop new methods for the preparation of special polypeptides and proteins of biomedical inter-est; furthermore, we have intensive programs in small molecule receptor ligand development, radioactive labeling, molecule spectroscopy and molecule modeling. These combined efforts result in protein derivatives enriched in chemical information, small ligand libraries with systematic structural changes and radiolabeled receptor lig-ands that are used as probes for protein function and receptor signaling. Beyond the biological studies, theoretical chemical techniques support the in-depth analysis of structure – activity/function relationships.

CHemICal bIoloGY(neURobIoloGY UnIT)

Csaba TÖMBÖLY / Principal Investigator Géza TóTH / Principal Investigator

Attila BORICS / staff scientistAttila KERESZTES / staff scientistMariann MAJER / research assistantJayapalreddy MALLAREDDY / Phd studentBalázs SCHÄFER / Phd studentÉva TóTHNÉ PAPP / technician

61

BIO

CHEM

ISTR

Y

szeged). This research will provide a new methodology for protein production and novel proteins presumably with designed properties.

Molecule structure investigation

structural investigations with various spectro-scopic and computational methods are focused on the opioid receptor—opioid ligand interactions, on the membrane embedding of semisynthetic PrP, and on the development of novel strategies for conformational analysis. structure-activity studies of the opioid recep-tor ligands help refine the theory on the proper three-dimensional arrangement of pharmacophore elements. for µ-opioid ligands the simultaneous fulfillment of four structural requirements were shown to result in high affinity binding. membrane environment studies are set up to give information in atomic details about the distribution and orientation of the PrP-anchor mi-metic conjugates embedded in a lipid bilayer.

our strategy development involves the theoreti-cal reproduction of Ir and vcd spectra using mo-lecular dynamics simulations and quantum chemical calculations. The advantage of this approach is that it provides atomic resolution information about all con-formational states present in the equilibrium, while other high resolution experimental techniques, such as nmr provide only time-averaged results when fast conformational transitions occur.

Radioactive labeling

In vitro or in vivo investigation of radiolabeled high affinity and selectivity ligands is an important step in

the characterization of the ligand – receptor interac-tions and in the investigation of the pharmacological properties of an administered drug. Preferred radiolig-ands or drugs for this purpose are H-3, c-14 and I-125 labeled compounds. The tritium and carbon isotopes are incorporated by total chemical synthesis, or in the case of tritium by reduction of halogenated or unsatu-rated precursor compounds. for peptides not contain-ing either aromatic amino acids or Pro or Leu, a new tritium labeling method has been developed that allows the incorporation of tritium into val or ala residues of peptides by the reduction of Pen or cys residues, re-spectively. The most important novel tritiated μ and δ opioid receptor selective ligands are: endomorphin 1, (tyr-Pro-trp-Phe-nH2), endomorphin 2, (tyr-Pro-Phe-Phe-nH2), taPP (tyr-d-ala-Phe-Phe-nH2), tIPP [Ψ] (tyr-ticΨ[cH2-nH]Phe-Phe-nH2), Ile5,6-deltorphin II (tyr-d-ala-Phe-Glu-Ile-Ile-Gly-nH2), nociceptin (Phe-Gly-Gly-Phe-Thr-Gly-ala-arg-Lys-ser-ala-arg-Lys-Leu-ala-asn-Gln-oH), nociceptin (1–13) amide and N,N-dimethyl-dmt-tic-oH. [3H]β-amyloid fragments and [3H]GnrH-III are further ex-amples of compounds prepared for receptor research. The description of the in vitro proteolytic degradation of endomorphins was achieved with the digestion of different H-3 isotopomers of endomorphins.

Being useful research tools, some of these radioli-gands have become commercial products, and our ra-diolabeling expertise is applied in numerous contract research tasks to prepare deuterium, tritium or carbon labeled compounds.


Csaba TÖMBÖLY, Géza TóTH CHemICal bIoloGY

62

BIO

CHEM

ISTR

Y

Modeling cardiovascular disease (atherosclerosis)

cholesterol is transported in a lipoprotein-bound form in the blood. The four major transporter mol-ecules are: high density lipoprotein (HdL), interme-diate density lipoprotein (IdL), low density lipopro-tein (LdL) and very low density lipoprotein (vLdL). among these lipoproteins, LdL is highly prone to deposition in the intima of blood vessels initiating atherosclerosis. Apolipoprotein B (apoB) is a major structural component of LdL and vLdL, therefore, high plasma levels of apoB-containing lipoproteins are associated with an increased risk of coronary ar-tery disease. The molecular and cellular mechanisms of the pathobiological changes that lead to the disease are still poorly understood. accumulating evidence during recent years has led to the concept that sub-endothelial retention of apoB100-containing lipopro-teins is the initial event in atherogenesis. This process might trigger local inflammation promoting vascular lesion formation.

We have developed hyperlipidemic mice by overex-pressing the human apoB-100 gene in transgenic mice. These mice showed significantly elevated serum level

of triglyceride when they were fed normal chow diet, and elevated serum cholesterol level when they were fed cholesterol-rich diet. transgenic mice kept on cho-lesterol-rich diet developed atherosclerosis by the age of 6-7 months. In the heart of these animals increased cardiac superoxide and peroxinitrite formation was detected, which resulted in cardiac dysfunction (3).

Modeling age-related neurodegeneration

recent studies show that the human neurodegen-erative disorder, alzheimer’s disease (ad) is accompa-nied by elevated apolipoprotein B concentration in the serum, and high serum level of apoB-100 modulates cerebral aβ deposition in vivo. ad is characterized by progressive memory loss and cognitive impairment accompanied by neural degeneration, formation of amyloid plaques and neurofibrillary tangles. abnor-mal accumulation of apolipoproteins and cholesterol in the brain of ad patients has been detected as core components of mature amyloid plaques. cholesterol content in neuronal membranes contributes to the maintenance of neuronal plasticity and might directly modulate the rate of amyloid precursor protein (aPP) processing. a disturbance or imbalance in the sterol

The main activity of our group is to develop disease models. We focus on cardiovascular disease (atheroscle-rosis), diabetes, neurodegeneration and psychiatric disorders. Mouse models provide excellent tools to study the pathomechanism of diseases in detail using genomics, proteomics, lipidomics, molecular biology, immunohisto-chemistry and electrophysiology. The major benefit of the animal models is their use in the development of effective drugs and therapies.

laboRaToRY of anImal GeneTICs anD moleCUlaR neURobIoloGY(neURobIoloGY UnIT)

Miklós SáNTHA / Principal Investigator, Group Leader

Melinda E. TóTH / staff scientistNikolett LÉNáRT / Phd studentSzilvia GONDA / technicianMónika MARI / technician

63

BIO

CHEM

ISTR

Y

metabolism has been found in ad and vascular de-mentia, evidenced by an increase in the cholesterol breakdown product 24s-hydroxycholesterol. It was re-cently demonstrated that elevation of plasma triglyc-eride level precedes amyloid deposition in alzheimer’s disease model mice. although apolipoprotein and cholesterol research was previously mainly focusing on cardiovascular diseases, the latest findings indicate that apoB-100 might be involved in the development of stroke and neurodegenerative processes.

Investigating the phenotype of apoB-100 transgen-ic mice we found that vascular lesions affect not only the cardiovascular but also the cerebrovascular sys-tem. apoB-100 overexpression induced alterations in the cerebral protein profile of transgenic mice, an ap-proximately two-fold increase in the level of signaling proteins involved in apoptosis, nonreceptor tyrosine kinase Pyk2, p38maPK, maPK (mitogen activated protein kinase) and brain “injury indicator” proteins (nnos, inos, s-100 β, glutamine synthetase, and cel-lular stress protein, Hsp70) were detected (2). mor-phological analysis using mrI and memrI revealed a large increase in the cavity size of the lateral and dor-sal ventricles and a moderate enlargement of the aq-

ueduct (fourth ventricle) in the brain of heterozygous transgenic animals (2). moreover, the enlargement of the ventricular system was transgene dose-dependent, which is more pronounced in homozygous than in heterozygous transgenic mice (see fig. below).


Miklós SáNTHA laboRaToRY of anImal GeneTICs anD moleCUlaR neURobIoloGY

64

BIO

CHEM

ISTR

Y

In joint laboratories located at the Brc and at the Biochemistry and molecular Biology department of szeged university we use biochemical and genetic ap-proaches to tackle problems related to transcription regulation. ongoing research projects of the labora-tory target different participants of the transcription process. some of the factors studied play a direct role in rna synthesis (Pol II ctd, rPB4), others regulate the activity of the Pol II complex (fcP1, p53), and fur-ther ones are modifiers of chromatin structure (saGa and atac complexes).

recent results of the group on the functional anal-ysis of genes encoding transcriptional coactivators (ada2a, ada2b, ada3) have lead to the discovery of two Drosophila histone acetyltransferase (Hat) com-plexes: saGa and atac, sharing a common acetyl-transferase subunit: Gcn5. By detailed analysis of the effects of mutations in shared and complex-specific subunits of saGa and atac we have shown that by acetylating histone H3 and H4 lysine residues these complexes regulate different sets of genes and various chromatin-associated processes. The demonstration of the functional interconnection of histone acetylation, phosphorylation and methylation is a fine in vivo ex-

ample for a molecular mechanism of complex histone modification pattern formation which contributes to epigenetic mechanisms.


Transcription of eukaryotic genes is a multistep process that involves the interaction of a large number of func-tionally different protein factors and requires the ordered assembly of giant multiprotein complexes in gene regula-tory regions. Understanding the role of functionally distinct classes of transcription regulatory proteins is a key to uncovering cellular processes and deciphering why and how those are de-regulated in diseases.

eUkaRYoTIC TRansCRIpTIon ReGUlaTIon (eUkaRYoTIC moleCUlaR bIoloGY UnIT)

Imre M. BOROS / Principal Investigator, Group Leader

István TOMBáCZ / staff scientistNorbert PARDI / staff sciencitstEdith váMOS / staff scientistGábor GRÉZAL / Phd studentKatalin ÖKRÖS / technician

65

BIO

CHEM

ISTR

Y

Ubiquitin-dependent protein degradation in Drosophila

orderly and timely progression through the cell di-vision cycle is essential to maintain genome stability in eukaryotes. errors in this process alter gene dosage and can result in developmental defects or cancer. Key transitions in the eukaryotic cell cycle are regulated through intracellular proteolysis by the ubiquitin-proteasome system (uPs). due to the irreversible na-ture of protein degradation, by sequentially removing regulators, this process generates directionality in the cell cycle and ensures proper timing and coordination of events.

degradation of proteins by the uPs involves two successive steps: tagging of substrate proteins by the covalent attachment of polyubiquitin chains and the subsequent degradation of the tagged protein by the 26s proteasome. Besides the ubiquitin conjugating pathway and the 26s proteasome, additional factors like deubiquitylating enzymes (duBs) and polyubiq-uitin binding proteins (uBPs) are involved in the ef-ficient degradation of target proteins.

The ubiquitin conjugating pathway involves an en-zymatic cascade of ubiquitin-activating (e1), ubiqui-tin-conjugating (e2), ubiquitin ligase (e3) enzymes, and at least sometimes, specific polyubiquitin chain elongation factors (e4), which catalyze the assembly of polyubiquitin chains on lysine residues of substrate

proteins. The key component of this protein degrada-tion machinery is the ubiquitin ligase, because it de-termines substrate specificity.

two critical transitions in mitosis, the metaphase to anaphase transition and the exit from mitosis are mediated by a large ubiquitin-protein ligase complex known as the anaphase-promoting complex or APC. The aPc is composed of at least twelve subunits; most

The main interest of our laboratory is to understand the role of ubiquitin-dependent proteolysis in the control of the eukaryotic cell cycle. We exploit genetic, molecular and cell biological techniques available in Drosophila melanogaster for mutant isolation and functional analysis of cell cycle regulation and ubiquitylation.

eUkaRYoTIC Cell CYCle ReGUlaTIon(eUkaRYoTIC moleCUlaR bIoloGY UnIT)

Péter DEáK / Principal Investigator, Group Leader

Margit PáL / staff scientistOlga NAGY / staff scientistKata vARGA / Phd student

66

BIO

CHEM

ISTR

Y

of them are evolutionally conserved in all eukaryotes from yeasts to humans.

Though the main function of aPc and key steps in its regulation are well established, many important questions related to the ubiquitylation state and func-tion of regulatory proteins remain unanswered. We don't yet know why so many subunits are present in the aPc or what their individual functions are. It is unclear how the aPc mediates the assembly of polyu-biquitin chains on substrates and how these tagged proteins are escorted and recognized by the 26s pro-teasome. Little is known about the regulatory role of

deubiquitylating enzymes (duBs) that remove ubiq-uitin molecules from proteins.

our group is focusing on studying the role of aPc subunits, duBs and uBPs in the proteasomal degra-dation of mitotic regulatory proteins in the fruit fly, Drosophila melanogaster. We use P element-based for-ward and reverse genetic techniques to isolate mutant alleles of genes involved in these processes and to de-termine their roles.


Péter DEáK eUkaRYoTIC Cell CYCle ReGUlaTIon

67

BIO

CHEM

ISTR

Y

selective intracellular protein degradation proceeds in two steps: first the protein to be degraded is marked by a specific posttranslational modification (ubiquiti-nation) performed by a series of ubiquitin conjugating enzymes, and this is followed by the proteolytic step performed by a very large multisubunit protease (26s proteasome), which cleaves only the ubiquitinated proteins. The 20s proteasome, a multicatalytic large protease complex was identified as the catalytic com-ponent of the 26s proteasome. The 20s proteasome alone has no specificity: it cleaves any non-ubiquiti-nated protein. The regulatory component of the 26s proteasome, responsible for the selective degradation of ubiquitinated proteins was identified, purified and characterized in my laboratory (J. Biol. chem. 268, 9055. 1993). We showed that the 26s proteasome can be reconstituted in vitro from the purified regulatory complex and the 20s proteasome, and the in vitro re-constituted 26s proteasome degrades selectively only ubiquitinated proteins. The regulatory complex is a large (800 kda) multiprotein particle. We have cloned and sequenced several subunits of this complex. Yeast homologs of two subunits of the regulatory complex were identified. mutation of these subunits in S. cerevi-siae stabilized the short-lived ubiquitin-proline-beta-

galactosidase fusion protein and the cLB2 and cLB3 cyclins, and arrested cell division in G2/metaphase (nature 366, 358. 1993). This was the first direct evi-dence that (i) the 26s proteasome is required for the in vivo degradation of ubiquitinated proteins, and (ii) cyclins are degraded in vivo by the ubiquitin-26s pro-teasome system. recently we have identified, cloned and sequenced all the subunits of the Drosophila reg-ulatory complex, and using a chemical cross-linking approach and a highly selective immunological de-tection system we have identified the closely-spaced subunits of the regulatory complex. to study the in vivo function of the multiubiquitin-binding subunit of the regulatory complex (subunit p54) we have deleted the single-copy gene of subunit p54. The homozygous Drosophila deletion results in pupal lethality, the large accumulation of multiubiquitinated proteins, severe mitotic defects and a co-ordinated upregulation of all the proteasomal genes (figs. 1 and 2). The function of the different domains of the multiubiquitin-binding subunit is analyzed recently by establishing transgenic Drosophila strains expressing the full-length subunit, its n-terminal half carrying the vWa domain or its c-terminal half carrying the uIm-domains. We have shown that the c-terminal half of the subunit in its

Cellular homeostasis is maintained by the dynamic balance of regulated expression of genes and the regu-lated degradation of proteins. The role of chromatin architecture in the regulation of gene expression was in the focus of our previous research activity. During the last years we concentrate on studying the structure and func-tion of the 26S proteasome, a large proteolytic complex responsible for the regulated intracellular degradation of proteins.

ReGUlaTIon of InTRaCellUlaR pRoTeIn DeGRaDaTIon (eUkaRYoTIC moleCUlaR bIoloGY UnIT)

Andor UDvARDY / Principal Investigator

Zoltán LIPINSZKI / staff scientistKatalin UDvARDY / technician

68

BIO

CHEM

ISTR

Y

extraproteasomal state is multiubiquitinated, the site of this modification is the cluster of lysines at the very c-terminal end of the subunit, and this modification is not a degradation signal. recently we analyze the developmentally regulated changes of the 26s protea-somes. We found that the stoichiometry of the multi-ubiquitin-binding subunit drops suddenly at the end of the embryogenesis, remains low through the larval stages, starts to increase at the end of the third instar larval stage and reaches a high level in pupae, adults

and embryos. a specific proteolytic system was iden-tified which is responsible for the selective degrada-tion of the ubiquitin-binding subunit during the lar-val stage. There is an inverse relationship between this larval-specific proteolytic system and the 26s protea-somes.


Andor UDvARDY ReGUlaTIon of InTRaCellUlaR pRoTeIn DeGRaDaTIon

69

BIO

CHEM

ISTR

Y

The function of ECM proteins in muscle development, muscle regeneration and carcinogenesis

By cloning the matrilin-1 and matrilin-2 genes, we discovered the matrilin family of noncollagenous proteins, which form collagen-dependent and inde-pendent filamentous networks in the ecm of vari-ous tissues. matrilin-1 and -3 are deposited only in cartilage and bone, whereas matrilin-2 and -4 are found in a large variety of tissues including skin, sev-eral epithelia, muscle and the nerve system. We study the expression and function of matrilins during dif-ferentiation and regeneration processes and under pathological conditions. to this end, we generated cell lines that produce a variety of matrilin-2 recom-binants to pinpoint the role of the various protein modules in macromolecular interactions during the organization of the ecm. In the framework of inter-national collaborations, transgenic mice have been generated that are deficient in the matrilin-2 gene. at present our efforts are focused on two projects based on national and international collaborations. one of the projects aims at studying the role of mat-rilin-2 in muscle differentiation and regeneration.

We observed transient activation of the gene in re-generating rat skeletal muscle. In vitro differentiation of myoblasts recapitulated the process. We perform comprehensive analysis to compare matrilin-2 pro-tein and rna expression with those of other marker genes during muscle development and skeletal mus-cle regeneration in wild type and matrilin-2 deficient mice as well as in differentiating myoblast cultures. It is under study whether matrilin-2 can directly or in-directly affect muscle cells, by interacting with other ecm components. We are interested in what signal transduction pathways are involved and how the dif-ferentiation is altered.

Multicellular organisms deposit a highly organized extracellular matrix (ECM) around the cells, which pro-vides physical support and the necessary milieu for normal cell metabolism and development, and delineates pathways during differentiation and tissue regeneration. The ECM performs essential, but very divergent functions in the various tissues and organs. We investigate how matrilins and other noncollagenous glycoproteins involved in the organization of the ECM contribute to tissue integrity, differentiation and carcinogenesis using molecular biology methods, cell culture and transgenesis. We also investigate how the tissue-specific expression of these genes is regulated in the developing musculo-skeletal system.

moleCUlaR bIoloGY of THe exTRaCellUlaR maTRIx(eUkaRYoTIC moleCUlaR bIoloGY UnIT)

Ibolya KISS / Principal Investigator, Group Leader

Ferenc DEáK / senior scientistErzsébet KÉNESI / staff scientistviktória SZŰTS / staff scientistYvonne ZSáK / Phd studentAnikó SIMON / technicianEmőke HORváTH / technicianKlaudia KávAI / technician

70

BIO

CHEM

ISTR

Y

The other project aims to study the role of matri-lin-2 in tumor formation. We have found previously that the matrilin-2 protein/rna level can be used as a diagnostic/prognostic marker in particular neopla-sias. Therefore we investigate tumor formation and the underlying molecular mechanisms in matrilin-2 defi-cient mice. We also participate in testing anticancer drugs.

Molecular basis of cartilage-specific gene expression and establishment of transgenic animal models for studying skeletal development and cartilage regeneration

The prevalence of joint diseases increases worldwide with the aging of the population. Inherited joint diseases disturbing longitudinal bone growth often cause dwarf-ism, which cannot be cured by current medical treat-ments. Arthritis is a chronic degenerative arthropathy that frequently leads to persistent pain and disability. Due to the large burden on the society, finding better treatments for arthritis is a major focus of medically oriented research. There is a great need for new ther-apies that address not only the reduction of pain and inflammation, but also the promotion of cartilage ma-trix regeneration. Development of new drugs requires the development of animal models suitable for testing cartilage-specific gene expression and cartilage regen-eration. Therefore, we work on establishing transgenic animal models for monitoring cartilage wear and re-generation.

Previously we cloned genes for cartilage link pro-tein and matrilin-1 specifically expressed in cartilage. The matrilin-1 gene has the unique property among cartilage protein genes that its expression is restricted to the late proliferative and prehypertrophic zones

of the growth plate, which play an important role in the regulation of bone growth. By means of recom-binant dna technology, in vitro dna-protein inter-action studies, transiently transfected cell cultures and transgenic mice, we characterized the regulatory regions and transcription factors responsible for the tissue- and zone-specific expression of the gene. our data demonstrate a complex regulation based on the modular arrangement of the tissue-specific control elements. We constructed cartilage-specific vectors and generated transgenic mice, which express a well-detectable marker gene in distinct zones of the growth plate. These animal models are suitable for testing the effect of drugs and drug candidates on skeletal devel-opment in safety pharmacology.

We also aim at inducing arthritis in transgenic mice expressing the reporter gene in each chondrocyte and developing an animal model suitable for testing the effect of anti-inflammatory drugs and potential antirheumaticums on articular cartilage degradation and regeneration.


Ibolya KISS moleCUlaR bIoloGY of THe exTRaCellUlaR maTRIx

71

BIO

CHEM

ISTR

Y

We studied reverse signaling in the tumor necrosis factor (tnf) – tnf receptor system. modification of this bidirectional signaling promised the attenuation of the autoimmune reactions and host versus graft diseases.

The new project of the group project targets age-re-lated macular degeneration (arm), the leading cause of blindness in developed countries. This disease af-fects a high proportion of elderly people leading to sig-nificant deterioration of life quality and generation of societal problems.

a number of risk alleles of many genes are identi-fied and environmental or behavioral factors, like dia-

betes, smoking, obesity and hypertension are also as-sociated with the development of arm. still, the exact mechanisms are far from being known.

our goal is to investigate the physiological and im-munological background leading to druzen forma-tion, neovascularization and loss of vision, to identify potential new targets for the treatment and especially the prevention of this debilitating disease.


The main subject of the group has been the investigation of reverse signaling, one of the fine-tuning mechanisms of the immune response and the inflammatory reaction.

ReveRse sIGnalInG of TUmoR neCRosIs faCToR-alpHa (Tnf)(eUkaRYoTIC moleCUlaR bIoloGY UnIT)

Ernő DUDA / Principal Investigator, Group Leader

Krisztina BUZáS / staff scientistTamás LETOHA / staff scientistKata JUHáSZ / Phd student

72

BIO

CHEM

ISTR

Y

Beside his participation in the main project of the eukaryotic molecular Biology unit, csaba vizler di-rects a pharmacological research sub-group that uses animal disease models and cell culture for mecha-nism of action studies and pre-clinical testing of drug candidates developed either in house or by Hungar-ian biotech companies. Three main research lines are pursued. (1) studies on the anti-tumoral effect, phar-macokinetics and mode of action of novel anti-cancer drugs; (2) development and testing of immunomodu-lating peptides and small molecules; (3) The trPv1 pain receptor (capsaicin receptor) is the major recep-tor involved in the generation of inflammatory pain and neuropathic pain. The research sub-group stud-ies the pharmacology and physiology of the trPv1

receptor and develops receptor antagonists and channel blockers for the treatment of cancer pain and neuropathies.

The portfolio of the group consists of numerous in vivo mouse disease models (transplantable tumors, graft-versus-host disease, dtH, streptozocin diabe-tes, analgesia assays, experimental allergic encepha-lomyelitis), mammalian tissue culture, in vitro im-munoassays, medium-throughput isotope influx- and luminescent reporter cell assays and hybridoma tech-nology.


pHaRmaColoGY laboRaToRY

Csaba vIZLER / Principal Investigator

László PECZE / staff scientist

73

BIO

CHEM

ISTR

Y

Stress protein molecular chaperones

Induced by a wide range of stressors, from tem-perature stress to hypoxia, inflammation, infections or environmental pollutants, stress proteins, also termed heat shock proteins (HsPs) play key roles in living sys-tems. The HsPs are named according to their molecular weights. HsP100, HsP90, HsP70, HsP60 and the “small heat shock proteins”, each define families of chaperones.

The HsP molecular chaperones are able to recog-nize damaged proteins and to redirect them to repair (refolding) or to proteolysis. By helping to stabilize partially unfolded proteins, HsPs aid in transporting proteins across membranes within the cell. HsPs can regulate the life or death of cells by directly modulat-ing certain apoptotic signaling events or indirectly, by participating in antigen processing.

Stress proteins in the membranes

Quite surprisingly, a subpopulation of HsPs is present either on the surface or within the cellular membranes. as we have shown in our studies de-scribed below, via their specific lipid and protein in-teractions certain HsPs can control major attributes of membranes. The membrane (raft or non-raft) associ-ated HsPs can also participate in the orchestration of distinct stress signaling platforms. In spite of lacking a secretory signal, some HsPs are mysteriously released from cells by various secretory mechanisms. Irrespec-tive of the secretory routes chosen (raft-, exosome- or secretary granule-mediated, etc.), these exogenous HsPs can then stimulate both the innate and the adap-tive immune system.

Our major goal is to understand those changes in lipid composition, fluidity- and microdomain organization of plasma and endo-membranes which alter the expression of the stress protein molecular chaperones (HSPs). Among the specific transcription factors, HSF1 plays a central role in this “membrane-controlled” boosting or silencing of HSPs. The relationship between the specific distribution of lipid rafts and the concomitant changes in the level, pro-file and cellular distribution of HSPs is currently determined via monitoring the surface membrane microdomains with confocal- and ultrasensitive single molecular microscopy. Through comparative lipidomics, key lipid molecu-lar species involved in the activation or attenuation of HSP signaling pathways are identified. We have shown that membrane-associated HSPs can control major attributes of the membranes, like fluidity, permeability, curvature, and the operation of raft-associated signaling platforms. Moreover, membrane association of HSPs can refine hsp gene expression. Applying these principles can yield novel pharmaceutical agents that have the potential of major therapeutic benefit to a number of diverse disease states.

moleCUlaR sTRess bIoloGY(membRane anD sTRess bIoloGY UnIT)

László vÍGH / Principal Investigator, Group Leader

Ibolya HORváTH / staff scientistZsolt TÖRÖK / staff scientistAttila GLATZ / staff scientistImre GOMBOS / staff scientistMária PÉTER / staff scientist

Tim CRUL / staff scientistBurcin GUNGOR / Phd student Gábor BALOGH / research assistantÉva DOBó / technicianGabriella BOGDáN / technician

74

BIO

CHEM

ISTR

Y

Stress response from membranes and back to membranes: lessons from unicellular stress models

an earlier “central dogma” suggested that stress-induced protein denaturation serves as a sole primary stress-sensing machinery, which triggers Hsp gene expression. From the past decade, we have introduced and experimentally verified a new but not exclusive cel-lular “thermosensor” model, which predicts the exist-ence of membrane-associated stress sensing and sign-aling mechanisms. as depicted below (see fig.1.), we propose that changes in the physical state (fluidity) and/or composition of lipid molecular species with the concomitant destabilization/reorganization of membrane microdomains may serve as the “primary molecular switch” for the operation of these “cellular thermometers”.

Figure 1. A model of the crosstalk between the stress sensory membrane, hsp gene expression, and the membrane association of a specific subset of HSPs (see vigh et al, TIBS, 2007). Temperature stress modifies the physical state of the membrane and this activates a membrane signal culminating in the transcription of hsp genes. In turn, as our group evidenced for various prokaryotic models, specific subsets of HSPs (e.g. GroESL and the small HSP called HSP17 in the blue-green alga Synechocystis) localize with specific membrane domains tempo-rarily via newly formed “heat shock lipids”, thus re-establishing proper membrane lipid order and phase-state that then may turn off hsp gene transcription.

The striking similarities of the human and Schizosac-charomyces pombe (fission yeast) signal transduction pathways prompted us to introduce S. pombe as a pow-erful and complementary “micromammal” model. We use it for elucidating early activation steps of the heat shock response including membrane-associated stress sensors, stress signaling pathways and the interplay of potential cellular stress survival strategies (i.e. the concerted and complementary actions of sugar (treha-lose), protein (HsP), and lipid chaperones). Thus, the S. pombe model may allow studying the individual members or the entire stress-protective network in a simple cellular context.

Membrane-regulated stress response in mammalian cells: lipid rafts at the crossroad

due to their multiple and vital functions, stress proteins play a fundamental role in the pathology of several human diseases. aberrantly high levels of certain HsP classes are characteristic in cancer cells and the converse situation applies for type-2 diabetes or neurodegeneration. In accordance, understanding the mechanism whereby mammalian cells can elicit a stress protein response is of key importance. In ad-dition, correcting the defects of membrane domains, engaged in the generation and transmission of stress signals may be of paramount importance for the de-sign of new drugs with the ability to induce or attenu-ate the level of a particular class of heat shock proteins (see fig.2.).

Figure 2. Membrane-mediated stress protein response and the cellular localization of HSPs (highlighted by HSP70) in mammalian cells (see Horváth et al, BBA, 2008). HSF1 is a key coordinator of the initiation of heat shock gene transcription, which is activated mainly by the appearance of denatured or misfolded proteins. In addition, stress sensing-signaling mechanisms operate through stress-induced mem-

László vÍGH moleCUlaR sTRess bIoloGY

75

BIO

CHEM

ISTR

Y

brane rearrangements. Such typical membrane-mediated changes that are evidenced to refine the expression of heat shock genes are the non-specific clustering of the growth factor receptors associated with mem-brane microdomains (“rafts”) (1) or the activation of phospholipases (2), which sequester themselves into unsaturated-rich microdomains and cleave arachidonic acid, a known HSP inducer. Stress activation of such pathways alters the nuclear accumulation and transactivation capacity of HSF1 (3) via its covalent post-translational modifications and ultimate-ly retailor the abundance and profile of HSPs. The function of individual HSPs (highlighted on the scheme by HSP70) depends on their intracel-lular, membrane bound or extracellular location. The major action of chaperone proteins in the cytosol is to maintain protein homeostasis (4). HSP70 can promote cell survival by inhibiting lysosomal membrane per-meabilization via the interaction with specific lipids (5). We are currently

studying the interaction of HSPs with cellular lipid droplets. Experimen-tal evidence is accumulating in favor of the presence of HSP70 (and other HSPs) in lipid rafts as components of signaling or trafficking platforms (6). HSPs can also associate with specific lipids and proteins in the plasma membrane, inducing “membrane stabilization” and/or exhibiting an im-munogenic potential (7). HSP70s of extracellular location (8) have immu-nomodulatory capacities and are potent agents in the activation of the innate and adaptive immune system.


László vÍGH moleCUlaR sTRess bIoloGY

76

BIO

CHEM

ISTR

Y

the group carries out its research activity in both the Biochemistry and the enzimology Institutes.

an ever-increasing number of diseases have been shown to originate from protein misfolding. among them, there are more than 20 diseases [including alzheimer's, Parkinson's, Huntington's and trans-missible spongiform encephalopathies (tses)] that are characterized by amyloidal protein deposits and thought to share common structural features and mechanism of action. (the amyloidal deposits usu-ally consist of fibres that contain misfolded proteins with a β-sheet conformation.) tses, which we chose as a model for the study of these conformational diseases, have drawn special attention due to the outbreak of a new variant of creutzfeldt-Jakob dis-ease (nvcJd) in the united Kingdom that seemed to be associated with eating beef from tse-infected cattle.

tse in humans may result from infection (nvcJd, iatrogenic cJd, kuru); can be inherited when a germ line mutation exists in the PrnP gene that encodes

the prion protein [familial cJd, Gerstmannstreüssler-scheinker syndrome (Gsss), fatal familial Insom-nia]; and can be sporadic (sporadic cJd) when the origin of the disease is neither determined nor un-derstood. tses are also observed in other mammals including sheep, goats, deer, elk, cattle, mink, cats and zoo animals.

The primary symptoms of tses usually include progressive dementia and ataxia, associated with spongiform degeneration of the brain and accumu-lation of an abnormal protease-resistant form of the prion protein (PrPres) in the central nervous system. (The normal, cellular form of this protein is denoted as PrPc.) after symptoms are first manifested, the progression of the disease can be dramatically rapid, leading inevitably to the death of the affected indi-vidual.

PrPc is a mainly α-helical, protease-sensitive, sol-uble monomer protein with a disordered n-terminus (approximate residues 23–127) and a structured c-terminal domain (residues 128–228, figure 1.).

Transmissible spongiform encephalopathies (TSEs) are deadly neuro-degenerative disorders among humans and mammals. The infectiousness is the most alarming feature of these diseases. It can be transmitted to humans from animals through the food chain and from humans by blood or organ donation or by infected medical devices. There is no cure or sufficient sensitive early detection for TSEs. The infectious agent, called prion, is believed to be a normal cell protein, the prion protein, with an unknown conformation that is different from its normal confor-mation. Our aim is to understand the conformational transition of the prion protein to disease-associated forms at the molecular level by combining the results of biochemical and biophysical measurements, cell-free conversion reactions, and experiments in cell and animal model systems.

ConfoRmaTIonal DIseases GRoUp(membRane anD sTRess bIoloGY UnIT)

Ervin WELKER / Principal Investigator, Group Leader

Frida FODOR / staff scientistAdrien BORSY / staff scientistAntal NYESTE / Phd studentEszter TóTH / Phd studentKrisztina HUSZáR / Phd studentPetra BENCSURA / Phd studentJudit BAUHOCH / technicianErika ZUKIC / technician

77

BIO

CHEM

ISTR

Y

Figure 1. PrP is a 208 amino-acid residue glycosylated protein, which is connected to the cell surface by a glycosyl phosphatidyl inositol (GPI) an-chor. However, only 110 residues (121-231) of the PrP domain are shown here. The two glycosylation sites are Asn 181 and Asn 197, which are both shown in yellow. One disulfide bond is present in the protein (colored gold here) connecting the second and third helices via Cys179-Cys214. The protein is colored by secondary structure. Alpha helices=magenta, beta sheets=blue.

By contrast, PrPres has a very compact structure, which is high in both α and β conformations, exists as oligomers and can be dissolved only by denaturing in GdnHcl or detergents. The disease-associated protein (PrPres) has partial protease-resistance; its n-termi-nal ~6 kda fragment is digested under conditions in which the highly compact c-terminal domain remains

intact. Both forms of the protein contain a single di-sulfide bond and two glycosylation sites. The generally accepted assertion that the difference between the cel-lular and disease-associated forms of the prion protein is purely conformational with respect to its disulfide bond patterns is based on our earlier experiments.

There is no cure for the disease, and early diagnosis is not available to discriminate between tse-infected unsymptomatic and uninfected individuals. such a sensitive, rapid, economical and non-invasive screen-ing test is vital with respect to animals destined for the human food chain, and to humans, who may partici-pate in tissue and blood donation programs. as with any other disease, a detailed mechanistic understand-ing of pathogenesis is the most effective approach for the development of sensitive predictive diagnostic and efficacious therapeutic regimens.

our aim is to understand tses at molecular and cellular levels as well as at the level of the whole organ-ism as a model for the formation of amyloid fibrils in conformational diseases.


Ervin WELKER ConfoRmaTIonal DIseases GRoUp

78

BIO

CHEM

ISTR

Y

Antal KissHeitman, J., Ivanenko, t. and Kiss, a. (1999). dna nicks in-flicted by restriction endonucleases are repaired by a reca- and recB-dependent pathway in Escherichia coli. Mol. Micro-biol. 33: 1141-1151.

raskó, t., finta, c. and Kiss, a. (2000). dna bending induced by dna (cytosine-5) methyltransferases. Nucleic Acids Res. 28: 3083-3091.

simoncsits, a., tjörnhammar, m.-L., raskó, t., Kiss, a. and Pongor, s. (2001). covalent joining of the subunits of a ho-modimeric type II restriction endonuclease: single-chain PvuII endonuclease. J. Mol. Biol. 309: 89-97.

Kiss, a., Pósfai, G., Zsurka, G., raskó, t. and venetianer, P. (2001). role of dna minor groove interactions in substrate recognition by the m.sinI and m.ecorII dna (cytosine-5) methyltransferases. Nucleic Acids Res. 29: 3188-3194.

roberts, r.J., Belfort, m., Bestor, t., Bhagwat, a.s., Bickle, t.a., Bitinaite, J., Blumenthal, r.m., degtyarev, s.K., dryden, d.t.f., dybvig, K., firman, K., Gromova, e.s., Gumport, r.I., Halford, s.e., Hattman, s., Heitman, J., Hornby, d.P., Janu-laitis, a., Jeltsch, a., Josephsen, J., Kiss, a., Klaenhammer, t.r., Kobayashi, I., Kong, H., Krüger, d.H., Lacks, s., marinus, m.G., miyahara, m., morgan, r.d., murray, n.e., nagaraja, v., Piekarowicz, a., Pingoud, a., raleigh, e., rao, d.n., re-ich, n., repin, v.e., selker, e.u., shaw, P.-c., stein, d.c., stod-dard, B.L., szybalski, W., trautner, t.a., van etten, J.L., vitor, J.m.B., Wilson, G.G. and Xu, s. (2003). a nomenclature for re-striction enzymes, dna methyltransferases, homing endonu-cleases and their genes. Nucleic Acids Res. 31: 1805-1812.

tímár, e., Groma, G., Kiss, a. and venetianer, P. (2004). chang-ing the recognition specificity of a dna-methyltransferase by in vitro evolution. Nucleic Acids Res. 32: 3898-3903.

rathert, P., raskó, t., roth, m., Ślaska-Kiss, K., Pingoud, a., Kiss, a. and Jeltsch, a. (2007). reversible inactivation of the cG-specific sssI dna-(cytosine-c5)-methyltransferase with a photocleavable protection group. ChemBioChem 8: 202-207.

chuluunbaatar, t., Ivanenko-Johnston, t., fuxreiter, m., me-leshko, r., raskó, t., simon, I., Heitman, J. and Kiss, a. (2007). an ecorI-rsrI chimeric restriction endonuclease retains pa-rental sequence specificity. BBA-Proteins Proteom. 1774: 583-594.

Kiss, a. and Weinhold, e. (2008). functional reassembly of split enzymes on-site: a novel approach for highly sequence-specific targeted dna methylation. ChemBioChem 9: 351-353.

van der Gun, B.t.f., Wasserkort, r., monami, m., Jeltsch, a., raskó, t., Ślaska-Kiss, K., cortese, r., rots, m.G., de Leij, L.f.m.H., ruiters, m.H.J., Kiss, a., Weinhold, e. and mcLaugh-lin, P.m.J. (2008). Persistent down-regulation of the pancarci-noma-associated epithelial cell adhesion molecule via active intranuclear methylation. Int. J. Cancer 123: 484-489.

Kiss, a., Balikó, G., csorba, a., chuluunbaatar, t., medzi-hradszky, K.f. and alföldi, L. (2008). cloning and charac-terization of the dna region responsible for megacin a-216 production in Bacillus megaterium 216. J. Bacteriol. 190: 6448-6457.

tímár, e., venetianer, P. and Kiss, a. (2008). In vivo dna protection by relaxed-specificity sinI dna methyltransferase variants. J. Bacteriol. 190: 8003-8008.

György PósfaiPósfai, G., Koob, m., Kirkpatrick, H. and Blattner, f.r. (1997). versatile insertion plasmids for targeted genome manipula-tions in bacteria: isolation, deletion and rescue of the patho-genicity island Lee of the Escherichia coli o157:H7 genome. J. Bacteriol. 179: 4426-4428.

Pósfai, G., Kolisnychenko, v., Bereczki, Z. and Blattner, f.r. (1999). markerless gene replacement in Escherichia coli stimu-lated by a double-strand break in the chromosome. Nucleic Ac-ids Res. 27: 4409-4415.

Kolisnychenko, v., fehér, t., Herring, c.d., Plunkett, G. III, Blattner, f.r. and Pósfai, G. (2002). engineering a reduced E. coli genome. Genome Research 12: 640-647.

Pósfai, G., Plunkett, G3rd., fehér, t., frisch, d., Keil, G., umen-hoffer, K., Kolisnychenko, v., stahl, B., arruda, m., sharma,

s.s., Burland, v., Harcum, s.W., Blattner, f.r. (2006). emer-gent properties of reduced-genome Escherichia coli. Science 312: 1044-1046.

fehér, t., cseh, B., umenhoffer, K., Karcagi, I. and Pósfai, G. (2006). characterization of cycA mutants of Escherichia coli. an assay for measuring in vivo mutation rates. Mutat. Res. 595: 184-190.

fehér, t., Papp, B., Pál, c. and Pósfai, G. (2007). systematic ge-nome reductions: Theoretical and experimental aspects. Chem. Rev. 107: 3498-3513.

fehér, t., Karcagi, I., Győrfy, Z., umenhoffer, K., csörgő, B. and Pósfai, G. (2008). scarless engineering of the Escherichia coli genome. Methods Mol. Biol. 416: 251-259.

Csaba PálPapp, B., Pál, c. and Hurst, L.d. (2003). dosage sensitivity and the evolution of gene family size in yeast. Nature 424: 194-197.

Papp, B., Pál, c. and Hurst, L.d. (2003). evolution of cis-regu-latory elements in duplicated genes of yeast. Trends Genet. 19: 417-422.

Pál, c. and Hurst, L.d (2003). evidence for co-evolution of gene order and recombination rate. Nature Genetics 33: 392-395.

Papp, B., Pál, c. and Hurst, L.d. (2004). metabolic network analysis of the causes and evolution of enzyme dispensability in yeast. Nature 429: 661-664.

Hurst, L.d., Pál, c. and Lercher, m.J. (2004). The evolutionary dynamics of eukaryotic gene order. Nature Reviews Genetics 200 5: 299-310.

Pál, c., Papp, B. and Lercher. m.J. (2005). adaptive evolution of bacterial metabolic networks by horizontal gene transfer. Na-ture Genetics 37: 1372-1375.

Pál, c., Papp, B. and Lercher, m.J. (2006). an integrated view on protein evolution. Nature Reviews Genetics 7: 337-348.

Pál, c., Papp, B., Lercher, m.J., csermely, P., oliver, s.G. and Hurst, L.d. (2006). chance and necessity in the evolution of minimal metabolic networks. Nature 440: 667-670.

Pál, c., macia, m., oliver, a., schacher, I. and Buckling, a. (2007). coevolution with viruses drives the evolution of bacte-rial mutation rates. Nature 450: 1079-1081.

InsTITUTe of bIoCHemIsTRYseleCTeD pUblICaTIons:

79

BIO

CHEM

ISTR

Y

Harrison, r., Papp, B., Pál, c., oliver, s.G. and delneri, d. (2007). Plasticity of genetic interactions in metabolic networks of yeast. Proc. Natl. Acad. Sci. U.S.A. 104: 2307

Balázs PappPál, c., Papp, B. and Hurst, L.d. (2001). Highly expressed genes in yeast evolve slowly. Genetics 158: 927-931.

Papp, B., Pál, c. and Hurst, L.d. (2003). dosage sensitivity and the evolution of gene families in yeast. Nature 424: 194-197. Pál, c., Papp B. and Hurst, L.d. (2003). Genomic function: rate of evolution and gene dispensability. Nature 421: 496-497.

Papp, B., Pál, c. and Hurst, L.d. (2004). metabolic network analysis of the causes and evolution of enzyme dispensability in yeast. Nature 429: 661-664.

Pál, c., Papp B. and Lercher, m.J. (2005). adaptive evolution of bacterial metabolic networks by horizontal gene transfer. Na-ture Genetics 37: 1372-1375.

Pál, c., Papp, B. and Lercher, m.J. (2006). towards an inte-grated view on protein evolution. Nature Reviews Genetics 7: 337-348.

Pál, c., Papp, B., Lercher, m.J., csermely, P., oliver, s.G. and Hurst, L.d. (2006). chance and necessity in the evolution of minimal metabolic networks. Nature 440: 667-670.

Harrison, r., Papp, B., Pál, c., oliver, s.G. and delneri, d. (2007). Plasticity of genetic interactions in metabolic networks of yeast. Proc. Natl. Acad. Sci. U.S.A. 104: 2307-2312.

Károly, K., Hurst, L.d. and Papp, B. (2009). stochasticity in protein levels drives colinearity of gene order in metabolic operons of E. coli. PloS Biology 7: e1000115.

Sándor Benyhe and Anna BorsodiPattee, P., Ilie, a.e., Benyhe, s., tóth, G., Borsodi, a., nagalla, s.r. (2003). cloning and characterization of Xen-dorphin pro-hormone from Xenopus laevis: a new opioid-like prohormone distinct from proenkephalin and prodynorphin. J. Biol. Chem. 278: 53098-53104.

Wollemann, m., Benyhe, s. (2004). minireview: non-opioid actions of opioid peptides. Life Sciences 75: 257-270.

tömböly, cs., Kövér, K., Péter, a., tourwè, d., Biyashev, d., Benyhe, s., Borsodi, a., al-Khrasani, m., rónai, a.Z., tóth, G. (2004). structure-activity study on the Phe side-chain arrange-ment of endomorphins using conformationally constrained analogues. J. Med. Chem. 47: 735-743.

Kocsis, L., orosz, G., magyar, a., al-Khrasani, m., Kató, e., rónai, a.Z., Bes, B., meunier, J.-c., Gündüz, Ö., tóth, G., Bor-sodi, a., Benyhe, s. (2004). nociceptin antagonism: Probing the receptor by n-acetyl oligopeptides. Regulatory Peptides 122: 199-207.

Gündüz, Ö., sipos, f., spagnolo, B., Kocsis, L., magyar, a., Bor-sodi, a., caló, G., Benyhe, s. (2006). structure-activity studies on ac-rYYrIK-nH2 hexapeptide analogues targeting noP receptor. Neurosignals 15: 91-101.

Gündüz, Ö., rizzi, a., Baldisserotto, a., Guerrini, r., spagnolo, B., Gavioli, e.c., Kocsis, L., magyar, a., Benyhe, s., Borsodi a., calo’, G. (2006). In vitro and in vivo pharmacological charac-terization of the nociceptin/orphanin fQ receptor ligand ac-rYYrIK-ol. Eur. J. Pharmacology 539: 39-48.

carmona-aparicio, L., Peña, f., Borsodi, a., rocha, L. (2007). effects of nociceptin on the spread and seizure activity in the rat amygdala kindling model: their correlations with 3H-leu-cyl-nociceptin binding. Epilepsy Res. 77: 75-84.

rocha, L., cuellar-Herrera, m., velasco, m., velasco, f., ve-lasco, a.L., Jiménez, f., orozco-suarez, s., Borsodi, a. (2007). opioid receptor binding in parahippocampus of patients with temporal lobe epilepsy: its association with the antiepileptic ef-fects of subacute electrical stimulation. Seizure 16: 645-652.

stevens, c.W., tóth, G., Borsodi, a., Benyhe, s. (2007). Xendorphin B1, a novel opioid-like peptide determined from a Xenopus laevis brain cdna library, produces opioid antinociception after spinal administration in amphibians. Brain Res. Bulletin 71: 628-632.

Páldy, e., Bereczki, e., sántha, m., Wenger, t., Borsodi, a., Zimmer, a., Benyhe, s. (2008). cB2 cannabinoid receptor an-tagonist sr144528 decreases mu-opioid receptor expression and activation in mouse brainstem: role of cB(2) receptor in pain. Neurochemistry International 53: 309-316.

Wollemann, m., Ioja, e., Benyhe, s. (2008). capsaicin inhibits the in vitro binding of peptides selective for mu- and kappa-opi-oid, and nociceptin-receptors. Brain Res. Bulletin 77: 136-142.

Bojnik, e., magyar, a., tóth, G., Bajusz, s., Borsodi, a., Benyhe, s. (2009). Binding studies of novel, non-mammalian enkepha-lins, structures predicted from frog and lungfish brain cdna sequences. Neuroscience 158: 867-874.

Mária Szűcsfábián, G., Bozó, B., szikszay, m., Horváth, G., coscia, c.J. and szűcs, m. (2002). chronic morphine-induced changes in mu-opioid receptors and G proteins of different subcellular loci in rat brain. J. Pharm. Exp. Ther. 302(2): 774-780.

szűcs, m., Boda, K. and Gintzler, a.r. (2004). dual effects of tyr-d-ala-Gly-(nme)Phe-Gly-ol (damGo) and d-Phe-cys-tyr-d-trp-arg-Thr-Pen-Thr-nH2 (ctaP) on adenylyl cyclase activity; implications for µ-opioid receptor Gs coupling. J. Pharm. Exp. Ther. 310/1: 256-262.

Keresztes, a., tóth, G., fülöp, f. and szűcs, m. (2006). synthe-sis, radiolabeling and receptor binding of [3H][(1S, 2R)acPc2]endomorphin-2. Peptides 27: 3315-3321.

Keresztes, a., szűcs, m., Borics, a., Kövér, K., forró, e., fülöp, f., tömböly, c., antal, P., Pahi, a., fábián, G., murányi, m. and tóth, G. (2008). new endomorphin analogs containing alicyclic β-amino acids: Influence on bioactive conformation and pharmacological profile. J. Med. Chem. 51: 4270-4279.

Birkás, e., Kertész, I., tóth, G., Bakota, L., Gulya, K. and szűcs, m. (2008). synthesis and pharmacological characterization of a nov-el, highly potent, peptidomimetic delta-opioid radioantagonist, [H]tyr-tic-(2s,3r)-β-mePhe-Phe-oH. Neuropeptides 42: 57-67.

cinar, r., freund, t.f., Katona, I., mackie, K. and szűcs, m. (2008). reciprocal inhibition of G-protein signaling is induced by cB1 cannabinoid and GaBaB receptor interactions in rat hippocampal membranes. Neurochem. Int. 52: 1402-1409.


80

BIO

CHEM

ISTR

Y

cinar, r. and szűcs, m. (2009). cB1 receptor-independent actions of sr141716 on G-protein signaling; co-application with the mu-opioid agonist damGo unmasks a novel, per-tussis-toxin insensitive opioid signaling in mor-cHo cells. J. Pharm. Exp. Ther. doI: 10.1124/jpet.109.152710.

Csaba Tömbölytömböly, cs., Péter, a. amd tóth, G. (2002). In vitro quanti-tative study of the degradation of endomorphins. Peptides 23: 1571-1578.

tömböly, cs., Kövér, K.e., Péter, a., tourwé, d., Biyashev, d., Benyhe, s., Borsodi, a., al-Khrasani, m., rónai, a.Z. and tóth, G. (2004). structure-activity study on the Phe side-chain arrangement of endomorphins using conformationally con-strained analogs. J. Med. Chem. 47: 735-743.

Keresztes, a., tóth, G., fülöp, f. and szűcs, m. (2006). synthe-sis, radiolabelling and receptor binding of [3H][(1S,2R)acPc2]endomorphin-2. Peptides 27: 3315-3321.

rónai, a.Z., szemenyei, e., Kató, e., Kocsis, L., orosz, Gy., al-Khrasani, m. and tóth, G. (2006). endomorphin synthesis in rat brain from intracerebroventicularly injected [3H]-tyr-Pro: a possible biosynthetic route for endomorphins. Regulatory Peptides 134: 54-60.

tóth, G., Ioja, e., tömböly, cs., Ballet, s., tourwé, d., Péter, a., martinek, t., chung n.n., schiller, P.W., Benyhe, s. and Borsodi, a. (2007). β-methyl substitution of cyclohexylalanine in dmt-tic-cha-Phe-peptides results in highly potent δ opioid antagonists. J. Med. Chem. 50: 328-333.

Borics, a., murphy, r.f. and Lovas, s. (2007). conformational analysis of ac-nPGQ-nH2 and ac-vPaH-nH2 by vibrational circular dichroism spectroscopy combined with molecular dy-namics and quantum chemical calculations. Protein Peptide Lett. 14: 353-359.

Knütter, I., Hartrodt, B., tóth, G., Keresztes, a., Kottra, G., mrestani, c., Born, I., daniel, H., neubert, K. and Brandsch, m. (2007). synthesis and characterization of a new and radi-olabelled high–affinity substrate for H+/peptide cotransport-ers”. FEBS Journal 274: 5905-5914.

tömböly, cs., Ballet, s., feytens, d., Kövér, K.e., Borics, a., Lovas, s., al-Khrasani, m., fürst, Zs., tóth, G., Benyhe, s. and tourwé, d. (2008). endomorphin-2 with a β-turn backbone constraint retains the potent µ opioid receptor agonist proper-ties. J. Med. Chem. 51: 173-177.

Keresztes, a., szűcs, m., Borics, a., forró, e., fülöp, f., Kövér, K.e., tömböly, cs., Péter, a., Páhi, a., fábián, G., murányi, m. and tóth, G. (2008). new endomorphin ana-logues with alicyclic β-amino acids: inf luence on bioactive conformation and pharmacological profile. J. Med. Chem. 51: 4270-4279.

Miklós SánthaGogos, J.a., morgan, m., Luine, v., sántha, m., ogawa, s., Pfaff, d. and Karayiorgou, m. (1998). catechol-o-methyl-transferase-deficient mice exhibit sexually dimorphic changes in catecholamine levels and behavior. Proc. Natl. Acad. Sci .U.S.A. 18; 95(17): 9991-9996.

Gogos, J.a., sántha, m., malinova, L., Lattal, K.m., abel, t. and Karayiorgou, m. (1999). The gene encoding proline dehy-drogen is a strong candidate gene for the 22q11-associated psy-chiatric phenotypes. Mol. Psychiatr. 4: s107-s107.

Gogos, J.a., sántha, m., takács, Z., Beck, K.d., Luine, v., Lu-cas, L.r., nadler, J.v. and Karayiorgou, m. (1999). The gene en-coding proline dehydrogenase modulates sensorimotor gating in mice. Nature Genet. 21(4): 434-439.

Huotari, m., sántha, m., Lucas, L.r., Karayiorgou, m., Gogos, J.a. and mannisto, P.t. (2002). effect of dopamine uptake in-hibition on brain catecholamine levels and locomotion in cat-echol-o-methyltransferase (comt) disrupted mice. J. Pharm. Exp. Ther. 303(3): 1309-1316.

csont, t., Bencsik, P., Bereczki, e., csonka, c., onody, a., fo-dor, G., Giricz, Z., sántha, m. and ferdinandy, P. (2004). cho-lesterol-enriched diet leads to increased oxidative stress and cardiac dysfunction in apoB100 transgenic mice. J. Mol. Cell. Cardiology, 37. p281. c63

csont, t., Bencsik, P., Bereczki, e., rigó, K., Kocsis, f.G., Giricz, Z., csonka, c., sántha, m. and ferdinandy, P. (2005). Hypercholesterolemia leads to cardiac dysfunction in apo100 transgenic mice: role of oxidative stress & mmPs. J. Mol. Cell. Cardiology 38. p.1015 P045.

csont, t., Bereczki, e., Bencsik, P., fodor, G., Görbe, a., Zvara, Á., csonka, c., Puskás, L.G., sántha, m. and ferdinandy, P. (2007). Hypercholesterolemia increases myocardial oxidative and nitrosative stress thereby leading to cardiac dysfunction in apoB-100 transgenic mice. Cardiovasc. Res. 76: 100-109.

Bereczki, e., Gonda, sz., csont, t., Korpos, e., Zvara, Á., fer-dinandy, P. and sántha, m. (2007). overexpression of biglycan in the heart of transgenic mice: an antibody microarray study. J. Proteome Res. 6: 854-861.

Bereczki, e., Bernát, G., csont, t., ferdinandy, P., scheich, H. and sántha, m. (2008). overexpression of human apolipopro-tein B-100 induces severe neurodegeneration in transgenic mice. J. Proteome Res.7(6): 2246-52.

Qiaozhu, su, Julie tsai, elaine Xu, Wei Qiu, Bereczki, e., sántha, m. and Khosrow adeli. (2009). apolipoprotein B100 acts as a molecular link between lipid-induced endoplasmic reticulum stress and hepatic insulin resistance. Hepatology may11. epub.

Imre M. Borosciurciu, a., Komonyi, o., Pankotai, t. and Boros, I. (2006). The Drosophila histone acetyltranferase gcn5 and transcriptional adaptor ada2a are involved in nucleosomal histone h4 acetyla-tion. Mol. Cell. Biol. 26(24): 9413-9423.

Bodai, L., Pardi, n., ujfaludi, Z., Bereczki, o., Komonyi, o., Bálint, É. and Boros, I.m. (2007). daxx-like protein of Dro-sophila interacts with dmp53 and affects longevity and ark mrna level. J. Biol. Chem. 282(50): 36386-36393.

carré, c., ciurciu, a., Komonyi, o., Jacquier, c., fagegaltier, d., Pidoux, tricoire, H., tora, L., Boros, I. and antoniewski, c. (2008). The nurf remodeling complex and the atac histone acetylase complex cooperate in the maintenance of high order chromosome structure. EMBO Rep. 9: 187-92.

B. Grau, B., Popescu, c., torroja, L., ortuño-sahagún, d., Boros, I. and ferrús, a. (2008). The transcriptional adaptor ada3 of Drosophila is required for histone modification, position effect variegation and transcription. Mol. Cell. Biol. 28(1): 376-85.


81

BIO

CHEM

ISTR

Y

Bereczki, o., ujfaludi, Z., Pardi, n., nagy, Z., tora, L., Boros, I.m. and Bálint, É. (2008). tata binding protein associated factor 3 (taf3) interacts with p53 and inhibits its function. BMC Molecular Biology 9: 57.

ciurciu, a., Komonyi, o. and Boros, I.m. (2008). Loss of atac-specific histone H4 lysine 12 acetylation reduces JIL-1 binding to chromatin and phosphorylation of histoneH3 at serine 10. J. Cell Science 121: 3366-3372.

ciurciu, a., tombácz, I., Popescu, c. and Boros, I. (2009). GaL4 induces transcriptionally active puff in the absence of JIL-1, dsaGa and atac specific chromatin modifications in the Drosophila melanogaster polytene chromosome. Chromo-soma, in press.

schauer, t., tombácz, I., ciurciu, a., Komonyi, o. and Boros, I. (2009). misregulated rna Pol II ctd phosphorylation results in apoptosis. Cell. Mol. Life. Sci. 66(5): 909-918.

Komonyi, o., schauer, t., Pápai, G., deák, P. and Boros, I.m. (2009). a product of the bicistronic cG31241 Drosophila mela-nogaster gene interplays with atr but acts independently of atm in telomere protection. J. Cell Science 122: 769-774.

Péter Deákdeák, P., donaldson, m. and Glover, d.m. (2003). mutations in mákos, a Drosophila gene encoding the cdc27 subunit of the anaphase promoting complex enhance centrosomal defects in polo and are suppressed by mutations in twins/aar encoding a regulatory sub-unit of PP2a. J. Cell Sci. 116: 4147-4158.

máthé, e., Kraft, c., Giet, r., deák, P., Peters, J.m. and Glover, d.m. (2004). The e2-c vihar is required for the correct spatio-temporal proteolysis of cyclin B and itself undergoes cyclical degradation. Current Biology 14(19): 1723-1733.

szabó, a., Pál, m., deák, P., Kiss, P., ujfaludi, Z., Pankotai, t., Lipinszki, Z. and udvardy, a. (2007). molecular charac-terization of the rpt1/p48B atPase subunit of the Drosophila melanogaster 26s proteasome. Mol. Genet. Genomics 278(1): 17-29.

Pál, m., varga, K., nagy, o. and deák, P. (2007). characteri-zation of the aPc10/doc1 subunit of the anaphase promot-ing complex in Drosophila melanogaster. Acta Biol. Hung. 58: 51-64.

Pál, m., nagy, o., ménesi, d., udvardy, a. and deák, P. (2007). structurally related tPr subunits contribute differently to the function of the anaphase promoting complex in Drosophila melanogaster. J. Cell Sci. 120: 3238-3248.

narbonne-reveau, K., senger, s., Pál, m., Herr, a., richardson, H.e., asano, m., deák, P. and Lilly, m.a. (2008). aPc/cfzr/cdh1 promotes cell cycle progression during the Drosophila endocy-cle. Development 135(8): 1451-1461.

Andor Udvardyudvardy, a. (1993). Purification and characterization of a mul-tiprotein component of the 26s (1500 kda) proteolytic com-plex. J. Biol. Chem. 268: 9055.

Ghislain, m., udvardy, a. and mann, c. (1993). S. cerevisiae 26s protease mutants arrest cell division in G2/metaphase. Na-ture 366: 358.

udvardy, a. and schedl, P. (1993). The dynamics of chromatin con-densation: redistribution of topoisomerase II in the 87a7 heat shock locus during induction and recovery. Mol. Cell. Biol. 13: 7522.

Haracska, L. and udvardy, a. (1995). cloning and sequencing a non-atPase subunit of the regulatory complex of the Dro-sophila 26s protease. Eur. J. Biochem. 231: 720.

udvardy, a. (1999). dividing the empire: boundary chromatin elements delimit the territory of enhancers. EMBO J. 18: 1-8.

Hölzl, H., Kapelari, B., Kellermann, J., seemüller, e., sümegi, m., udvardy, a., medalia, o., sperling, J., müller, s.a., engel, a. and Baumeister, W. (2000). The regulatory complex of Drosophila melanogaster 26s proteasomes: subunit compsition and locali-zation of a deubiquitinating enzyme. J. Cell Biol. 150: 119-129.

szlanka, t., Haracska, L., Kiss, I., deák, P., Kurucz, É., andó, I., virágh, e. and udvardy, a. (2003). deletion of proteasomal subunit s5a/rpn10/p54 causes lethality, multiple mitotic de-fects and overexpression of proteasomal genes in Drosophila melanogaster. J. Cell Sci. 116: 1023-1033.

sümegi, m., Hunyadi-Gulyás, É., medzihradszky, K.f. and udvardy, a. (2003). 26s proteasome subunits are o-linked n-acetylglucosamine-modified in Drosophila melanogaster. Bio-chem. Biophys. Res. Commun. 312: 1284-1289.

Kiss, P., szabó, a., Hunyadi-Gulyás, É., medzihradszky, K.f., Lipinszki, Z., Pál, m. and udvardy, a. (2005). Zn2+-induced reversible dissociation of subunit rpn10/p54 of the Drosophila 26 s proteasome. Biochem. J. 391: 301-310.

Pál, m., nagy, o., ménesi, d., udvardy, a. and deák, P. (2007). structurally related tPr subunits contribute differently to the function of the anaphase-promoting complex in Drosophila melanogaster. J. Cell Sci. 120: 3238-3248.

szabó, a., Pál, m., deák, P., Kiss, P., ujfaludi, Z., Pankotai, t., Lipinszki, Z. and udvardy a. (2007). molecular characteriza-tion of the rpt1/p48B atPase subunit of the Drosophila mela-nogaster 26s proteasome. Mol. Gen. Genomics 278(1): 17-29.

Lipinszki, Z., Kiss, P., Pál, m., deák, P., szabó, Á., Hunyadi-Gu-lyás, É., Klement, É., medzihradszky, K.f. and udvardy, a. (2009). developmental-stage-specific regulation of the polyubiquitin re-ceptors in Drosophila melanogaster. J. cell Sci. 122: 3083-3092.

Ibolya KissKiss, I., deák, f., mestric, s., delius, H., soós, J., dékány, K., argraves, W.s., sparks, K.J. and Goetinck, P.f. (1987). struc-ture of the chicken link protein gene: exons correlate with the protein domains. Proc. Natl. Acad. Sci. U.S.A. 84: 6399-6403.

Kiss, I., deák, f., Holloway, r.G., Jr., delius, H., mebust, K.a., frimberger, e., argraves, W.s., tsonis, P.a., Winterbottom, n. and Goetinck, P.f. (1989). structure of the gene for cartilage matrix protein, a modular protein of the extracellular matrix. J. Biol. Chem. 264: 8126-8134.

szabó, P., moitra, J., rencendorj, a., rákhely, G., rauch, t. and Kiss, I. (1995). Identification of a nuclear factor-I family pro-tein-binding site in the silencer region of the cartilage matrix protein gene. J. Biol. Chem. 270: 10212-10221.

deák, f., Piecha, d., Bachrati, c., Paulsson, m. and Kiss, I. (1997). Primary structure and expression of matrilin-2, the closest rela-tive of cartilage matrix protein within the von Willebrand factor type a-like module superfamily. J. Biol. Chem. 272: 9268-9274.


82

BIO

CHEM

ISTR

Y

deák, f., Wagener, r., Kiss, I. and Paulsson, m. (1999). The matrilins: a novel family of oligomeric extracellular proteins. Matrix Biol. 18: 55-64.

Piecha, d., muratoglu, s., mörgelin, m., Hauser, n., studer, d., Kiss, I., Paulsson, m., deák, f. (1999). matrilin-2, a large oli-gomeric matrix protein, is expressed by a great variety of cells and forms fibrillar networks. J. Biol. Chem. 274: 13353-13361.

mátés, L., deák, f., Korpos, P.G.É., Liu, Z., Beier, d.r., aszódi, a. and Kiss, I. (2002). comparative analysis of the mouse and human genes (matn2 and natn2) for matrilin-2, a filament-forming protein widely distributed in extracellular matrices. Matrix Biol. 21: 163-174.

Karcagi, I., rauch, t., Hiripi, L., rentsendorj, o., nagy, a., Bősze, Zs. and Kiss, I. (2004). functional analysis of the regu-latory regions of the matrilin-1 gene in transgenic mice reveals modular arrangement of tissue-specific control elements. Ma-trix Biol. 22: 605-618.

mátés, L., mörgelin, m., deák, f., Kiss, I. and aszódi, a. (2004). mice lacking the extracellular matrix adaptor protein matrilin-2 develop without obvious abnormalities. Matrix Biol. 23: 195-204.

Korpos, É., molnár, a., Papp, P., Kiss, I., orosz, L. and deák, f. (2005). expression pattern of matrilins and other extracellular matrix proteins characterize distinct stages of cell differentia-tion during antler development. Matrix Biol. 24: 124-135.

rentsendorj, o., nagy, a., sinkó, I., daraba, a., Barta, e. and Kiss, I. (2005). Highly conserved proximal promoter element harbouring paired sox9-binding sites contributes to the tis-sue- and developmental stage-specific activity of the matrilin-1 gene. Biochem. J. 389: 705-716.

sharma, m.K., Watson, m.a., Lyman, m., Perry, a., aldape, K.d., deák, f. and Gutmann, d.H. (2006). matrilin-2 expres-sion distinguishes clinically relevant subsets of pylocytic as-trocytoma. Neurology 66: 127-130.

szabó, e., Lódi, c., Korpos, É., Batmunkh, e., rottenberger, Z., deák, f., Kiss, I., tőkés, a., Lotz, G., László, v., Kiss, a., schaff, Z. and nagy, P. (2007). expression of matrilin-2 in oval cells during rat liver regeneration. Matrix Biol. 26: 554-560.

Ernő Dudademeter, K., Herberth, B., duda, e., domonkos, a., Jaffredo, t., Herman, J.P. and madarász, e. (2004). fate of cloned em-bryonic neuroectodermal cells implanted into the adult, new-born and embryonic forebrain. Exp. Neurol. 188(2): 254-267.

Paszt, a., takács, t., rakonczay, Z., Kaszaki, J., Wolfard, a., tis-zlavicz, L., Lázár, G., duda, e., szentpáli, K., czakó, L., Boros, m., Balogh, a. and Lázár G. Jr. (2004). The role of the glucocorticoid-dependent mechanism in the progression of sodium taurochola-te-induced acute pancreatitis in the rat. Pancreas 29(1): 75-82.

Letoha, t., somlai, c., takács, t., szabolcs, a., Jármay, K., ra-konczay, Z. Jr, Hegyi, P., varga, I., Kaszaki, J., Krizbai, I., Boros, I., duda, e., Kusz, e. and Penke, B. (2005). a nuclear import in-hibitory peptide ameliorates the severity of cholecystokinin-in-duced acute pancreatitis. World J. Gastroenterol. 11(7): 990-999.

Letoha, t. somlai, c., takács, t., szabolcs, a., rakonczay, Z., Jármay, K., szalontai, t., varga, I., Kaszaki, J., Boros, I., duda, e., Hackler, L., Kurucz, I. and Penke, B. (2005). The protea-some inhibitor mG132 protects against acute pancreatitis. Free Radic. Biol. Med. 39(9): 1142-1151.

fejér, G., szalay, K., Győry, I., fejes, m., Kusz, e., nedieanu, s., Páli, t., schmidt, t., siklódi, B., Lázár, Gy. jr., Lázár, Gy. sr. and duda, e. (2005). adenovirus infection dramatically augments lipopolysaccharide-induced tnf production and sensitizes to lethal shock. J. Immunol. 175(3): 1498-1506.

Letoha, t., Gaál, s., somlai, c., venkei, Z., Glavinas, H., Kusz, e., duda, e., czajlik, a., Peták, f. and Penke, B. (2005). Inves-tigation of penetratin peptides. Part 2. In vitro uptake of pen-etratin and two of its derivatives. J. Pept. Sci. 11(12): 805-811.

Kis, K., Bodai, L., Polyanka, H., Éder, K., Pivarcsi, a., duda, e., soós, G., Bata-csörgő, Z. and Kemény, L. (2006). Budesonide, but not tacrolimus, affects the immune functions of normal human keratinocytes. Int. Immunopharmacol. 6(3): 358-368.

Letoha, t., Kusz, e., Pápai, G., szabolcs, a., Kaszaki, J., varga, I., takács, t., Penke, B. and duda, e. (2006). In vitro and in vivo nuclear factor-kappaB inhibitory effects of the cell-pene-trating penetratin peptide. Mol. Pharmacol. 69(6): 2027-2036.

Gyurján, I. Jr. molnár, a., Borsy, a., stéger, v., Hackler, L. Jr, Zomborszky, Z., Papp, P., duda, e., deák, f., Lakatos, P., Puskás L.G. and orosz, L. (2007). Gene expression dynamics in deer antler: mesenchymal differentiation toward chondro-genesis. Mol. Genet. Genomics 277(3): 221-235.

Éder, K. Guan, H., sung, H.Y., Ward, J., angyal, a., Janas, m., sarmay, G., duda, e., turner, m., dower, s.K., francis, s. e., crossman, d. c. and Kiss-tóth, e. (2008). tribbles-2 is a novel regulator of inflammatory activation of monocytes. Int. Immu-nol. 20(12): 1543-1550.

Ábrahám, s. szabó, a., Kaszaki, J., varga, r., Éder, K., duda, e., Lázár, G., tiszlavicz, L., Boros, m. and Lázár, G. (2008). Kupffer cell blockade improves the endotoxin-induced micro-circulatory inflammatory response in obstructive jaundice. Shock 30(1): 69-74.

Paszt, a., Éder, K., szabolcs, a., tiszlavicz, L., Lázár, G., duda, e., takács, t. and Lázár, G. (2008). effects of gluco-corticoid agonist and antagonist on the pathogenesis of L-arginine-induced acute pancreatitis in rat. Pancreas 36(4): 369-376.

tubak, v., Határvölgyi, e., Krenács, L., Korpos, e., Kusz, e., duda, e., monostori, e. and rauch, t. (2009). expression of immunoregulatory tumor necrosis factor-like molecule tL1a in chicken chondrocyte differentiation. Can. J. Vet. Res. 73(1): 34-38.

Csaba Vizlerdelarasse, c., daubas, P., mars, L.t., vizler, c., Litzenburger, t., Iglesias, a., Bauer, J., della Gaspera, B., schubart, a., decker, L., dimitri, d., roussel, G., dierich, a., amor, s., dautigny, a., Liblau, r. and Pham-dinh, d. (2003). myelin/oligodendrocyte glycoprotein-deficient (moG-deficient) mice reveal lack of im-mune tolerance to moG in wild-type mice. J Clin. Invest. 112: 544-553.

fantin, m., Quintieri, L., Kusz, e., Kis, e., Glavinas, H., floreani, m., Padrini, m., duda, e. and vizler, c. (2006). Pentoxifylline and its major oxidative metabolites exhibit different pharmacological properties. Eur. J. Pharmacol. 535: 301-309.

Quintieri, L., fantin, m. and vizler, c. (2007). Identification of molecular determinants of tumor sensitivity and resistance to anticancer drugs. Adv. Exp. Med. Biol. 593: 595-104.


83

BIO

CHEM

ISTR

Y

Pecze, L., szabó, K., széll, m., Jósvay, K., Kaszás, K., Kusz, e., Letoha, t., Prorok, J., Koncz, I., tóth, a., Kemény, L., vizler, c. and oláh, Z. (2008). Human keratinocytes are vanilloid resis-tant. PLoS ONE.3(10): e3419.

Pecze, L., Pelsőczi, P., Kecskés, m., Winter, Z., Papp, a., Kaszás, K., Letoha, t., vizler, c. and oláh, Z. (2009). resiniferatoxin mediated ablation of trPv1+ neurons removes trPa1 as well. Can. J. Neurol. Sci. 36: 234-241.

szabó, a., varga, r., Keresztes, m., vizler, c., németh, I., ráz-ga, Z. and Boros, m. (2009). Ischemic limb preconditioning downregulates systemic inflammatory activation. J. Orthop. Res. 27(7):897-902.

László Víghvígh, L., Literati, n.P., Horváth, I., török, Z., Balogh, G., Glatz, a., Kovács, e., Boros, I., ferdinandy, P., farkas, B., Jaszlits, L., Jednakovits, a., Korányi, L. and maresca, B. (1997). Bimoclo-mol: a novel,non-toxic, hydroxylamine derivative with stress protein inducing activity and wide cytoprotective effects. Na-ture Med. 3: 1150-1154.

török, Z., Horváth, I., Goloubinoff, P., Kovács, e., Glatz, a., Balogh, G. and vígh, L. (1997). evidence for a lipochaperonin: association of active protein-folding GroesL oligomers with lipids can stabilize membranes under heat shock conditions. Proc. Natl. Acad. Sci. U.S.A. 94: 2192-2197.

Glatz, a., Horváth, I., varvasovszki, v., Kovács, e., török, Z. and vígh, L. (1997). chaperonin genes of the Synecosystis Pcc 6803 are differentially regulated under light-dark tran-sition during heat stress. Biochem. Biophys. Res. Commun. 239: 291-297.

vígh, L., maresca, B. and Harwood, J.L. (1998). does the mem-brane physical state control the expression of heat shock and other genes? trends Biochem. sci. 23: 369-374.

Horváth, I., Glatz, a., varvasovszki, v., török, Z., Páli, t., Ba-logh, G., Kovács, e., nádasdi, L., Benkő, s., Joó, f. and vígh, L. (1998). membrane physical state controls the signaling mecha-nism of the heat shock response in Synechocystis Pcc 6803: Identification of hsp17 as a "fluidity gene". Proc. Natl. Acad. Sci. U.S.A. 95: 3513-3518.

Glatz, a., vass, I., Los, d. and vígh, L. (1999). The Synechocystis model of stress: from molecular chaperons to membranes. Plant Physiol. Biochem. 37: 1-12.

török, Z., Goloubinoff, P., Horváth, I., tsvetkova, n.m., Glatz, a., Balogh, G., varvasovszki, v., Los, d,a., vierling, e., crowe, J.H. and vígh, L. (2001). Synechocystis HsP17 is an amphitrop-ic protein that stabilizes heat-stressed membranes and binds denatured proteins for subsequent chaperone-mediated refold-ing. Proc. Natl. Acad. Sci. U.S.A. 98: 3098-3103.

Kovács, e., van der vies, s.m., Glatz, a., török, Z., varvas-ovszki, v., Horváth, I. and vígh, L. (2001). the chaperonins of Synechocystis Pcc 6803 differ in heat inducibility and chaperone action. Biochem. Biophys. Res. Commun. 289: 908-915.

tsvetkova, n.m., Horváth, I., török, Z., Wolkers, W.f., Balogh, Z., shigapova, n., crowe, L.m., tablin, f., vierling, e., crowe, J.H. and vígh, L. (2002). small heat-shock proteins regulate membrane lipid polymorphism. Proc. Natl. Acad. Sci. U.S.A. 99: 13504-13509.

török, Z., tsvetkova, n.m., Balogh, G., Horváth, I., nagy, e., Pénzes, Z., Hargitai, J., Bensaude, o., csermely, P., crowe, J.H., maresca, B. and vígh, L. (2003). Heat shock protein coinducers with no effect on protein denaturation specifically modulate the membrane lipid phase. Proc. Natl. Acad. Sci. U.S.A. 100: 3131-3136.

vígh, L., escriba, P.v., sonnleitner, a., sonnleitner, m., Piotto, s., maresca, B., Horváth, I. and Harwood, J.L. (2005). The signifi-cance of lipid composition for membrane activity: new concepts and ways of assessing function. Prog. Lipid Res. 44: 303-344.

soti, c., nagy, e., Giricz, Z., vígh, L., csermely, P. and ferdi-nandy, P. (2005). Heat shock proteins as emerging therapeutic targets. Brit. J. Pharmacol. 146: 769-780.

shigapova, n., török, Z., Balogh, G., Goloubinoff, P., vígh, L. and Horváth, I. (2005). membrane fluidization triggers mem-brane remodeling which affects the thermotolerance in Es-cherichia coli. Biochem. Biophys. Res. Commun. 328: 1216-1223.

Balogi, Z., török, Z., Balogh, G., Josvay, K., shigapova, n., vi-erling, e., vígh, L. and Horváth, I. (2005). “Heat shock lipid” in cyanobacteria during heat/light-acclimation. Arch. Biochem. Biophys. 436: 346-354.

Balogh, G., Horváth, I., nagy, e., Hoyk, Z., Benkő, s., Bensau-de, o. and vígh, L. (2005). The hyperfluidization of mamma-lian cell membranes acts as a signal to initiate the heat shock protein response. FEBS J. 272: 6077-6086.

csermely, P. and vígh, L. eds. (2006). molecular aspects of the stress response: chaperones, membranes and networks. Ber-lin, new York: sPrInGer, 201p.

vígh, L., Horváth, I., maresca, B. and Harwood, J.L. (2007). can the stress protein response be controlled by 'membrane-lipid therapy'? Trends Biochem. Sci. 32(8):357-363.

nakamoto, H. and vígh, L. (2007). The small heat shock pro-teins and their clients. Cell. Mol. Life Sci. 64(3): 294-306.

nagy, e., Balogi, Z., Gombos, I., akerfelt, m., Bjorkbom, a., Balogh, G., török, Z., maslyanko, a., fiszer Kierzkowska, a., Lisowska, K., slotte, P.J., sistonen, L., Horváth, I. and vígh, L. (2007). Hyperfluidization-coupled membrane microdomain reorganization is linked to activation of the heat shock re-sponse in a murine melanoma cell line. Proc. Natl. Acad. Sci. U.S.A. 104(19): 7945-7950.

Horváth, I., multhoff, G., sonnleitner, a. and vígh, L. (2008). membrane-associated stress proteins: more than simply chap-erones. BBA-Biomembranes 1778(7-8): 1653-1664.

escriba, P.v., Gonzalez-ros, J.m., Goni, f.m., Kinnunen, P.K.J., vígh, L., sanchez-magraner, L., fernandez, a.m., Busquets, X., Horváth, I. and Barcelo-coblijn, G. (2008). membranes: a meeting point for lipids, proteins and therapies. J. Cell. Mol. Med. 12(3): 829-875.

chung, J., nguyen, a.K., Henstridge, d.c., Holmes, a.G., chan, m.H.s., mesa, J.L., Lancaster, G.I., southgate, r.J., Bruce, c.r., duffy, s.J., Horváth, I., mestril, r., Watt, m.J., Hooper, P.L., Kingwell, B.a., vígh, L., Hevener, a. and febbraio, m.a. (2008). HsP72 protects against obesity-induced insulin resist-ance. Proc. Natl. Acad. Sci. U.S.A. 105(5): 1739-1744.

Balogi, Z., cheregi, o., Giese, K.c., Juhász, K., vierling, e., vass, I., vígh, L. and Horváth, I. (2008). a mutant small heat shock protein with increased thylakoid association provides an elevated resistance against uv-B damage in Synechocystis 6803. J. Biol. Chem. 283(34): 22983-22991.


84

BIO

CHEM

ISTR

Y

Ervin WelkerWelker, e., narayan, m., volles, m.J. and scheraga, H.a. (1999). two new structured intermediates in the oxidative folding of rnase a. FEBS Lett. 460: 477-479.

narayan, m., Welker, e., Wedemeyer, W. J. and scheraga, H. a. (2000). oxidative folding of proteins. Acc. Chem. Res. 33: 805-812.

Welker, e., narayan, m., Wedemeyer, W. J. and scheraga, H. a. (2001). structural determinants of oxidative folding in pro-teins. Proc. Natl. Acad. Sci. U.S.A. 98: 2312-2316.

Welker, e., Wedemeyer, W.J. and scheraga, H.a. (2001). a role for Intermolecular disulfide Bonds in Prion diseases? Proc. Natl. Acad. Sci. U.S.A. 98: 4334-4336.

Welker, e., Wedemeyer, W.J., narayan, m. and scheraga, H.a. (2001). coupling of conformational folding and disulfide-bond reactions in oxidative folding of proteins. Biochemistry 40: 9059-9064.

saito, K., Welker, e. and scheraga, H.a. (2001). folding of a disulfide-bonded protein species with free thiol(s): competi-tion between conformational folding and disulfide reshuffling

in an intermediate of bovine pancreatic ribonuclease a. Bio-chemistry 40: 15002-15008.

Welker, e., raymond, L.d., scheraga, H.a. and caughey, B. (2002). Intramolecular versus intermolecular disulfide bonds in prion proteins. J. Biol. Chem. 277: 33477-33481.

Wedemeyer, W.J., Welker, e. and scheraga, H.a. (2002). Pro-line cis-trans isomerization and protein folding. Biochemistry 41: 14637-14645.

Welker, e., Hathaway, L. and scheraga, H.a. (2004). a new method for rapid characterization of the folding pathways of multi-disulfide-containing proteins. J. Am. Chem. Soc. 126: 3720-3721.

Welker, e., maki, K., shastry, m.c.r., Juminaga, d., Bhat, r., scheraga, H.a. and roder, H. (2004). ultra-rapid mixing ex-periments shed new light on the characteristics of the initial conformational ensemble during the folding of ribonuclease a. Proc. Natl. Acad. Sci. u.s.a. 101: 17681-17686.

narayan, m., Welker, e., Zhai, H., Han, X., Xu, G., mcLafferty, f.W. and scheraga, H.a. (2008). detecting native-folds in mix-tures of disulfide-bond-containing proteins. Nature Biotech-nology 26: 427-429.


85

ENZY

MO

LOG

Y

Institute of EnzymologyH-1113 Budapest, Karolina út 29.H-1518 Budapest, P.O. Box 7, Hungary

Brc

86

ENZY

MO

LOG

Y

Our newly established laboratory wishes to study signal transduction pathways, particularly tyrosine kinase signaling. These pathways play key roles in diverse cellular processes, such as cell division or cell movement as well as immune reactions.

sIGnal TRansDUCTIon laboRaToRY

László BUDAY / Principal Investigator, Group Leader

Julianna SÜDI / staff scientist

Scaffold proteins in the signal transduction pathways of tyrosine kinases

signaling pathways utilizing tyrosine kinases regulate many aspects of cell function, including cell division, cell movement, the general metabolism, and the function of the immune system. However, their impaired function may lead to endemic diseases such as human cancer and diabe-tes mellitus. Therefore, investigation of tyrosine kinases can lead us not just to a better understanding of cell biology or biochemistry, but it can also give us some hope of success-ful treatment of cancer or type II diabetes mellitus.

In the last 10-15 years it has become clear in the understanding of cell signaling that not only enzymes and substrates exist, but also other kind of proteins that promote the assembly of large multiprotein complexes. In this way substrates can be targeted to their enzymes, localization of proteins can be changed etc. specialised proteins play important roles in the assembly of the multiprotein complexes. Those proteins do not possess any enzymatic activity but contain certain domains that play important roles in protein-protein interactions.

our group focuses on scaffold proteins, such as cort-actin. It was identified originally as a substrate for the protein tyrosine kinase src. cortactin comprises bind-ing domains for f actin and arp2/3, and via simultane-ous interactions with f-actin and the arp2/3 complex it is specifically localised to sites where branching actin polymerisation is nucleated. It can enhance both the ac-tin nucleating activity of arp2/3 and the stability of the dendritic actin network. We showed earlier that cortac-

tin has an important role in the integrin signaling path-ways, and cortactin phosphorylation was also investi-gated. We recently demonstrated that in hepatocytes stimulated by phorbol esters cortactin translocates to the lamellipodia of migrating cells, and this transloca-tion is independent of rac- and PI 3-kinase activity. In addition, tyrosine-phosphorylated cortactin was seen to be colocalized with focal adhesion complexes along with paxillin and focal adhesion kinase.

caskin1 and its closely related isoform, caskin2, are multidomain proteins possessing six n-terminal an-kyrin repeats, two sterile α motifs (sam) and a single sH3 domain followed by a long proline-rich sequence. recently, we have identified more than 20 binding part-ners of caskin1 in a yeast two-hybrid system. Investi-gating the structure of caskin1, we have identified some potential phosphorylation sites, against which phospho-specific polyclonal antibodies were raised. We have been able to demonstrate that both threonin 1065 and serine 1067 are good in vivo phosphorylation sites for both protein kinase c and protein kinase a. We wish to study whether the interaction of caskin1 with its bind-ing partners is regulated by these phosphorylations. to study the physiological role of caskin proteins in the animal brain, generation of caskin1 and 2 conditionally knock-out mice was started in 2007. Hopefully, the first caskin1 Ko mice will be born in 2009. We would like to study whether the lack of caskin proteins leads to mor-phologic and/or functional changes in the animals.


87

ENZY

MO

LOG

Y

Our group is engaged in a multifaceted research on the relationship of the structure and function of the calpain superfamily, at the levels of biochemistry, biophysics, molecular and cell biology. Deeper understanding of the mechanism of action of calpains is expected to pave the way toward a deeper insight into various pathological proc-esses. At the atomic level we scrutinize the structural details of enzyme activation. At the cell and tissue levels we look for in vivo targets of calpain action, and the consequences of specific activation and inhibition of calpains.

sTRUCTURe anD fUnCTIon of CalpaIns

Péter FRIEDRICH / research Professor, Group Leader

Attila FARKAS / staff scientistágnes TANTOS / staff scientistZoltán BOZóKY / staff scientist

Research topics

calpains are intracellular (cytoplasmic) sH-proteases widely distributed in living cells, found in all eukaryo-tes. as opposed to digestive proteases, calpains as a rule exert limited proteolysis on their substrates. This is re-garded as a regulatory step in an intracellular pathway. Though our knowledge of calpain-induced regulation is far from complete, strong evidence supports their in-volvement in cell division, differentiation, cell adhesion, cell motility tumor metastasis and apoptosis.

studying calpains is particularly warranted by their likely involvement in human pathological conditions such as alzheimer and Parkinson diseases, muscle dys-trophy and type II diabetes.

our research on calpains (mammalian and Dro-sophila) is centered over three issues:

• mapping the fine structural details of calcium-induced activation: signal propagation within the calpain molecule,

• Identification of calpain substrate proteins in in-tact cells,

• development of specific, membrane permeable calpain activators and inhibitors: these are general tools for pinpointing calpain effects in vivo.


88

ENZY

MO

LOG

Y

Lysophospholipids are emerging mediators physiologically relevant in the regulation of fundamental cellular functions, including survival, proliferation, migration and differentiation of cells. The best-characterized members of this recently recognized lipid mediator family are lysophosphatidic acid (LPA), sphingosine-1-phosphate (S1P) and sphingosylphosphorylcholine (SPC). The unique feature of lysophospholipid mediator signaling is that they exert their activities as autocrine/paracrine mediators acting on plasma membrane G protein-coupled receptors (GPCR), but can be second messengers as well. We have shown that lysophospholipids can selectively interact with intra- and extracellular proteins besides GPCR. Our aim is to characterize their cell-surface and intracellular re-ceptors as well as lysophospholipid–protein interactions.

lYsopHospHolIpID ReCepToRslYsopHospHolIpID–pRoTeIn InTeRaCTIons

Károly LILIOM / Principal Investigator, Group Leader

Attila BAKSA / Phd studentErika KOváCS / Phd studentHenriett PáL-GáBOR / Phd student

G protein-coupled receptors selective for lysophospholipids

LPa, s1P and sPc elicit numerous cellular respons-es through the activation of several sets of heptahelical receptors. The first identified endothelial differentia-tion gene family comprised five GPcr exclusively se-lective for s1P and three for LPa. In a collaboration led by Gabor tigyi (university of tennessee, memphis), homology models based on the 3d structure of rho-dopsin were developed and validated experimentally.

Binding of S1P to its G protein-coupled receptor S1P1 The extracellular side is facing upward. The key ionic interactions coordinating S1P are shown enlarged.

We found that the main features of ligand recognition by these receptors are as that the apolar hydrocarbon chain of the lipid ligands are immersed into a cavity formed by the seven transmembrane helices, while their polar head-groups are kept by ionic interactions close to the outer surface of the receptors. The anionic phosphate groups of the lipids are coordinated by two salt-bridges formed by cationic residues of the third and seventh transmembrane helices. a third polar interaction takes place between the lipids’ polar side-groups and the third helix, also provid-ing the source for ligand-selectivity: the five s1P receptors contain glutamic acid in this position forming a salt-bridge with s1P’s amino group, while the three LPa receptors contain glutamine hydrogen-bonding with the oH-group of LPa. We also developed a set of fatty alcohol phosphates selectively interacting with the LPa receptors. We found that the phosphate head-group of the compounds are co-ordinated with the same ionic interactions as LPa, and the agonists' apolar hydrocarbon chains are immersed in the helices, whereas those of the antagonists' are bound to the extracellular loops of the receptors. We now develop novel binding and functional assay systems to character-ize GPcrs' ligand recognition and activation processes, which can be used in drug development.

89

ENZY

MO

LOG

Y

Intracellular lysophospholipid receptors

s1P and sPc were first identified as second mes-sengers capable of liberating ca2+ from intracellular stores. s1P is formed inside cells from sphingosine by the action of sphingosine kinases, while the metabolism of sPc is unknown. The signaling roles of intracellu-lar s1P and sPc were questioned by the discovery of GPcr selectively activated by these sphingophospholi-pids. In a collaborative effort led by dagmar meyer zu Heringdorf (Goethe university, frankfurt) we proved that s1P, liberated intracellularly from an inactive membrane-permeable cage by photocleavage, elevates mobilized ca2+ from thapsigargin-sensitive ca2+ stores of the endoplasmic reticulum. The next step to explore the second messenger functions of lysophospholipid mediators could be the identification of their intracellu-lar target proteins. It has been published that sPc acti-vates ryanodine receptors and that these ca2+ channels are regulated by calmodulin (cam). We hypothesized that cam interacts with sPc, we therefore carried out a set of biophysical and biochemical assays with puri-fied calf brain cam and the signaling lysophospholi-pids including their structural analogs. We found that sPc selectively binds to cam with high affinity, leading to inhibition of the protein's function. We successfully crystallized the complex and determined its structure refined to 1.6 Å resolution. The cam backbone's confor-mation resembles that found in its complex with triflu-operazine, the classical cam antagonist:

X-ray structure of the CaM-SPC complex.

The protein is wrapped around a bunch of lipids (4–6) resulting in the inhibition of its function.

The significance of our findings is that sPc is the first known endogenous compound capable of inhibit-ing cam, raising the possibility of a novel regulation of intracellular ca2+ signaling. We have started to ex-plore these possibilities.

Lysophospholipid-protein interactions

We have shown that cam binds sPc only if it is clus-tered but not as an individual lipid molecule. This, we think, represents a novel class of binding mode we are characterizing now. We started to explore the interac-tions of lysophospholipid mediators with proteins other than GPcr, and found another interesting case biochem-ically similar to the cam-sPc system. We identified β2-microglobulin (β2m) as a selective high affinity binding partner for LPa. In this system the protein also interacts only with the clustered lipid, resulting in the destabiliza-tion of its structure leading to an amyloidogenic confor-mation of β2m, which promotes fibril formation under physiological conditions. This is important, because it is not known what is the pathomechanism of fibril deposi-tions of β2m observed in long-term dialysis patients, con-sidering that β2m is a stable monomeric protein even at the highest concentrations found in patients.

LPa is a blood-born lipid mediator, whose level is also elevated in dialysis patients, thereby providing a possible explanation for fibril formation of the protein by LPa-induced amyloidogenicity.

We believe these novel interactions we identified between lysophospholipid mediators and proteins are of broader occurrence and importance, the study of which is the platform of our ongoing efforts.


Károly LILIOM lYsopHospHolIpID ReCepToRslYsopHospHolIpID–pRoTeIn InTeRaCTIons

90

ENZY

MO

LOG

Y

Cell aRCHITeCTURe GRoUp

Judit OváDI / Principal Investigator, Group Leader

Ferenc OROSZ / senior scientistJudit OLáH / senior scientistAttila LEHOTZKY / staff scientistIstván HORváTH / staff scientistOrsolya vINCZE / staff scientistNatália TŐKÉSI / Phd studentágnes ZOTTER / Phd studentEmma HLAvANDA / technician

TPPP proteins: from unstructured proteins to neurodegeneration

The most frequent dementias of the aging popula-tion are the neurodegenerative disorders, involving the important group of conformational diseases such as Parkinson's, alzheimer's and Huntington's diseases. unfolded/misfolded proteins and their pathological aggregation via multistep processes have been pro-posed to play a critical role in the pathogenesis of neu-rodegenerative disorders. obviously, the identification of the pathomechanisms of these processes is one of the major targets of the recent molecular neurology.

We have suggested by means of molecular biologi-cal, ultrastructural as well as system biological ap-proaches that the tubulin Polymerization Promoting Protein (tPPP/p25), a newly discovered unstructured microtubule-targeting protein plays an important role in certain neurological disorders. translation products of the TPPP paralogous human genes have been iden-tified recently, and reported to display distinct struc-

tural and functional features in vitro. The function of tPPP/p25 is regulated by phosphorylation due to the action of specific kinases at serine and threonine sites of the unfolded n-terminal segment of tPPP/p25. The expression of the protein enhances the stability of mi-crotubules, inhibits cell proliferation, but tPPP/p25 is not expressed in neoplastic brain tissues. Physiologi-cally tPPP/p25 is specifically expressed in differenti-ated oligodendrocytes, which develop the protecting myelin sheet surrounding neuronal projections; tPPP/p25 affects microtubule dynamics, which is necessary for oligodendrocyte differentiation. consequently, the dysfunction of this protein could have a crucial role in the etiology of sclerosis multiplex due to the damage of myelin sheet.

our investigations with human cell lines and transgenic mice performed at system level rendered it possible to evaluate a rational mathematical model for the characterization of the effect of overexpres-sion of unfolded proteins on the intracellular energy metabolism. We provided evidence that the activation and microcompartmentation of certain glycolytic en-zymes and metabolites results in enhanced atP levels counteracting the pathological effects. our immuno-histochemical studies on post mortem human brain tissues suggest that tPPP/p25 can be a biomarker of synucleinopathies, as well as a potential drug-target in tPPP/p25-related cns diseases or a marker for their recognition in early state.


Group homepage: http://www.enzim.hu/labs/Ovadi/

91

ENZY

MO

LOG

Y

We perform complex bioinformatic analyses of genomic and protein sequence data to develop more reliable gene prediction methods and to predict the structure and function of novel genes/proteins and protein domains.

We combine the results of our in silico predictions with wet lab practice to determine the 3D structure and study the function of novel domains/proteins.

fUnCTIonal GenomICs GRoUp

László PATTHY / research Professor, Group Leader

László BáNYAI / senior scientist Mária TREXLER / senior scientist Alinda NAGY / staff scientistIldikó NAGY / Phd studentKatalin KONDáS / Phd studentKornél TAKáTS / Phd studentGyörgy SZLáMA / Phd studentKatalin WODZINSKY / technician

recent analyses of the performance of various computational gene-finding methods have revealed that no prediction method correctly identified greater than 45% of the coding transcripts of human genes. The poor performance of current gene prediction tools thus suggests that a significant proportion of genes of higher eukaryotes is likely to be mispredicted.

as a participant of the Biosapiens network of excel-lence (www.biosapiens.info) funded by the european union’s 6th framework Program, we have developed the misPred computational pipeline that provides an efficient means for the quality control of gene predic-tions. The rationale of the misPred project is that a pro-tein-coding gene is likely to be mispredicted if some of

its features conflict with our current knowledge about protein-coding genes and proteins. Behind each mis-Pred tool there are well-established rules about the structure and function of proteins: predicted proteins that conflict with one of the rules are identified as er-roneous and their genes as mispredicted. analysis of various databases with the misPred computational pipeline has revealed that the proportion of erroneous sequences is significantly higher than anticipated.

We also perform complex bioinformatic analyses of genomic and protein sequence data to predict the structure and function of novel genes and proteins, primarily from metazoa. These analyses include iden-tification of novel genes/proteins, definition of novel

1jbi.pdb 1uap.pdb 2d3j.pdb

Figure 1. Three dimensional structure of the LCCL domain of human cochlin (1jbi.pdb), the NTR domain of human type I procollagen C-proteinase enhancer (1uap.pdb) and the WIF domain of human WIF-1 protein (2d3j.pdb).

92

ENZY

MO

LOG

Y

domain-families, prediction of their structure and function etc. as a result of such analyses we have identified some novel multidomain proteins—e.g. WfIKKn1, WfIKKn2—and several novel protein domain-families, e.g. the ntr, Pan, LccL and WIf domain families.

The WfIKKn2 protein was implicated in the regu-lation of muscle growth. furthermore, we have shown that both WfIKKn1 and WfIKKn2 bind myostatin and Gdf11 with high affinity. The interaction is medi-ated primarily through their follistatin domains, but their ntr domains also participate in binding.

In collaboration with the nmr group of Gottfried Ötting (Karolinska Institute, stockholm and the aus-tralian national university, canberra) we have deter-mined the 3d structures of ntr-, LccL- and WIf-domains (figure 1). These studies revealed that LccL and WIf domains are novel folds, whereas characteri-zation of the structure of the ntr domain confirmed our prediction that this domain is related to the n-terminal domain of tImPs.

structural characterization of the LccL module of the inner ear protein cochlin is of major medical interest, since mutations affecting the LccL module of cochlin cause dfna9 deafness disorder. We have shown that five of the six mutations that cause hear-ing loss affect structurally important residues and the

mutations lead to misfolding of the protein. our find-ing that the second vWfa domain of cochlin interacts with type II collagen, the major collagenous compo-nent of the inner ear has obvious relevance for the bio-logical function of this protein; the structural organi-zation of this matrix protein is severely perturbed in dfna9 patients.

Wnt proteins play important roles during verte-brate and invertebrate development, in adult homeos-tasis and in the maintenance of stem cell pluripotency. aberrant activation of the Wnt signaling pathway is frequently associated with different human cancers. different types of leukemias are characterized by de-creased levels of WIf-1, a Wnt antagonist that binds to Wnts through its n-terminal WIf domain. structural characterization of the WIf domain of this protein is of major interest, since it may help in the identification of the structural determinants of the Wnt-WIf inter-action. our finding that the extracellular region of the ’orphan’ ryk receptor tyrosine kinases belongs to the WIf-domain family led us to predict that ryks serve as Wnt receptors. This prediction was confirmed by sub-sequent studies, identifying a third pathway through which Wnts exert their diverse biological functions.


László PATTHY fUnCTIonal GenomICs GRoUp

93

ENZY

MO

LOG

Y

We have discovered a new serine peptidase family, unrelated to the trypsin and subtilisin families. The enzymes of this group, which was called prolyl oligopeptidase family, cannot hydrolyze proteins or peptides containing more than about 30 amino acid residues. Members of the prolyl oligopeptidase family are important targets for drug design, as they are involved in several diseases, such as mental disorders (prolyl oligopeptidase), diabetes type 2 (dipeptidyl peptidase IV), trypanosomiasis (oligopeptidase B) and carcinoma (acylaminoacyl peptidase).

sTRUCTURe anD fUnCTIon of olIGopepTIDases

László POLGáR / Principal Investigator, Group Leader

Zoltán SZELTNER / senior scientistKlarissza DOMOKOS / staff scientistZsuzsanna NAGY / staff scientistIlona SZAMOSI / technician

The structure of prolyl oligopeptidase

The first crystal structure in the family was deter-mined for prolyl oligopeptidase. It was shown that the enzyme consists of a serine peptidase domain with an α/β-hydrolase fold and a seven-bladed regulatory β-propeller domain. The propeller covers the active site of the catalytic domain (ser554, His680, asp641), thus preventing the access for large, structured pep-tides and proteins (figure 1).

Figure 1. Structure of prolyl oligopeptidase. The ribbon diagram is colour-ramped blue to red from the amino to the carboxyl terminus. The catalytic residues are shown in ball and stick representation

our kinetic and structural studies indicated that substrate binding is restricted to the P4-P2’ region and offered a method for the determination of the real pKa of the catalytic histidine residue. The contribution of asp641, the third member of the catalytic triad, is very much dependent on the substrate leaving-group, which was not the case for the classic serine peptidases, such as trypsin. We have also delineated the catalytic role of the oxyanion binding site, using the tyr473Phe enzyme variant.

The source of oligopeptidase activity

Proteases have a variety of strategies for selecting substrates in order to prevent uncontrolled protein deg-radation. In prolyl oligopeptidase the narrow entrance of the propeller opposite to the active site is much smaller than the diameter of an average peptide. a large conformational change in the enzyme is necessary to allow the substrate to enter the active site region. cross-linking the two domains with engineered disulphide bonds, studying the stability of the isolated propeller, and using molecular dynamics and molecular frame-work approaches we suggested that an opening must be formed between the domains. The large opening was revealed by X-ray crystallography with a bacterial prolyl oligopeptidase, as well as our current structure deter-mination of an acylaminoacyl peptidase.

94

ENZY

MO

LOG

Y

Searching for the possible biological roles of prolyl oligopeptidase

several lines of evidences indicate that prolyl oli-gopeptidase has important intracellular role in cellu-lar signalling and cellular transport processes mediat-ed through the inositol-phosphate pathway involving various protein-protein interactions. at present, we are investigating the interaction of prolyl oligopepti-dase with the microtubular system and with GaP43, a protein important in neuronal growth cone forma-tion, axonal growth and apoptosis.

Acylaminoacyl peptidase

In contrast to the monomeric prolyl oligopepti-dase, acylaminoacyl peptidase is a tetrameric enzyme, and the assembly of the subunits is of primary interest. unfortunately, the crystal structure of the mammalian acylaminoacyl peptidase has not yet been solved. our studies on the oxyanion binding site, however, demon-strated that this catalytic entity is different from that of the prolyl oligopeptidase or oligopeptidase B. This result was confirmed with the orthologous bacterial enzyme from the thermophilic aeropyrum pernix,

the crystal structure of which was known. using site-specific mutagenesis, kinetic analysis and structure determination of the modified bacterial enzyme, the contribution of the oxyanion binding site to catalytic efficacy was evaluated. furthermore, a major specifici-ty difference was found between the mammalian and the bacterial enzymes. While the mammalian enzymes are true exopeptidases cleaving an acyl-dipepide from the n-terminus of the substrate, the bacterial enzyme also displays endopeptidase activity. determination of the crystal structure of substrate/inhibitor com-plexes formed with the bacterial enzyme revealed the structural basis of the endopeptidase activity. It is very likely that the acylaminoacyl peptidase originally was less specialized and became a genuine acylaminoacyl peptidase only at a later stage of evolution. a further difference among acylaminoacyl peptidases relates to their oligomerization states. In addition to the dimer-ic and tetrameric enzymes, we have recently found a hexameric structure. The crystal structure determina-tion of the hexameric enzyme is underway.


László POLGáR sTRUCTURe anD fUnCTIon of olIGopepTIDases

95

ENZY

MO

LOG

Y

Genome projects have significantly extended the scope of investigated proteins. First, transmembrane proteins have been shown to be of much higher proportion among proteins than ever thought before. Later the existence of partially or fully disordered proteins and their surprisingly high representation in eukaryotic organisms have been detected, as well as their key role in signal transduction and regulation processes. Our group applies and develops theoretical and computational tools to investigate the organization of functionally relevant states and stability of proteins, as well as the physical background of their functionality. We are keeping up with new scientific trends and our group is engaged in the research of globular, transmembrane and disordered proteins. Last, but not least significant efforts are made to clarify the basic principles determining proteins’ physiologically relevant states. Not only have the results been published in journals, but also over a dozen web servers have been established for structure analysis and prediction.

pRoTeIn sTRUCTURe ReseaRCH GRoUp

István SIMON / Principal Investigator, Group Leader

Zsuzsanna DOSZTáNYI / senior scientistMónika FUXREITER / senior scientistCsaba MAGYAR / senior scientistGábor E. TUSNáDY / senior scientistÉva TÜDŐS / staff scientistBálint MÉSZáROS / staff scientistDániel KOZMA / staff scientistLetif MONES / Phd student

Globular Proteins

ever since our group was established, we have been engaged in the research of the largest protein family. numerous structure prediction methods have been developed based on protein sequence and structure analysis; such methods can be found on the cYsre-doX web server for the prediction of cystein resi-dues participating in disulphide bonds. The concept of stabilization centers has been introduced for non-covalent crosslink playing a dominant role in protein structure stability. a new method has been developed for the prediction and identification of amino acids in these centers based on the amino acid sequence and on the protein structure, respectively (i.e. scide, scpred, sride web servers). among others, these results have been successfully applied for the analysis of the func-tionally regulated stability of mHc proteins and of the properties of tIm barrel structural elements, as well as for the demonstration of the divergent evolution of Pd-(d/e)XK restriction endonucleases. The structur-al properties of several proteins have been determined by molecular mechanics and molecular dynamics

tools. The role of the hydration sphere in protein-dna recognition processes has been shown. also, the role of metal ions in the functionality of endonucleases has been identified. our most recent results are in the de-velopment of novel methodology for the modeling of biochemical reactions.

Figure 1. Flexible regions surround the possible active site of prolyl oli-gopeptidase

96

ENZY

MO

LOG

Y

Transmembrane Proteins

In the second half of the 90’s the focus of our research shifted towards the study of transmembrane proteins. We have shown that the sequence alignment of trans-membrane proteins requires special methods and that the topology of transmembrane proteins corresponds to a state of maximum likelihood, which can be identified by a hidden markov algorithm. These findings are the basis of our widely used das and HmmtoP topology prediction algorithms, the das-tmfilter method, used for the identification of transmembrane proteins, and partially the Psort-B method, used for the prediction of the subcellular localization of proteins. furthermore, we have developed an algorithm for determining the exact location and orientation of the membrane in 3d protein structures (see tmdet web server). The weekly updated PdBtm database is assembled using this al-gorithm. two additional databases, toPdB and toP-dom containing chemical and biochemical data were constructed as well. all three databases are equipped with search engines and are publicly available through the internet. apart from general theoretical and meth-odological works, the group has conducted significant studies concerning aBc transporter proteins and un-veiled the transmembrane origin of prion proteins.

Figure 2. The TOPDB (Topology Data Bank of Transmembrane Proteins) server

Disordered Proteins

our most recognized achievement of the past five years was the realization that disordered proteins have a higher mean energy than globular proteins. Based on this observation, the partially or fully disordered pro-teins or protein segments can be distinguished using the protein sequence alone (see IuPred web server).

Figure 3. The estimated pairwise energy content discriminates be-tween folded (red) and disordered (blue) proteins

This allowed to gain valuable insights into the inter-molecular interactions of disordered proteins, and un-covering the role preformed structural elements and certain motifs play in protein-protein interactions. furthermore, it enabled the verification of the abun-dance of disordered proteins among hubs in protein interaction networks and the significantly high occur-rence of protein disorder in essential proteins. These fundamental results enabled the characterization of interacting regions in disordered proteins and the de-velopment of a prediction method called ancHor for the prediction of such disordered binding sites. We have also demonstrated the functional role of protein disorder in numerous cases, including the analysis of dutPase and transglutaminase substrate preference and the mediator complex involved in transcriptional regulation.


István SIMON pRoTeIn sTRUCTURe ReseaRCH GRoUp

97

ENZY

MO

LOG

Y

laboRaToRY of Cell bIoloGY

Gergely SZAKáCS / Principal Investigator, Group Leader

Dóra SZEGŐ / staff scientistNóra KUCSMA / staff scientistKatalin KISS / Phd studentDóra TÜRK / Phd studentZsuzsanna SEBESTYÉN / technician

1. Development of compounds targeting multidrug resistant cancer

despite considerable advances in drug discovery, resistance to chemotherapy confounds the effective treatment of patients. cancer cells can become re-sistant to a single drug or they may acquire broad cross-resistance to mechanistically and structurally unrelated drugs (multidrug resistance (mdr)). atP-binding cassette (aBc) proteins, present in most living organisms from prokaryotes to mammals, include the best known mediators of mdr. In par-ticular, mdr pumps aBcB1 (mdr1-Pgp), aBcc1-mrP1, and aBcG2-mXr actively extrude many types of drugs from cancer cells, thereby conferring resistance to those agents. Known substrates of Pgp include natural-product antineoplastics such as an-thracyclines, vinca alkaloids, taxanes, and epipodo-phyllotoxins. Pgp expression, frequently detected in human solid and hematological cancers, is a marker of chemoresistance or decreased survival in leukemi-as, lymphomas, osteosarcomas, small cell lung can-cers and breast cancers, among other malignancies. Thus, innate or acquired expression of Pgp is a major problem in cancer chemotherapy.

despite promising in vitro results, successful modulation of clinical mdr through the chemical inhibition of drug efflux from cancer cells remains elusive. Interestingly, expression and function of some aBc transporters, most notably Pgp, may cause an increase in the sensitivity of cancer cells to some drugs (“mdr1-inverse compounds”). This increase is unexpected, as Pgp is perhaps the best-known multi-drug resistance protein. a central hypothesis arising

from this observation is that mdr1-inverse com-pounds, whose toxicity is potentiated, rather than antagonized by the activity of Pgp, might provide an entirely novel way to fight multidrug resistant can-cer.

Preliminary data indicate that increased sensitiv-ity of otherwise multidrug resistant cells is not due to nonspecific factors arising during the selection of the cells. rather, toxicity of such “mdr1-inverse” com-pounds is directly enhanced by Pgp function. our general aim is to further characterize mdr1-inverse compounds showing increased toxicity in cells that are otherwise notoriously resistant to treatment. our objective is three-fold: 1) to identify further mdr1-inverse compounds; 2) to understand molecular mechanisms causing hypersensitivity of mdr cells and 3) to determine if and how these compounds can be used to overcome clinical Pgp-mediated multid-rug resistance.

98

ENZY

MO

LOG

Y

2. Study of the mitochondrial ABC transporter protein ABCB6

aBc transporter proteins are located in the plasma membrane of the cells, or in the membranes of dif-ferent cellular organelles, and mediate the transloca-tion of various molecules across these barriers. some aBc proteins facilitate the transport of inorganic ions, whereas others pump organic compounds, in-cluding lipids, bile acids, glutathione and glucuronide conjugates, or even short peptides. most aBc proteins utilize the energy of atP hydrolysis for this transport activity (active transporters), but some aBc trans-porters form specific membrane channels. Their func-tional significance is suggested by the observation that they form one of the largest protein families. Based on sequence homology, 48 different aBc transport-ers (grouped into seven subfamilies ranging from a to G) have been defined in the human genome. our overall goal is to understand the pathophysiology of mitochondrial aBc transporters. In particular, we are interested in the role of aBcB6 in protecting the cells against xenobiotic toxicity.

aBcB6 belongs to the B [mdr/taP] sub-family of atP-binding cassette transporters. The B subfamily consists of 11 members, with functions ranging from peptide transport (aBcB2-3) to secretion of bile ac-ids (aBcB11). aBcB1-mdr1, the first human aBc transporter cloned and characterized through its ability to confer an mdr phenotype to cancer cells, stands out among aBc transporters by conferring the highest resistance against the greatest variety of com-pounds. In addition to the four full-length transport-ers, the aBcB subfamily contains seven half trans-porters, of which four (aBcB6, aBcB7, aBcB8 and

aBcB10) are believed to reside in the mitochondria. The function of the human mitochondrial aBc pro-teins is unknown. The outer membrane of mamma-lian mitochondria is freely permeable to most small molecules, whereas the inner membrane forms an impermeable barrier between the mitochondrial ma-trix and the cytosol. Based on the general role of aBc transporters, members expressed in the mitochondria are likely to play a role in transport processes through the inner membrane (thus regulating the intramito-chondrial milieu), or in the communication of signals transmitted from the mitochondria to the rest of the cell. although sequence conservation alone does not allow the exact determination of function, possible roles of mitochondrial aBc transporters include the export of toxic substances that accumulate in the mi-tochondrial matrix (cf. aBcB1), export of peptides (cf. aBcB2-3) or transport of phospholipids (cf. aBcB4). furthermore, depending on their orientation in the membrane, aBc transporters may also be involved in the import of certain constituents.

our general aim is to establish a comprehensive experimental system combining biochemical, ge-netic and cell biological approaches that will lead to the identification of the function and structure of mi-tochondrial aBc transporters and the nature of the compounds they transport. our studies are designed to address the following critical questions: first, what substrate(s) does aBcB6 transport? second, where is aBcB6 located in the cell? Third, what is the function-al form of aBcB6? candidate multidrug transporter?


Gergely SZAKáCS laboRaToRY of Cell bIoloGY

99

ENZY

MO

LOG

Y

InTRInsICallY DIsoRDeReD pRoTeIns GRoUp

The group studies the structure-function relationship of intrinsically disordered proteins (IDPs) by a variety of techniques including bioinformatics, molecular biology, cell biology and structural biology. Our aim is to arrive at general conclusions through studies of selected IDPs, such as the plant stress protein ERD14 and human cell-cycle regulatory protein securin.

Péter TOMPA / Principal Investigator, Group Leader

Hedvig HEGYI / senior scientistLajos KALMáR / staff scientistDénes KOváCS / staff scientistBeáta SZABó / staff scientistágnes TANTOS / staff scientistÉva SCHáD / staff scientistEszter SáGINÉ HáZY / Phd studentBianka áGOSTON SZALAINÉ / Phd student

Research activity

The classical structure-function paradigm states that a well-defined 3d structure is the prerequisite of protein function. recently, it has been recognized that this view portrays too simple a picture, as many proteins or regions of proteins are intrinsically disor-dered/unstructured (IdPs/IuPs) under native, physi-ologiocal conditions. Protein disorder has undergone evolutionary expansion, and it reaches high propor-tions in the proteomes of higher eukaryotes, with as many as 12% of proteins being fully disordered, and 50% of proteins containing at least one long (>30 con-secutive amino acids) disordered region in the human proteome. structural disorder correlates with func-tions of signaling and regulation of transcription, thus mutations of IdPs are often involved in debilitat-ing diseases, such as cancer, cardiovascular diseases and neurodegenerative disorders. The prevalence and functional importance of structural disorder demands the structure-function paradigm to be re-assessed and extended.

In the group, we pursue research in different di-rections carrying out both experimental structure-function studies and bioinformatics analyses. our molecular biological and structural studies are aimed

at a better understanding of the recognition function of IdPs, their function as chaperone proteins and also their functional promiscuity (moonlighting). We also address the properties of their surface hydrate layer. Bioinformatic approaches are used to understand the molecular mechanism of their interactions, the role they play in diseases and the regularities of their evo-lution. These studies enable us to draw general conclu-sions on the structure-function relationship of IdPs, their evolutionary advance and prevalence in eukary-otic proteomes. In all, our studies contribute to re-as-sessing the structure-function paradigm of proteins.

Potential applications

mutations of IdPs are often involved in disease, whereas their special modes of interactions with mac-romolecular partners (another protein, rna or dna) enable the development of inhibitors against them. This direction of research may lead to the development of a novel generation of drug candidates to be used in treating cancer and neurodegenerative diseases.


100

ENZY

MO

LOG

Y

folDInG anD fUnCTIon of moDUlaR pRoTeIns

Mária vAS / Principal Investigator, Group Leader

Andrea vARGA / staff scientistÉva GRáCZER / Phd student

Co-operation of PGK domains

3-Phosphoglycerate kinase (PGK) is an essential enzyme for the carbohydrate metabolism of all living systems. Besides, PGK contributes to phosphorylation of nucleotides important in antiviral and antitumour therapy.

Previously we evidenced sequential refolding of the two domains, supporting the essential role of domains in the formation of the native three-dimensional struc-ture. In unfolding experiments, however, we demon-strated highly co-operative disruption of the two do-mains. This can be due to the fact that – in contrast to refolding – unfolding starts from the native structure with well-established inter-domain interactions, re-sulting in a stronger coupling between domains. We intend to get a deeper insight into the co-operation of PGK domains both in the stabilisation of native con-formation and in the achievement of catalysis.

several lines of experimental evidence support the participation of a hinge-bending type domain closure in PGK catalysis, as illustrated by comparing the open and the variously closed structures (fig. 1). for the mecha-nism of domain closure we localised the main molecu-lar hinge at the interdomain region and delineated its

operation as a double-sided molecular switch under the concerted action of the two substrates that bind sepa-rately to each domain. using site-directed mutagenesis we have identified conserved residues at the substrate binding pockets to be responsible for transmission of the effects of substrates towards the main hinge.

Figure 1. PGK structures with the two domains in open (red and orange) and in partially (blue) or completely (green) closed conformations are superimposed according to the nucleotide binding domain. The bound substrates are shown as stick models. The main hinge is highlighted by ribbons.

However, the role of the side-chains at the nucle-otide sub-site, where the base is bound is not yet clari-fied. such studies would be particularly timely, since

The rules of self-organization of protein building blocks (modules, domains or subunits) into three-dimensional structure have not been clarified: they may fold either independently or co-operatively into the native molecule. Another unresolved problem is the role of modular structure in the performance of biological function. The de-tails of co-operation between modules, domains or subunits (mediated by protein structural flexibility) and its functional consequences have not yet been delineated. These questions are investigated on two individual proteins (enzymes), namely the monomeric 3-phosphoglycerate kinase (PGK), consisting of two structural domains and the dimeric 3-isopropylmalate dehydrogenase (IPMDH) with two identical subunits.

101

ENZY

MO

LOG

Y

it is the base that determines the specificity of nucle-otides, and the low specificity of PGK towards nu-cleotides is especially remarkable due to its ability to phosphorylate various nucleotides, including the ones applied in HIv therapy.

Therefore, our further aim is twofold: i. to map the role of the side-chains located in the hydrophobic binding pocket of the nucleoside sub-site in transmis-sion of the nucleotide-effect towards the main hinge; ii. to specify the structural basis of the broad nucle-otide specificity of human PGK. In this way, molecu-lar details of transmission of the substrate-triggered conformational changes from one domain to the other will be better clarified.

for these purposes enzymological (kinetic and equilibrium), site-directed mutagenesis and biophysi-cal (fluorescence and cd spectroscopy, saXs, micro-calorimetry etc.) studies are conducted in our labora-tory in close connection with X-ray crystallography and molecular graphical analysis.

Subunit-subunit Interactions of IPMDH

Isopropylmalate dehydrogenase (IPmdH) is one of the essential enzymes of leucine biosynthesis. Its dimeric structure provides a suitable model for in-vestigating the role of subunit-subunit interactions in folding and function. The complementary formation of the active site upon association of the two identical subunits represents a unique type of folding pattern (fig. 2).

We have clarified the importance of subunits in the reconstruction of native IPmdH structure from three different sources: the bacterial heat-stable Ther-mus thermophilus, the mesophilic E. coli and the cold-active Vibrio sp. I5 enzymes. our in vitro unfolding-refolding studies have revealed for all three enzyme forms that association of the two polypeptide chains is a prerequisite of the reconstruction of the native struc-ture. It occurs at the very beginning of folding, but it does not constitute a rate-determining step. further-more, the time course of changes in enzyme activity and in spectroscopic signals (fluorescence and cd)

during refolding are found to be biphasic first order processes, indicating the presence of a molten globule-like dimeric intermediate. folding of the two domains within each subunit also occurs in a symmetrical way, as indicated by the refolding studies carried out with single trp mutants, reporting about reconstruction of the native structure of the individual domains. Thus, both domains and subunits exhibit a high extent of co-operativity during formation of the three-dimensional structure of the IPmdH molecule.

Figure 2. Dimeric structure of IPMDH: the subunits are coloured differ-ently (green and red). The bound IPMs are indicated by black stick mod-els. The active site residues are shown by ball-and-stick models.

our further aim is the elucidation of the role of domain-domain and subunit-subunit cooperativity in performing the enzymatic function of IPmdH. Pre-vious structural studies have shown that the relative position of the two domains may vary between open and closed states. This suggests an essential role of domain closure during the catalytic cycle, similar to the mechanisms assumed also for other multidomain enzymes. Therefore, we intend to identify the details of the conformational changes occurring in all bina-ry and ternary enzyme substrate complexes. for this purpose systematic X-ray crystallographic structural analysis as well as complementary single crystal mi-cro-spectrophotometric studies are underway in our laboratory.


Mária vAS folDInG anD fUnCTIon of moDUlaR pRoTeIns

102

ENZY

MO

LOG

Y

aCTIve TRanspoRT pRoTeIns GRoUp

András váRADI / Principal Investigator, Group Leader

Éva BAKOS / senior scientistTamás ARáNYI / senior scientistZalán SZABó / staff scientistCaroline BACQUET / staff scientistKrisztina FÜLÖP / Phd student

Flóra SZERI / Phd studentviola POMOZI / Phd studentHugues de BOUSSAC / Phd studentGyörgyi vERMES / technician

ABC proteins: from multidrug resistance to genetic disease

following the cloning of the first cancer multid-rug-resistance aBc-transporter, mdr1/P-glycopro-tein in the late eighties, there was a boom in aBc-protein research. The field is progressing quickly, and there are some 500–1000 papers on aBc transport-ers every year. With the completion of the Human Genome Project, forty-eight genes encoding aBc-proteins were identified, many of which are associ-ated with genetic disorders. our group joined the international scene in the early nineties, working in close collaboration with the research group of Balázs sarkadi. In addition to the traditional structure-func-tion studies of transport proteins, our group initiated a research project focusing on the expressional regu-lation of aBc transporter genes. our work also has important physiological relevance, since one of the aBc transporters studied in our laboratory, aBcc6, is related to a rare genetic disorder, pseudoxanthoma elasticum.

aBc proteins share the same basic molecular ar-chitecture: they are composed of combinations of transmembrane domains (tmd) and aBc (atP-Binding cassette) domains. most of the characterized members of the family are involved in transmembrane transport; the process of atP binding and hydrolysis (which happen in the aBc-domains) is a crucial part of their transport-related function. on the basis of amino acid sequence similarities, the human aBc-protein family can be divided into seven subfamilies (aBca trough aBcG).

numerous xenobiotics are eliminated from the cells by the sequence of oxidation, conjugation to an anionic group (glutathione, glucuronate or sulfate) and transport across the plasma membrane into the extracellular space. These transport processes are me-diated by specific transport proteins, by multidrug re-sistance proteins (MDRs). together with the sarkadi group, we have recently developed the “chemoimmu-nity system” theory of the network of aBc-proteins as an early defence mechanism of cells and multicellular organisms.

mrPs also contribute to drug resistance in cancer cells that is the major obstacle of successful chemo-therapy. They belong to the aBcc subfamily of aBc proteins. our group was the first to demonstrate that in case of the so called “long” mrPs (aBcc1, aBcc2, aBcc3 and aBcc6) the core structure (tmd1, aBc1, tmd2, aBc2) is extended with an n-terminal transmembrane domain (tmd0) including 5 trans-membrane helices and a connecting cytoplasmic loop (L0). We have established the correct membrane to-pology model and domain arrangement of the mem-bers of the mrP family. We have discovered that the tmd0 domain is dispensable for the function of hu-man aBcc1, since a truncated mutant that lacks this domain is functional with respect to transport activi-ty, assuming basolateral localization in polarized cells. However, extending the deletion to L0 abrogates the activity of the pump, indicating that the L0 domain is essential for function. We have shown that L0 in-teracts with the core region of aBcc1. We have ex-amined the details of the catalytic cycle in the major multidrug transporter aBcc1.

103

ENZY

MO

LOG

Y

Phylogenetic analysis revealed that the Drosophi-la MRP protein (DMRP) is the orthologue of hu-man “long” multidrug resistance-associated proteins aBcc1, aBcc2, aBcc3 and aBcc6. our labora-tory demonstrated that dmrP possesses combined features of its human orthologues; however, we found dmrP to exhibit an extremely high turnover rate for substrate transport as compared to its human ortho-logues. These features make dmrP a valuable model protein for further studies to reveal the mechanistic features of mrPs, especially for investigations that re-quire high turnover.

aBc genes have been identified in each genome that has been sequenced so far. The high number of aBc genes demonstrates their important, however mostly unknown physiological role. according to the current hypothesis, different members have a wide ex-pression pattern and partially overlapping functions, which account for the relatively complex phenotypes upon their loss of function. We have embarked upon a research program that involves a wide spectrum of experimental strategies aimed to elucidate the mo-lecular basis of pseudoxanthoma elasticum (PXE). PXe is caused by mutations in the ABCC6 gene. Pseu-doxanthoma elasticum is a heritable disorder, affect-ing several different elastic tissues, including the skin, the eye and the arterial system. While a spectrum of mutations within the ABCC6 gene is clearly respon-sible for PXe, the functional relationship between al-tered ABCC6 gene products and the PXe phenotype is still unknown. similarly, very little is known about the transcriptional regulation of the aBcc6 gene. In-vestigating the molecular basis of monogenic inher-ited diseases provides invaluable information about normal human physiology. Basic research of this type usually does not yield direct therapeutic interventions, but may open avenues towards such applications.

similarly to the general strategy in the case of aBc transporters, the basic aim of our research on this dis-ease-associated protein is to identify and characterize transported substrates. one of the unique features of the proposed strategy is that potential physiological substrate candidates identified by functional in vitro assays will direct us to metabolic pathways for further candidates, and such compounds will also be tested

as inducers/inhibitors of gene expression. discovery of the physiological substrate(s) of the transporter will help understand its physiological role in the given tis-sue. In the case of the human aBcc6 protein another important goal of the research project is to establish genotype-phenotype relationships of the PXe-asso-ciated missense mutant forms of aBcc6 in order to initiate further effort to correct these mutations. for this purpose we utilize various heterologous expres-sion systems and, in addition to transport biochemis-try, experimental strategies of cell biology.

We have demonstrated that aBcc6 is a primary active transporter of organic anions and also revealed that the loss of atP-dependent transport resulted in PXe phenotype through direct influence on the trans-port activity of three missense mutants of aBcc6 protein we studied. It has been also shown that the hu-man aBcc6 protein is exclusively localized to the ba-solateral membrane of kidney-derived epithelial cells (mdcKII).

A homology model of ABCC6 transporter has been built by our laboratory. The large number of PXe-associated missense mutations provides an op-portunity to analyze their distribution within the pre-dicted three-dimensional structure. We have analyzed this distribution, paying special attention to potential domain–domain interactions. significant clustering of the missense mutations has been found at complex do-main-domain interfaces, namely at the transmission interface that involves four intracellular loops and the two aBc domains as well as at the aBc - aBc inter-acting surfaces. These data provide a genetic proof for the importance of these domain-domain interactions in the aBcc6 transporter.

our group published the first study on the tran-scriptional regulation of the ABCC6 gene. In this initial work we determined the aBcc6 transcription initiation site (tIs) and carried out phylogenetic foot-printing on a 10kb long 5’ region preceding the tIs. This approach identified two evolutionarily conserved regions and suggested their implication in the regula-tion of the gene. Interestingly, each of them contains a cpG island, which is characteristic of regulatory re-gions and is generally the site of potential tissue-specif-ic epigenetic regulation. dna methylation analysis of

András váRADI aCTIve TRanspoRT pRoTeIns GRoUp

104

ENZY

MO

LOG

Y

the cpG island of the proximal promoter revealed an inverse correlation between the tissue-specific expres-sion and the methylation of the gene. In the following experiments we focused on this region and identified in luciferase reporter gene assays a dna methylation dependent activator and a repressor sequence in the proximal promoter. currently, we are searching for transcriptional modulators of aBcc6 and analyzing the way of action of the identified molecules.

aBcc6 has two human specific pseudogenes, which share 99% sequence identity with the disease-

causing gene. In order to better understand how aBcc6 pseudogenes were evolved, we analyze the segmental duplications containing the aBcc6-like sequences. our results show that the pseudogenes are only present in primates and the ones found in man are human-specific. The duplication and presence of these long, more than 99% identical sequences might have important consequences.


András váRADI aCTIve TRanspoRT pRoTeIns GRoUp

105

ENZY

MO

LOG

Y

Genome meTabolIsm anD RepaIR

Maintenance of stable genetic information together with strict metabolic and developmental control of bio-synthesis of the nucleotide building blocks is essential for life. Uracil, a close thymine analog, is traditionally considered as a mistake to be corrected in DNA. Repair of uracil as a spontaneous cytosine deamination product is essential, but the repair mechanism eliminates thymine-replacing, "innocent" uracils as well. An elevated cel-lular dUTP/dTTP ratio, due to the suboptimal specificity of DNA polymerases, leads to incorporation of thymine-replacing uracils to an extent that overloads the repair apparatus and induces apoptosis (thymine-less cell death). This research field is of interest for therapeutical applications against viral and microbial pathogens as well as in cancer treatment. Perturbation of cellular dUTP/dTTP ratios is an especially promising chemotherapeutic strategy in Mycobacteria and Plasmodia, the causative agents of tuberculosis and malaria. Several studies indicate that uracil-DNA may be at least transiently tolerated in different organisms, and it may possess a physiological or de-velopmental role. Novel experimental data together with results from our laboratory argue for re-interpretation of the traditional view on uracil being solely a mistake in DNA.

Beáta G. vÉRTESSY / Principal Investigator, Group Leader

Judit TóTH / Principal InvestigatorAngéla BÉKÉSI / staff scientist Enikő TAKáCS / staff scientistvillő MUHA / staff scientistMária PUKáNCSIK / staff scientistImre ZAGYvA / research assistant

Preventive DNA repair via dUTPase action

dutPase is the unique and ubiquitous enzyme cata-lysing dutP hydrolysis to yield dumP and pyrophos-phate. organisms benefit from dutPase activity in two ways, both of which contribute to the maintenance of dna integrity: 1) continuous removal of cellular dutP to prevent excessive uracil incorporation into dna; 2) dumP supply for dttP biosynthesis. Lack of dutPase activity induces the transformation of base excision re-pair into a hyperactive futile cycle leading to thymine-less cell death. a main goal in my lab is to identify novel compounds for the promising chemotherapeutic strat-egy of dutPase antagonism, for selective killing of cells with ongoing dna synthesis, such as in neoplasia or virus infection, and with special interest in mycobac-teria and Plasmodium, where dutPase serves the sole biosynthetic route to dttP.

We study dutPase at several different levels ranging from 3d structural studies at high-reso-

lution in the crystal phase to phenotypic changes of dutPase deficient model organisms (human tumor cell lines, Drosophila, C. elegans). In pub-lished studies, we have identified the molecular mechanism of action of dutPases from bacteria, humans and retroviruses using a complex test sys-tem of X-ray crystallography, mutagenesis, fast and steady-state kinetics and thermodynamics (figure 1). sirna silencing of dutPase in transgenic Dro-sophila led to lethality at the prepupal/pupal transi-tion, in agreement with our working hypothesis (see below). Interestingly, sirna silencing of dutPase in HeLa cells rendered a viable phenotyope with sig-nificantly increased levels of other factors involved in thymidylate de novo biosynthesis, identifying targets for a combined chemotherapy. our current aims are to reveal the cellular interaction network of dutPases, including metabolic interactions, and to design novel drug-like compounds for perturba-tion of dutPase function.

András HORváTH / Phd studentEmese KóNYA / Phd studentIbolya LEvELES / Phd studentGábor MERÉNYI / Phd studentIldikó PÉCSI / Phd student

Extra thyminum non est vita; si est vita, non est ita

106

ENZY

MO

LOG

Y

Figure 1. dUTPase mechanism revealed by X-ray crystallography and kinetics. The intricate and unique fold of homotrimeric dUTPases is shown for the human enzyme in color-coded ribbon models. For clarity, the substrate dUTP (stick model) is shown only in one of the three ac-tive sites. Inset displays key residues surrounding the substrate. Among these, Phe158 was engineered into Trp for an active site label (main pan-el). Fluorescent time course on a fast kinetics scale (yellow recording and red fit curve) reports on cognate interactions within the active site and allows insights into the elementary steps of the catalytic reaction.

Uracil-DNA signaling / A novel Uracil-DNA degrading enzyme

uracil in dna is usually regarded to be a mis-take and two enzymes are essential for its exclusion: dutPase and uracil-dna-glycosylase (unG), prod-ucts of dut and ung genes. Interestingly, however, uracil in dna may also be recognized as a signal for immunoglobuline gene diversification in mam-mals or as a developmental signal in pupating in-sects (figure 2). We have recently identified a novel uracil-dna degrading factor (abbreviated as ude) in Drosophila melanogaster that possesses unique specificity to degrade uracil-substituted dna and is under strong developmental control. The specific activity of ude can be used as a key starting point to develop molecular biotechnological tools of new character. We initiated such studies and preliminary results indicate that the enzyme may be used in mo-lecular diagnostics and in genetic engineering. for successful design of efficient applications and to de-scribe the novel nuclease function we apply a wide-

spread structural biology repertoire for in-depth un-derstanding of the protein structure and function.

The gene ude encoding ude protein is present in each pupating insect genome available to date, and these genomes simultaneously lack the ung gene that encodes the major uracil-dna glycosylases, respon-sible for cleaving uracil out of dna. outside the evo-lutionary lineage encompassing pupating insects, no close homologues of ude could be indentified; these other genomes, however, do contain ung. In our recent preliminary experiments, efficient silencing of either ude or dutPase by sirna in transgenic flies led to lethality at the pupation stage. The essential character of ude and its exclusive presence in pupating insects identify this protein as a novel and promising target for insect control, a task that has much biomedical impor-tance as several insects are either agricultural pests or vectors for a number of deadly transmissible diseases (Anopheles mosquitoes/malaria or Aedes mosquitoes/dengue fever).

Figure 2. Uracil-DNA signaling in insect development. dUTPase, re-sponsible for preventing uracil incorporation into DNA, is present only in the imaginal discs of fruitfly larvae (left to right: immunofluorescence micrographs of eye-antenna, leg and wing discs, progenitors of adult fly organs). dUTPase presence correlates to disc survival during develop-ment. Lack of dUTPase in other larval tissues, sentenced to apoptosis, may result in highly-uracil substituted DNA. At the onset of metamor-phosis, UDE gets expressed and its degradation of uracil-DNA contrib-utes to cell death during development.


Beáta G. vÉRTESSY Genome meTabolIsm anD RepaIR

107

ENZY

MO

LOG

Y

sTRUCTURal bIopHYsICs

Proteins constitute a unique form of matter characterized by well-defined three dimensional structures on one hand and conformational flexibility on the other. This dual characteristic is a consequence of the dominance of non-covalent interactions in the stabilization of their spatial structures and the multifunctionality of their amino acid side chains. The individual proteins are very similar at first sight, but they possess unique surface patterns. Their functional roles include recognition, signal transduction and execution. Our research group is focusing on the exploration of the mechanism of these functions at the level of atomic and molecular interactions.

Péter ZávODSZKY / research Professor, Group Leader

Ferenc vONDERvISZT / senior scientistAndrás SZILáGYI / staff scientistSzilárd KAMONDI / staff scientistIstván HAJDÚ / staff scientistLászló BARNA / staff scientistGergely GYIMESI / Phd studentRáchel SAJó / Phd studentDániel GYÖRFFY / Phd student

Physical aspects of protein function, flexibility, dynamics, recognition and signal transduction

The macromolecular recognition event in itself is not the first step of the biological function only. In order to fulfill their biological function, proteins must undergo structural rearrangements, which can range from local fluctuations or loop movements to large-scale conformational changes, such as the rela-tive movement of structural domains. allostery, i.e. a change in properties at one side elicited by an interac-tion at a distant location is one of the most remark-able functions of enzymes. The classical induced fit model of such transitions does not give a mechanistic explanation of the phenomena. The approach known today as “population shift” model was initiated in our laboratory as the “fluctuation fit” mechanism (straub, 1964). This model has gained popularity during the last decade, as a consequence of the accumulating in-formation on conformational motions on the micro- and millisecond time scales.

our working hypothesis is that the unique sur-face patterns and conformational fluctuation spec-tra of proteins have a major role in recognition and the transmission of information. Well-defined signal transduction pathways exist in proteins, which trans-fer the information in a dynamic way from one func-

tional site to another. The existence of such pathways is confirmed by the conserved sequences in homolo-gous enzymes and the concerted motions involved in enzymatic catalysis.

Figure 1. The three-dimensional structure of the allosteric GAPDH en-zyme. The molecule was colored by the difference in Cα B-factors be-tween rabbit muscle GAPDH and Thermotoga maritima GAPDH (ΔB = BRmGAPDH -BTmGAPDH. Blue ΔB < 9; green 9 < ΔB < 20; red ΔB > 20 Å2). Regions showing the highest difference in flexibility are the NAD binding and substrate binding sites.

our goal is the proper description of the time course of signal transduction events based on struc-tural information. The time scales of relevant motions vary from the nanosecond to second scale; the decon-volution of these relevant motions and the statistical

108

ENZY

MO

LOG

Y

fluctuations is an essential task. only the initial and the final states of functionally relevant conformational transitions are usually known from crystal structures, and despite their functional significance, the confor-mational motions associated with allosteric regulation are extremely difficult to characterize at the quanti-tative level. since conformational flexibility strongly depends on temperature, temperature-dependent en-zyme kinetic studies combined with measurements related to conformational dynamics (H-d exchange, fret, nmr), in combination with molecular dynam-ics simulations can give us insight into the mechanism of allosteric signal propagation between distant sites of a protein molecule, at the atomic level.

our model systems include several types of inter-acting macromolecular systems of various complexi-ties: a typical two-domain, hinge-bending, monomeric enzyme, 3-phosphoglycerate kinase (PGK); 3-isopro-pylmalate dehydrogenase (IPmdH), a homodimeric enzyme; the early components of an immunologically important protein cascade, the complement system; and self-assembling and secretion-mediating proteins of the bacterial flagellar system.


Péter ZávODSZKY sTRUCTURal bIopHYsICs

109

ENZY

MO

LOG

Y

sTRUCTURal bIopHYsICs(moleCUlaR ImmUnoloGY UnIT)

Our research activity is focused on the characterization of serine proteases, which play important roles in the complement system of innate immunity. Using structural-biochemical methods such as X-ray diffraction and NMR, we determine the 3D structure of the enzymes. The structures help us reveal the mechanisms of the activa-tion of the proteases. We design and select specific inhibitors against the early proteases of the complement system, which can serve as lead molecules of anti-inflammatory drug development. Recently, we have discovered a new mechanism by which complement proteases stimulate immune cells.

Péter ZávODSZKY / research Professor, Group Leader Péter GáL / Principal Investigator

József DOBó / staff scientistKatalin CSEH-SZILáGYI / staff scientistAndrea KOCSIS / Phd studentLászló BEINROHR / Phd studentMárton MEGYERI / Phd studentBalázs MAJOR / Phd studentJúlia BALCZER / technician

Protease enzymes in the immune defense

The immune system protects the integrity of our body against invading pathogens, and it also main-tains the immune homeostasis through eliminating the altered self structures, such as cancer- and apoptotic cells. The immune system fulfils its role using a sophis-ticated network of cells and molecules. our research is focused on protease enzymes that play a crucial role in the development and regulation of the immune re-sponse. These serine protease enzymes have multido-main structures and they can cleave only a few target proteins. The complement system is a major component of the innate immunity consisting of a network of about 30 proteins. The main components of the system are serine proteases, which activate each other in a cascade-like manner. activation of the complement system re-sults in the destruction of the target cells via molecu-lar mechanisms, whereas the spilt products generated during the activation process stimulate the cellular im-mune system initiating inflammatory reaction. The in-tact complement system is required for maintaining the internal inflammatory homeostasis; its uncontrolled activation, however, can cause severe disease condi-tions. enhanced, pathologic complement activation is potentially harmful: it can damage self-tissues and

initiate autoimmune inflammation. This phenomenon contributes to cell death following myocardial infarc-tion and stroke, as well as to the development of age-related macular degeneration. It is therefore extremely important to understand the activation and regulation of the complement system and to identify the potential points of intervention.

Figure: The crystal structure of the C1r enzyme. In the crystal lattice the protease molecules form enzyme-product complexes. Based on this structural information we can build a functional model for the autoacti-vation of C1r inside the multimolecular C1 complex.

our research group studies the initial steps of the activation of the classical and lectin activation path-

110

ENZY

MO

LOG

Y

ways of complement. The serine protease enzymes par-ticipating in this process (c1r, c1s, masP-1, masP-2) form multimolecular complexes with pattern recog-nition molecules (c1q, mBL, ficolins). We use X-ray diffraction and nmr to determine the 3d structure of the proteases. The structures help us elucidate the activation mechanisms at the atomic level (figure) and design specific inhibitors. We also use in vitro evolution techniques (e.g. phage display) for inhibitor selections. We managed to solve the crystal structure of c1-inhibitor, the physiological inhibitor of the early complement proteases. using the structure we can ex-

plain the effect of disease- causing (angioedema) muta-tions, and we proposed a model for the potentiation of c1-inhibitor activity by heparin. We have discovered recently that the activation of the complement system can stimulate leucocytes and endothelial cells not only through proteolytic fragments, but also through direct stimulation of protease-activated receptors on the cell surface. The characterization of this new mechanism of cell stimulation is a major field of our research.


Péter ZávODSZKY sTRUCTURal bIopHYsICs

111

ENZY

MO

LOG

Y

László BudayBuday, L. and downward, J. (1993). eGf regulates p21ras through the formation of a complex of receptor, Grb2 adapter protein and sos guanine nucleotide exchange factor. Cell 73: 611-620.

Buday, L., Khwaja, a., sipeki, s., faragó, a. and downward, J. (1996). Interactions of cbl with two adaptor proteins, Grb2 and crk, upon t cell activation. J. Biol. Chem. 271: 6159-6163.

Wunderlich, L., faragó, a., downward, J. and Buday, L. (1999). association of nck with tyrosine-phosphorylated sLP-76 in activated t lymphocytes. Eur. J. Immunol. 29: 1068-1075.

tamás, P., solti, Z., Bauer, P., Illés, a., sipeki, s., Bauer, a., faragó, a., downward, J. and Buday, L. (2003). mechanism of eGf regulation of vav2, a guanine nucleotide exchange factor for rac. J. Biol. Chem. 278: 5163-5171.

tompa, P., szász, c. and Buday, L. (2005). structural disorder throws new light on moonlighting. Trends Biochem. Sci. 30: 484-489.

Illés, a., enyedi, B., tamás, P., Balázs, a., Bőgel, G. and Buday, L. (2006). Inducible phosphorylation of cortactin is not neces-sary for cortactin-mediated actin polymerisation. Cell Signal. 18: 830-840.

Buday, L. and downward, J. (2007). roles of cortactin in tumor pathogenesis. BBA – Reviews on Cancer 1775:263-273.

Buday, L. and downward, J. (2008). many faces of ras activa-tion. BBA – Reviews on Cancer 1786: 178-187.

Péter Friedrichfriedrich, P. (2004). The intriguing ca2+ requirement of cal-pain activation. Biochem. Bioph. Res. Co. 323(4): 1131-1133.

farkas, B., tantos, Á., schlett, K., vilagi, I. and friedrich, P. (2004). Ischemia-induced increase in long-term potentiation is warded off by specific calpain inhibitor Pd150606. Brain Res. 1024(1-2): 150-158.

friedrich, P., tompa, P. and farkas, a. (2004). The calpain-system of Drosophila melanogaster: coming of age. Bioessays, 26(10): 1088-1096.

friedrich, P. and Bozóky, Z. (2005). digestive versus regula-tory proteases: on calpain action in vivo. Biol. Chem. 386(7): 609-612.

Bozóky, Z., alexa, a., tompa, P. and friedrich, P. (2005). mul-tiple interactions of the 'transducer' govern its function in cal-pain activation by ca2+. Biochem J. 388(Pt 3): 741-744.

Banoczi, Z., tantos, Á., farkas, a., tompa, P., friedrich, P. and Hudecz, f. (2007). synthesis of cell-penetrating conjugates of calpain activator peptides. Bioconjug. Chem. 18(1): 130-137.

vilagi, I., Kiss, d.s., farkas, a., Borbély, s., tarnok, K., Halasy, K., Banoczi, Z., Hudecz, f. and friedrich, P. (2008). synthetic calpain activator boosts neuronal excitability without extra ca2+. Mol. Cell. Neurosci. 38(4): 629-636.

Banoczi, Z., alexa, a., farkas, a., friedrich, P. and Hudecz, f. (2008). novel cell-penetrating calpain substrate. Bioconjug. Chem. 19(7): 1375-1381.

tantos, a., friedrich, P. and tompa, P. (2009). cold stability of intrinsically disordered proteins. FEBS Lett. 583(2): 465-469.

Bozóky, Z., alexa, a., dancsok, J., Gogl, G., Klement, e., medzihradszky, K.f. and friedrich, P. (2009). Identifying cal-pain substrates in intact s2 cells of Drosophila. Arch. Biochem. Biophys. 481(2): 219-225.

Károly LiliomGuo, Z., Liliom, K., fischer, d.J., Bathurst, I.c., tomei, L.d., Kiefer, m.c. and tigyi, G. (1996). molecular cloning of a high-affinity receptor for the growth factor-like lipid mediator lyso-phosphatidic acid from Xenopus oocytes. Proc. Natl. Acad. Sci. U.S.A. 93(25): 14367-14372.

Bünemann, m., Liliom, K., Brandts, B.K., Pott, L., tseng, J.L., desiderio, d.m., sun, G., miller, d. and tigyi, G. (1996). a novel membrane receptor with high affinity for lysosphingo-myelin and sphingosine 1-phosphate in atrial myocytes. EMBO J. 15(20): 5527-5534.

Liliom, K., fischer, d.J., virág, t., sun, G., miller, d.d., tseng, J.L., desiderio, d.m., seidel, m.c., erickson, J.r. and tigyi, G. (1998). Identification of a novel growth factor-like lipid, 1-o-cis-alk-1'-enyl-2-lyso-sn-glycero-3-phosphate (alkenyl-GP) that is present in commercial sphingolipid preparations. J. Biol. Chem. 273(22): 13461-13468.

Liliom, K., Guan, Z., tseng, J.L., desiderio, d.m., tigyi, G. and Watsky, m.a. (1998). Growth factor-like phospholipids gener-ated after corneal injury. Am. J. Physiol. 274(4): c1065-1074.

Parrill, a.L., Wang, d., Bautista, d.L., van Brocklyn, J.r., Lőrincz, Z., fischer, d.J., Baker, d.L., Liliom, K., spiegel, s. and tigyi, G. (2000). Identification of edg1 receptor residues that recognize sphingosine 1-phosphate. J. Biol. Chem. 275(50): 39379-84.

Wang, d.a., Lőrincz, Z., Bautista, d.L., Liliom, K., tigyi, G. and Parrill, a.L. (2001). a single amino acid determines lyso-phospholipid specificity of the s1P1 (edG1) and LPa1 (edG2) phospholipid growth factor receptors. J. Biol. Chem. 276(52): 49213-49220.

Liliom, K., sun, G., Bünemann, m., virág, t., nusser, n., Baker, d.L., Wang, d.a., fabian, m.J., Brandts, B., Bender, K., eickel, a., malik, K.u., miller, d.d., desiderio, d.m., tigyi, G. and Pott, L. (2001). sphingosylphosphocholine is a naturally occur-ring lipid mediator in blood plasma: a possible role in regu-lating cardiac function via sphingolipid receptors. Biochem. J. 355(Pt 1): 189-97.

meyer zu Heringdorf, d., Liliom, K., schaefer, m., danneberg, K., Jaggar, J.H., tigyi, G. and Jakobs, K.H. (2003). Photolysis of intracellular caged sphingosine-1-phosphate causes ca2+ mo-bilization independently of G-protein-coupled receptors. FEBS Lett. 554(3): 443-449.

InsTITUTe of enZYmoloGYseleCTeD pUblICaTIons:

112

ENZY

MO

LOG

Y

virág, t., elrod, d.B., Liliom, K., sardar, v.m., Parrill, a.L., Yokoyama, K., durgam, G., deng, W., miller, d.d. and tigyi, G. (2003). fatty alcohol phosphates are subtype-selective ago-nists and antagonists of lysophosphatidic acid receptors. Mol. Pharmacol. 63(5): 1032-1042.

Liliom, K., tsukahara, t., tsukahara, r., Zelman-femiak, m., swiezewska, e. and tigyi, G. (2006). farnesyl phosphates are endogenous ligands of lysophosphatidic acid receptors: inhibi-tion of LPa GPcr and activation of PPars. BBA – Mol. Cell Biol. L. 1761(12): 1506-1514.

Kovács, e. and Liliom, K. (2008). sphingosylphosphorylcho-line as a novel calmodulin inhibitor. Biochem J. 410(2):427-37.

Kovacs, e., toth, J., vertessy, B.G. and Liliom, K. (2009). dis-sociation of calmodulin – target peptide complexes by the lipid mediator sphingosylphosphorylcholine: implications in cal-cium signaling. J. Biol. Chem. [epub ahead of print]

Judit Ováditirián, L., Hlavanda, e., oláh, J., Horváth, I., orosz, f., szabó, B., Kovács, J., szabad, J. and ovádi, J. (2003). tPPP/p25 pro-motes tubulin assemblies and blocks mitotic spindle forma-tion. Proc. natl. acad. sci. u.s.a. 100: 13976-13981.

vincze, o., tőkési, n., oláh, J., Hlavanda, e., Zotter, Á., Hor-váth, I., Lehotzky, a., tirián, L., medzihradszky, f.K., Kovács, J., orosz, f. and ovádi, J. (2006). tPPP proteins: members of a new family with distinct structures and functions. Biochemis-try 45: 13818-13826.

Hlavanda, e., Klement, É., Kókai, e., Kovács, J., vincze, o., tőkési, n., orosz, f., medzihradszky, f.K., dombrádi, v. and ovádi, J. (2007). Phosphorylation blocks the activity of tubulin polymerization-promoting protein (tPPP): identification of sites targeted by different kinases. J. Biol. Chem. 282: 29531-29539.

Preusser, m., Lehotzky, a., Budka, H., ovádi, J. and Kovács, G.G. (2007). tPPP/p25 in brain tumours: expression in non-neoplastic oligodendrocytes but not in oligodendroglioma cells. Acta Neuropathol. (Berl.), 113: 213-215.

Kovács, G.G., Gelpi, e., Lehotzky, a., Höftberger, r., erdei, a., Budka, H. and ovádi, J. (2007). The brain-specific protein tPPP/p25 in pathological protein deposits of neurodegenera-tive diseases. Acta Neuropathol. (Berl.). 113: 153-161.

Lehotzky, a., tőkési, n., Gonzalez-alvarez, I., merino, v., Ber-mejo, m., orosz, f., Lau, P., Kovács, G.G. and ovádi, J. (2008). Progress in the development of early diagnosis and a drug with unique pharmacology to improve cancer therapy. Philos. transact. a. math. Phys. eng. sci. 366: 3599-3617.

oláh, J., Klivényi, P., Gardián, G., vécsei, L., orosz, f., Kovacs, G.G., Westerhoff, H.v. and ovádi, J. (2008) Increased glucose metabolism and atP level in brain tissue of Huntington's dis-ease transgenic mice. feBs J. 275: 4740-4755.

ovádi, J. (2008). The tubulin polymerization promoting pro-tein, tPPP/p25. IuBmB Life 60: 637-642.

ovádi, J. and orosz, f. (2009). an unstructured protein with destructive potential: tPPP/p25 in neurodegeneration. Bioes-says 31(6):676-86.

László PatthyBányai, L. and Patthy, L. (1999). The ntr module: domains of netrins, secreted frizzled related proteins, and type I procolla-gen c-proteinase enhncer protein are homologous with tissue inhibitors of metalloproteinases. Protein Science 8: 1636-1642.

tordai, H., Bányai, L. and Patthy, L. (1999). The Pan mod-ule. The n-terminal domains of plasminogen and hepatocyte growth factor are homologous with the apple domains of the prekallikrein family and with a novel domain found in numer-ous nematode proteins. FEBS Letters 461: 63-67.

trexler, m., Bányai, L. and Patthy, L. (2000). The LccL-mod-ule, Eur. J. Biochem. 267(18): 5751-5757.

Patthy, L. (2000). The WIf module. Trends Biochem. Sci. 25: 13-14.

trexler, m., Bányai, L. and Patthy, L. (2001). a human protein containing multiple types of protease-inhibitory modules. Proc. Natl. Acad. Sci. U.S.A. 98: 3705-3709.

Liepinsh, e., trexler, m., Kaikkonen, a., Weigelt, J., Bányai, L., Patthy, L. and otting, G. (2001). nmr structure of the LccL domain and implications for dfna9 deafness disorder. EMBO J. 20: 5347-5353.

trexler, m., Bányai, L. and Patthy, L. (2002). distinct expres-sion pattern of two human proteins containing multiple types of protease-inhibitory modules. Biol. Chem. 383: 223–228.

Liepinsh, e., Bányai, L., Pintacuda, G., trexler, m., Patthy, L. and otting, G. (2003). nmr structure of the netrin-like do-main (ntr) of human type I procollagen c-proteinase en-hancer defines structural consensus of ntr domains and assesses potential proteinase inhibitory activity and ligand binding. J. Biol. Chem. 278: 25982-25989.

Liepinsh, e., Bányai, L., Patthy, L. and otting, G. (2006). nmr structure of the WIf domain of the human Wnt-inhibitory factor-1. J. Mol. Biol. 357: 942-950.

tress, m.L., martelli, P.L., frankish, a., reeves, G.a., Wesselink, J.J., Yeats, c., olason, P.L., albrecht, m., Hegyi, H., Giorgetti, a., raimondo, d., Lagarde, J., Laskowski, r.a., López, G., sadowski, m.I., Watson, J.d., fariselli, P., rossi, I., nagy, a., Kai, W., stør-ling, Z., orsini, m., assenov, Y., Blankenburg, H., Huthmacher, c., ramírez, f., schlicker, a., denoeud, f., Jones, P., Kerrien, s., orchard, s., antonarakis, s.e., reymond, a., Birney, e., Brunak, s., casadio, r., Guigo, r., Harrow, J., Hermjakob, H., Jones, d.t., Lengauer, t., orengo, c.a., Patthy, L., Thornton, J.m., tramon-tano, a. and valencia, a. (2007). The implications of alternative splicing in the encode protein complement. Proc. Natl. Acad. Sci. U.S.A. 104: 5495-5500.


113

ENZY

MO

LOG

Y

nagy, a., Hegyi, H., farkas, K., tordai, H., Kozma, e., Bányai, L. and Patthy, L. (2008). Identification and correction of ab-normal, incomplete and mispredicted proteins in public data-bases. BMC Bioinformatics 9: art. nr. 353.

nagy, a., Hegyi, H., farkas, K., tordai, H., Kozma, e., Bányai, L. and Patthy, L. (2008). Quality control of gene predictions, In: modern Genome annotation. The Biosapiens network, frishman, dmitrij, valencia, alfonso (eds.), springer 41-54.

Kondás, K., szláma, G., trexler, m. and Patthy, L. (2008). Both WfIKKn1 and WfIKKn2 have high affinity for growth and differentiation factors 8 and 11. J. Biol. Chem. 283: 23677-23684.

nagy, I., trexler, m. and Patthy, L.(2008). The second von Wil-lebrand type a domain of cochlin has high affinity for type I, type II and type Iv collagens. FEBS Letters 582: 4003-4007.

Harrow, J., nagy, a., reymond, a., alioto, t., Patthy, L., an-tonarakis, s.e. and Guigó, r. (2009). Identifying protein-cod-ing genes in genomic sequences. Genome Biol. 10(1): art. nr. 201.

László Polgárrawlings, n.d., Polgár, L. and Barrett, a.J. (1991). a new fam-ily of serine-type peptidases related to prolyl oligopeptidase. Biochem. J. 279: 907-911.

fülöp, v., Böcskei, Z. and Polgár, L. (1998). Prolyl oligopepti-dase: an unusual β-propeller domain regulates proteolysis. Cell 94: 161-170.

szeltner, Z., renner, v. and Polgár, L. (2000). substrate- and pH-dependent contribution of oxyanion binding site to the catalysis of prolyl oligopeptidase, a paradigm of the serine oli-gopeptidase family. Protein Sci. 9: 353-360.

fülöp, v., szeltner, Z., renner, v. and Polgár, L. (2001). struc-tures of prolyl oligopeptidase substrate/inhibitor complexes. use of inhibitor binding for titration of the catalytic histidine residue. J. Biol. Chem. 276: 1262-1266.

szeltner, Z., rea, d., Juhász, t., renner, v., mucsi, Z., orosz, G., fülöp, v. and Polgár, L. (2002). substrate-dependent com-petency of the catalytic triad of prolyl oligopeptidase. J. Biol. Chem. 277: 44597-44605.

szeltner, Z., rea, d., renner, v., Juhász, t., fülöp, v. and Polgár, L. (2004). concerted structural changes in the peptidase and the propeller domains of prolyl oligopeptidase are required for substrate binding. J. Mol. Biol. 340: 627-637.

Kiss, a.L., szeltner, Z., fülöp, v. and Polgár, L. (2004). His507 of acylaminoacyl peptidase stabilizes the active site conforma-tion, not the catalytic intermediate. FEBS Lett. 571: 17-20.

Juhász, t., szeltner, Z., fülöp, v. and Polgár, L. (2005). un-closed β-propellers display stable structures: Implications for substrate access to the active site of prolyl oligopeptidase. J. Mol. Biol. 346: 907-917.

fuxreiter, m., magyar, c., Juhász, t., szeltner, Z., Polgár, L. and simon, I. (2005). flexibility of prolyl oligopeptidase: molecular dynamics and molecular framework analysis of the potential substrate pathways. Proteins 60: 504-512.

Kiss, a.L., Hornung, B., rádi, K., Gengeliczki, Z., sztáray, B., Juhász, t., szeltner, Z., Harmat, v. and Polgár, L. (2007). The acylaminoacyl peptidase from aeropyrum pernix K1 thought to be an exopeptidase displays endopeptidase activity. J. Mol. Biol. 368: 509-520.

Kiss, a.L., Palló, a., náray-szabó, G., Harmat, v. and Polgár, L. (2008). structural and kinetic contributions of oxyanionm binding site to the catalytic activity of acylaminoacyl pepti-dase. J. Structural Biology 162: 312-323.

szeltner, Z., Kiss, a.L., domokos, K., Harmat, v., náray-szabó, G. and Polgár, L. (2009). characterization of a novel acylami-noacyl peptidase with hexameric structure and endopeptidase activity. Biochim. Biophys. Acta 1794: 1204-1210.

István Simonsimon, I. and cserző, m. (1990). rapid evolution of the amino acid composition of proteins. Trends Biochem. Sci. 15: 135-136.

dosztányi, Z., fiser, a. and simon, I. (1997). stabilization cent-ers in proteins: identification, characterization and predic-tions. J. Mol. Biol. 272: 597-612.

cserző, m., Wallin, e., simon, I., von Heijne, G. and elofs-son, a. (1997). Prediction of transmembrane alpha-helices in prokaryotic membrane proteins: the dense alignment surface method. Protein Eng. 10: 673-676.

tusnády, G.e. and simon, I. (1998). Principles governing ami-no acid composition of integral membrane proteins: applica-tion to topology prediction. J. Mol. Biol. 283: 489-506.

tompa, P., tusnády, G.e., cserző, m. and simon, I. (2001). Prion protein: evolution caught en route. Proc. natl. acad. sci. u.s.a. 98: 4431-4436.

tusnády, G.e. and simon, I. (2001). The HmmtoP transmem-brane topology prediction server. Bioinformatics 17: 849-850.

tusnády, G.e., dosztányi, Z. and simon, I. (2005). PdB_tm: selection and membrane localization of transmembrane proteins in the protein data bank. Nucleic Acids Res. 33: d275-d278.

dosztányi, Z., csizmók, v., tompa, P. and simon, I. (2005). The pairwise energy content estimated from amino acid composi-tion discriminates between folded and intrinsically unstruc-tured proteins. J. Mol. Biol. 347: 827-839.

fuxreiter, m., tompa, P. and simon, I. (2007). Local structural disorder imparts plasticity on linear motifs. Bioinformatics 23: 950-956.

fuxreiter, m., tompa, P., simon, I., uversky, v.n., Hansen, J.c. and asturias, f.J. (2008). malleable machines take shape in eukaryotic transcriptional regulation. Nature Chemical Biology 4: 728-737.


114

ENZY

MO

LOG

Y


Gergely Szakácsszakács, G., annereau, J.P., Lababidi, s., shankavaram, u., arciello, a., Bussey, K.J., reinhold, W., Guo, Y.P., Kruh, G.d., reimers, m., Weinstein, J.n. and Gottesman, m.m. (2004). Predicting drug sensitivity and resistance: Profil-ing aBc transporter genes in cancer cells. cancer cell 6(2): 129-137.

Ludwig, J.a., szakács, G., martin, s.e., chu B.f., cardarelli, c., sauna, Z.e., caplen, n.J., fales, H.m., ambudkar, s.v., Wein-stein, J.n. and Gottesman, m.m. (2006). selective toxicity of nsc73306 in mdr1-positive cells as a new strategy to circum-vent multidrug resistance in cancer. Cancer Res. 66(9):4808-4815.

szakács, G., Paterson, J.K., Ludwig, J.a., Booth-Genthe, c. and Gottesman, m.m. (2006). targeting multidrug resistance in cancer. nat. rev. drug discov. 5(3): 219-234.

Gottesman, m.m., Ludwig, J., Xia, d. and szakács, G. (2006) defeating drug resistance in cancer. Discov. Med. 6(31): 18-23.

sarkadi, B., Homolya, L., szakács, G. and váradi, a. (2006). Physiological role of the human multidrug resistance aBc transporters: The chemoimmunity network. Phys. Rev. 86(4): 1179-1236.

Paterson, J.K., shukla, s., Black, c.m., tachiwada, t., Garfield, s., Wincovitch, s., ernst, d.n., agadir, a., Li, X., ambudkar, s.v., szakács, G., akiyama, s.I. and Gottesman, m.m. (2007). Human aBcB6 localizes to both the outer mitochondrial membrane and the plasma membrane. Biochemistry 46(33): 9443-9452.

Péter Tompatompa, P., tusnády, G. e., cserző, m. and simon, I. (2001). Prion protein: evolution caught en route. Proc. natl. acad. sci. u.s.a. 98: 4431-4436.

tompa, P. (2002). Intrinsically unstructured proteins. trends Biochem. sci. 27: 527-533.

tompa, P. (2003). Intrinsically unstructured proteins evolve by repeat expansion. Bioessays 25: 847-855.

fuxreiter, m., simon, I., friedrich, P. and tompa, P. (2004). Preformed structural elements feature in partner recognition by intrinsically unstructured proteins. J. mol. Biol. 338: 1015-1026.

tompa, P. and csermely, P. (2004). The role of structural dis-order in the function of rna- and protein chaperones. faseB J. 18: 1169-1175.

tompa, P. (2005). The interplay between structure and func-tion in intrinsically unstructured proteins. feBs Lett. 579: 3346-3354.

tompa, P., szász, cs. and Buday, L. (2005). structural disorder throws new light on moonlighting. trends Biochem. sci. 30: 484-489.

csizmók, v., szőllősi, e., friedrich, P. and tompa, P. (2006). a novel 2d electrophoresis technique for the identification of intrinsically unstructured proteins. mol. cell. Proteomics 5: 265-273.

tompa, P. and fuxreiter, m. (2008). fuzzy complexes: poly-morphism and structural disorder in protein-protein interac-tions. trends Biochem. sci. 33: 2-8.

Mária Vasszilágyi, a.n. and vas, m. (1998) sequential domain folding of pig muscle 3-phosphoglycerate kinase: kinetic analysis of reac-tivation. Folding and Design 3: 565-575

szilágyi, a.n., Ghosh, m. Garman, e. and vas, m. (2001) a 1.8 Å resolution structure of pig muscle 3-phosphoglycerate kinase with bound mgadP and 3-phosphoglycerate in open conformation: new insight into the role of the nucleotide in domain closure. J. Mol. Biol. 306: 499-511.

merli, a., szilágyi, a.n., flachner, B., rossi,G.L. and vas, m. (2002) nucleotide binding to pig muscle 3-phosphoglycerate kinase in the crystal and in solution: relationship between substrate antagonism and interdomain communication. Bio-chemistry 41: 111-119.

Kovári, Z., flachner, B., náray-szabó, G. and vas, m. (2002) crystallographic and thiol-reactivity studies on the complex of pig muscle phosphoglycerate kinase with atP-analogues: cor-relation between nucleotide binding mode and helix flexibility. Biochemistry 41: 8796-8806.

flachner, B., Kovári, Z., varga, a., Gugolya, Z., vonderviszt, f., náray-szabó, G. and vas, m. (2004) role of phosphate chain mobility of mgatP in completing the 3-phosphoglyc-erate kinase catalytic site: Binding, kinetic and crystallo-graphic studies with atP and mgatP. Biochemistry 43: 3436-3449.

varga, a., flachner, B., Gráczer, É., osváth, s., szilágyi, a.n. and vas, m. (2005) correlation between conformational sta-bility of the ternary enzyme-substrate complex and domain closure of 3-Phosphoglycerate Kinase. FEBS J. 272: 1867-1885.

flachner, B., varga, a., szabó, J., Barna, L., Hajdú I., Gyimesi, G., Závodszky, P. and vas, m. (2005) substrate-assisted move-ment of the catalytic Lys 215 during domain closure: site-directed mutagenesis studies of Human 3-Phosphoglycerate Kinase. Biochemistry 44: 16853-865.

varga, a., flachner, B., Konarev, P., Gráczer, É., szabó, J., sver-gun, d., Závodszky, P. and vas, m. (2006) substrate-Induced double sided H-bond network as a means of domain closure in 3-Phosphoglycerate Kinase. FEBS Lett. 260: 2698-2706.

Gráczer, É., varga, a., Hajdú, I., melnik, B., szilágyi, a., semisotnov, G., Závodszky, P. and vas, m. (2007) rates of unfolding, rather than refolding, determine thermal stabilities of thermophilic, mesophilic and Psychrotroph-ic 3-Isopropylmalate dehydrogenases. Biochemistry 46: 11536-11549.

115

ENZY

MO

LOG

Y


varga, a., szabó, J., flachner, B., roy, B., Konarev, P., svergun, d., Závodszky, P., Périgaud, c., Barman, t., Lionne, c. and vas, m. (2008) Interaction of Human 3-Phosphoglycerate Kinase with L-adP, the mirror Image of d-adP. Biochem. Biophys. Res. Comm. 366: 994-1000.

szabó, J., varga, a., flachner, B., Konarev, P.v., svergun, d.I, Závodszky, P. and vas, m. (2008) role of side-chains in the operation of the main molecular Hinge of 3-Phosphoglycerate Kinase. FEBS Lett. 582: 1335-1340.

szabó, J., varga, a., flachner, B., Konarev, P.v., svergun, d.I, Závodszky, P. and vas, m. (2008) communication between the nucleotide site and the main molecular Hinge of Phos-phoglycerate Kinase Biochemistry 47: 6735-6744.

Gráczer, É., varga, a., melnik, B., semisotnov, G., Závodszky, P. and vas, m. (2009) symmetrical refolding of Protein domains and subunits: example of the dimeric two-domain 3-Isopro-pylmalate dehydrogenases. Biochemistry 48: 1153-1154.

András VáradiBakos, É., Hegedűs, t., Holló, Z., Welker, e., tusnády, G.e., Za-man, G.J., flens, m.J., váradi, a. and sarkadi, B. (1996). mem-brane topology and glycosylation of the human multidrug re-sistance-associated protein. J. Biol. Chem. 271: 12322-12326.

tusnády, G.e., Bakos, É., váradi, a. and sarkadi, B. (1997). membrane topology distinguishes a subfamily of the atP-binding cassette (aBc) transporters. FEBS Lett. 402: 1-3.

Bakos, É., evers, r., szakács, G., tusnády, G.e., Welker, e., szabó, K., de Haas, m., van deemter, L., Borst, P., váradi, a. and sarkadi, B. (1998). functional multidrug resistance pro-tein (mrP1) lacking the n-terminal transmembrane domain. J. Biol. Chem. 273: 32167-32175.

Bakos, É., evers, r., calenda, G., tusnády, G.e., szakács, G., váradi, a. and sarkadi, B. (2000). characterization of the ami-no-terminal regions in the human multidrug resistance pro-tein (mrP1). J. Cell Sci. 113(Pt 2): 4451-4461.

Iliás, a., urbán, Z., seidl, t.L., Le saux, o., sinkó, e., Boyd, c.d., sarkadi, B. and váradi, a. (2002). Loss of atP-dependent trans-port activity in pseudoxanthoma elasticum-associated mutants of human aBcc6 (mrP6). J. Biol. Chem. 277: 16860-16867.

sinkó, e., Iliás, a., ujhelly, o., Homolya, L., scheffer, G.L., Bergen, a.a., sarkadi, B. and váradi, a. (2003). subcellular lo-calization and n-glycosylation of human aBcc6, expressed in mdcKII cells. Biochem. Biophys. Res. Commun. 308: 263-269.

szentpétery, Z., Kern, a., Liliom, K., sarkadi, B., váradi, a. and Bakos, e. (2004). The role of the conserved glycines of atP-binding cassette signature motifs of mrP1 in the com-munication between the substrate-binding site and the cata-lytic centers. J. Biol. Chem. 279: 41670-41678.

szentpétery, Z., sarkadi, B., Bakos, e. and váradi, a. (2004). functional studies on the mrP1 multidrug transporter: char-acterization of aBc-signature mutant variants. Anticancer Res. 24: 449-455.

Kern, a., szentpétery, Z., Liliom, K., Bakos, e., sarkadi, B. and váradi, a. (2004). nucleotides and transported substrates modulate different steps of the atPase catalytic cycle of mrP1 multidrug transporter. Biochem. J. 380: 549-560.

arányi, t., ratajewski, m., Bardóczy, v., Pulaski, L., Bors, a., tordai, a. and váradi, a. (2005). Identification of a dna meth-ylation-dependent activator sequence in the pseudoxanthoma elasticum gene, aBcc6. J. Biol. Chem. 280: 18643-18650.

sarkadi, B., Homolya, L., szakács, G. and váradi, a. (2006). Human multidrug resistance aBcB and aBcG transporters: participation in a chemoimmunity defense system. Physiol. Rev. 86: 1179-236.

symmons, o., váradi, a. and arányi, t. (2008). How segmen-tal duplications shape our genome: recent evolution of aBcc6 and PKd1 mendelian disease genes. Mol. Biol. Evol. 25: 2601-2613.

szeri, f., Iliás, a., Pomozi, v., robinow, s., Bakos, e. and vára-di, a. (2009). The high turnover drosophila multidrug resist-ance-associated protein shares the biochemical features of its human orthologues. BBA-Biomembranes 1788: 402-409.

fülöp, K., Barna, L., symmons, o., Závodszky, P. and vára-di, a. (2009). clustering of disease-causing mutations on the domain-domain interfaces of aBcc6. Biochem. Biophys. Res. Commun. 379: 706-709.

Beáta G. VértessyBarabás, o., rumlová, m., erdei, a., Pongrácz, v., Pichová, I. and vértessy, B.G. (2003). dutPase and nucleocapsid polypep-tides of the mason-Pfizer monkey virus form a fusion protein in the virion with homotrimeric organization and low cata-lytic efficiency. J. Biol. Chem. 278(40): 38803-38812.

mustafi, d., Békési, a., vértessy, B.G. and makinen, m.W. (2003). catalytic and structural role of the metal ion in dutP pyrophosphatase. Proc. Natl. Acad. Sci. U.S.A. 100(10):5670-5675.

Barabás, o., Pongrácz, v., Kővári, J., Wilmanns, m. and vértessy, B.G. (2004). structural insights into the catalytic mechanism of phosphate ester hydrolysis by dutPase. J. Biol. Chem. 279(41): 42907-42915.

Békési, a., Zagyva, I., Hunyadi-Gulyás, e., Pongrácz, v., Kovári, J., nagy, a.o., erdei, a., medzihradszky, K.f. and vértessy, B.G. (2004). developmental regulation of dutPase in Drosophila melanogaster. J. Biol. Chem. 279(21): 22362-22370.

dubrovay, Z., Gáspári, Z., Hunyadi-Gulyás, É., medzihrad-szky, K.f., Perczel, a. and vértessy, B.G. (2004). multidimen-sional nmr identifies the conformational shift essential for catalytic competence in the 60-kda Drosophila melanogaster dutPase trimer. J. Biol. Chem. 279(17): 17945-17950.

Lari, s.u., chen, c.Y., vértessy, B.G., morré, J. and Bennett, s.e. (2006). Quantitative determination of uracil residues in Escherichia coli dna: contribution of ung, dug, and dut genes to uracil avoidance. DNA Repair (amst). 5(12): 1407-1420.

116

ENZY

MO

LOG

Y

tóth, J., varga, B. Kovács, m., málnási-csizmadia, a. and vértessy, B.G. (2007). Kinetic mechanism of human dut-Pase, an essential nucleotide pyrophosphatase enzyme. J. Biol. Chem. 282(46): 33572-33582.

varga, B., Barabás, o., Kovári, J., tóth, J., Hunyadi-Gulyás, É., Klement, É., medzihradszky, K.f., tölgyesi, f., fidy, J. and vértessy, B.G. (2007). active site closure facilitates juxtapo-sition of reactant atoms for initiation of catalysis by human dutPase. FEBS Lett. 581(24): 4783-4788.

Békési, a., Pukáncsik, m., muha v, Zagyva, I., Leveles, I., Hunyadi-Gulyás, e., Klement, e., medzihradszky, K.f., Kele, Z., erdei, a., felföldi, f., Kónya, e. and vértessy, B.G. (2007). a novel fruitfly protein under developmental control de-grades uracil-dna. Biochem. Biophys. Res. Commun. 355(3): 643-648.

németh-Pongrácz, v., Barabás, o., fuxreiter, m., simon, I., Pi-chová, I., rumlová, m., Zábranská, H., svergun, d., Petoukhov, m., Harmat, v., Klement, É., Hunyadi-Gulyás, É., medzihrad-szky, K.f., Kónya, e. and vértessy, B.G. (2007). flexible seg-ments modulate co-folding of dutPase and nucleocapsid pro-teins. Nucleic Acids. Res. 35(2): 495-505.

varga, B., Barabás, o., takács, e., nagy, e., nagy, P. and vértessy, B.G. (2008). active site of mycobacterial dutPase: structural characteristics and a built-in sensor. Biochem. Bio-phys. Res. Commun. 373(1): 8-13.

vértessy, B.G. and tóth, J. (2009). Keeping uracil out of dna: physiological role, structure and catalytic mechanism of dut-Pases. Acc. Chem. Res. 42(1): 97-106.

muha, v., Zagyva, I., venkei, Z., szabad, J. and vértessy, B.G. (2009). nuclear localization signal-dependent and -independ-ent movements of Drosophila melanogaster dutPase isoforms during nuclear cleavage. Biochem. Biophys. Res. Commun. 381(2): 271-275.

Péter ZávodszkyZávodszky, P., Kardos, J., svingor, Á. and Petsko, G.a. (1998). adjustment of conformational flexibility is a key event in the thermal adaptation of proteins. Proc. Natl. Acad. Sci. U.S.A. 95: 7406-7411.

szilágyi, a. and Závodszky, P. (2000). structural differences between mesophilic, moderately thermophilic and extremely thermophilic protein subunits: results of a comprehensive sur-vey. Structure 8: 493-504.

Gál, P., végh, B., Závodszky, P. and vonderviszt, f. (2006). ex-port signals. Nat. Biotechnol. 24: 900-901.

Kamondi, s., szilágyi, a., Barna, L. and Závodszky, P. (2008). engineering the thermostability of a tIm-barrel enzyme by rational family shuffling. Biochem. Biophys. Res. Commun. 374: 725-730.

Hajdú, I., Bothe, c., szilágyi, a., Kardos, J., Gál, P. and Závod-szky, P. (2008). adjustment of conformational flexibility of glyceraldehyde-3-phosphate dehydrogenase as a means of thermal adaptation and allosteric regulation. Eur. Biophys. J. 37: 1139-1144.

szilágyi, a., Györffy, d. and Závodszky, P. (2008). The twilight zone between protein order and disorder. Biophys. J. 95: 1612-1626.

Hajdú, I., szilágyi, a., Kardos, J. and Závodszky, P. (2009). a link between hinge-bending domain motions and the tem-perature dependence of catalysis in IPmdH. Biophys. J. 96: 5003-5012.

Péter Gálambrus, G., Gál, P., Kojima, m., szilágyi, K., Balczer, J., antal, J., Gráf, L., Laich, a., moffat, B.e., schwaeble, W., sim, r.B. and Závodszky, P. (2003). natural substrates and inhibitors of mannan-binding lectin-associated serine protease 1 and 2: a study on recombinant catalytic fragments. J. Immunol. 170: 1374-1382.

Harmat, v., Gál, P., Kardos, J., szilágyi, K., ambrus, G., végh, B., náray-szabó, G. and Závodszky, P. (2004). The structure of masP-2 reveals that nearly identical substrate specificities of c1s and masP-2 are realized through different sets of enzyme-substrate interactions. J. Mol. Biol. 342: 1533-1546.

Gál, P., Harmat, v., Kocsis, a., Bián, t., Barna, L., ambrus, G., végh, B., Balczer, J., sim, r.B., náray-szabó, G. and Závodszky, P. (2005). a true autoactivating enzyme. structural insight into mannose-binding lectin-associated serine protease-2 activa-tion. J. Biol. Chem. 280: 33435-33444.

Beinrohr, L., Harmat, v., dobó, J., Lőrincz, Zs., Gál, P. and Závodszky, P. (2007). c1 inhibitor serpin domain structure re-veals the likely mechanism of heparin potentiation and confor-mational disease. J. Biol. Chem. 282: 21100-21109. doi:10.1074/jbc.m700841200.

Kardos, J., Harmat, v., Palló, a., Barabás, o., szilágyi K., Gráf, L., náray-szabó, G., Goto, Y., Závodszky, P. and Gál, P. (2008). revisiting the mechanism of the autoactivation of the comple-ment protease c1r in the c1 complex: structure of the active catalytic region of c1r. Mol. Immunol. 45: 1752-1760.

Beinrohr, L., dobó, J., Závodszky, P. and Gál, P. (2008). c1, mBL-masPs and c1-inhibitor: novel approaches for target-ing complement-mediated inflammation. Trends Mol. Med. 14: 511-521.

megyeri, m., makó, v., Beinrohr, L., doleschall, Z., Prohászka, Z., cervenak, L., Závodszky, P. and Gál, P. (2009) complement protease masP-1 activates human endothelial cells: Par4 ac-tivation is a link between complement and endothelial func-tion J. Immunol. 183: 3409-3416.


117

GEN

ETIC

S

Institute of GeneticsH-6726 Szeged, Temesvári krt. 62.H-6701 Szeged, P.O. Box 521, Hungary

Brc

118

GEN

ETIC

S

In multicellular organisms, the division of a single totipotent cell, the fertilized zygote produces all the differ-ent types of cells with different shapes, structures and functions. The basis of this diversity is the differential gene expression of each cell type. Once the gene expression pattern is established, it has to be transmitted to the next cell generation of the lineage. This is provided by an epigenetic layer of regulation of gene expression. By combining genetic, transgenic and site-directed mutagenesis approaches we study the epigenetic regulation of the homeotic bithorax complex of the fruit fly, Drosophila melanogaster.

epIGeneTIC Gene ReGUlaTIon bY HIGHeR oRDeR CHRomaTIn sTRUCTURe

Henrik GYURKOvICS / Principal Investigator, Group Leader

Izabella BAJUSZ / staff scientistAnita KISS / technician

The homeotic bithorax gene-complex furnishes an excellent model system for studying the maintenance of gene activity. The Drosophila homeotic genes are regulated by complex segment-specific cis-regulatory regions, the activity patterns of which are set up in the early embryo and have to be maintained throughout later development. Homeotic genes control the mor-phogenetic differentiation of body segments; their misexpression produces easily detectable transforma-tions of different segments. Genes that are involved in maintaining the expression pattern of homeotic genes fall into two antagonistic groups: the Polycomb- and the trithorax-group. Polycomb-group (PcG) genes are required for maintaining the inactive state, whereas the trithorax-group (trxG) of genes is needed for the ac-tivity of homeotic genes. Proteins encoded by the two groups have specific target regions, called Polycomb response elements (Pres) and trithorax response elements (tres), respectively. Homologs of Drosophi-la PcG and trXG proteins have similar functions in other eukaryotes. our group studies three different aspects of this epigenetic maintenance system.

first, we would like to identify unknown players of this system. We use transgenic lines containing differ-ent fragments from a Pre (the iab-7 Pre). These lines

show pairing-sensitive silencing of the miniwhite re-porter gene: the eye color of transgenic flies is lighter in homozygous than in heterozygous conditions. This si-lencing effect is weakened by the introduction of a PcG mutation, whereas it is strengthened in a trxG mutant background. Based on this phenomenon, we screened over a million flies and identified previously unknown PcG and trxG genes, whose nature we analyze by ge-netic means. We found that different fragments of the iab-7 Pre respond to different sets of PcG and trxG mutations. to understand the contribution of each sub-region, we have generated transgenic lines carry-ing different fragments of the Pre. We use XchiP and in vivo footprinting in order to identify the positions of various PcG proteins on these fragments.

second, we would like to understand the role of the repressing elements, Pres in the open/active chroma-tin domains. We approach this question by manipu-lating the number of potentially interacting Pres (us-ing internal deletions) and the dosage of different PcG genes (using mutant versions) simultaneously. This approach is based on our previous observation that Pres do interact even in the open domains, suggest-ing a function for repressing elements different from that they serve for in closed chromatin domains.

119

GEN

ETIC

S

Henrik GYURKOvICS EPIGENETIC GENE REGULATION BY HIGHER ORDER CHROMATIN STRUCTURE

Third, using a site-directed mutagenesis approach, we would like to unravel the complex mechanism whereby cis-regulatory elements, such as Pres and enhancers can specifically interact with their proper target promoter in a situation where different promot-ers are located very far and nearly equidistantly from these elements.

In addition to these investigations, we are currently exploring the possibility of using the differentiation of Drosophila blood cell types as a model system for studying epigenetic regulation. This possibility is

suggested by the finding that some mutations in PcG genes, including some isolated by us, result in altered differentiation pattern of blood cells.

These studies are done in collaboration with the laboratory of francois Karch (university of Geneva, Geneva, switzerland) and the laboratories of István andó, József mihály and László sipos (Brc)


120

GEN

ETIC

S

Those who study transcriptional regulation in eukaryotes have come to realize that it’s all about chromatin structure. The biochemistry of histone modification is the topic of the day, but it is not clear how these modifica-tions alter nucleosome packing, or how the spatial limits of the modifications are defined. One of the primary systems for study is the long-term and long-range repression imposed by proteins of the Polycomb-Group. We are dissecting the DNA-sites required for Polycomb-Group repression in the natural context of the chromosome. Our emphasis is not on naming the biochemical players, but on defining the logic of how these sites are switched to repressing mode and how repression is imposed on neighboring sequences.

In sITU DIsseCTIon of CIs-aCTInG elemenTs In THe bITHoRax-Complex of DRoSopHiLA(CHRomaTIn sTRUCTURe anD Gene expRessIon ReseaRCH GRoUp)

László SIPOS / Principal Investigator, Group Leader

Gabriella KOZMA / staff scientistAnikó BERENTE / technicianAnna REHáK / technician

Figure 1. Targeting of the Gal4VP16 reporter gene into the bxd PRE re-sults in a segmentally restricted expression pattern of UASeGFP in larval stage.

many genes that respond to developmental cues must maintain their responding states long after the cues have disappeared. In Drosophila, the maintenance of repressed states of many developmentally important genes is accomplished by a protein called PoLYcomB and by interacting proteins of the Polycomb-Group. Homologous proteins are found in mammals, where they appear to have analogous functions. Long-term

repression by the Polycomb-Group is accompanied by a change in chromosome structure making it less ac-cessible to transcription factors and polymerases.

Figure 2. Removal of about 700 bp region in the bxd PRE results in a significant change in the chromatin structure of the bithorax-complex. The resulting ectopic gene activation leads to the partial transformation of wing towards haltera (compare left and right panels).

The Polycomb-Group proteins act through dna-sites called Polycomb Response Elements (PREs). The best examples of such sites are in the Drosophila bitho-rax-complex, a cluster of genes that control segmental differentiation. although one of these sites (bxd Pre) has been localized to a segment of about 1500 base

121

GEN

ETIC

S

László SIPOS IN SITU DISSECTION OF CIS-ACTING ELEMENTS IN THE BITHORAx-COMPLEx OF DROSOPHILA

pairs, it is not yet clear how it regulates the genes of the bithorax-complex, because there are no mutations that surgically target this Pre. our goal is to use gene con-version to generate a series of small deletions in and around the bxd Pre and to study the consequences for the genes of the bithorax-complex. We hope to learn whether the Pre is required for activation as well as repression, how the element receives its initial cue, and whether it is redundant or cooperative with other such elements in the bithorax-complex. The deletions may

permit a functional subdivision of the element, and further conversion experiments will target binding sites for particular Polycomb-Group proteins.

a second Polycomb response element (mcp Pre) in the bithorax-complex is also being dissected by gene conversion and its properties compared.


122

GEN

ETIC

S

The regulation of growth cone actin dynamics is a critical aspect of axonal growth control. However, the proteins that are directly involved in the regulation of actin dynamics in developing neurons have so far not been clearly identified. Recently, we have shown that the Drosophila formin DAAM plays a critical role in axonal morphogen-esis by promoting filopodia formation at the growth cone periphery. Moreover, we have collected several lines of evidence suggesting that the function of DAAM in developing neurons has been conserved during evolution. Cur-rently, we are using genetical, cell biological and biochemical approaches to understand the molecular mechanisms of how formin proteins of the DAAM family contribute to axonal growth regulation in Drosophila and mouse.

DRoSopHiLA DevelopmenTal bIoloGY

József MIHáLY / Principal Investigator, Group Leader

Tibor KALMáR / staff scientistRita GOMBOS / staff scientistTamás MATUSEK / staff scientistCsilla PATAKI / staff scientistAnita GEDAI / Phd studentImre MOLNáR / Phd studentAnikó BERENTE / technicianSzilvia BOZSó / technician

Formin proteins of the DAAM subfamily play a role in axonal growth

In the developing nervous system, axons are guid-ed to their targets by highly motile growth cones lo-cated at their distal tips. directed growth cone mo-tility in response to extracellular cues is produced by the coordinated regulation of peripheral f-actin and central microtubule networks. The peripheral f-actin is organized into long bundled actin fila-ments underlying the finger-like filopodia and dif-fuse networks of shorter actin filaments contained in the veil-like lamellipodia. Key regulators of actin dy-namics are the so-called nucleation factors, such as the arp2/3 complex and formins, which use different mechanisms to seed new actin filaments. These types of actin assembly factors are well-defined in migrat-ing cells; in growth cones, however, prior to our work the essential nucleators have not been unambigu-ously identified.

By using a number of different model systems, our group has recently provided compelling evi-dence that formin proteins of the daam subfamily play a pivotal role during axonal growth regulation. We found that the Drosophila daam protein is

highly enriched in the embryonic neurites, in par-ticular, the protein localizes in dots throughout the f-actin rich growth cones including the filopodia. the loss of dDAAM function in embryonic neurons results in reduced neurite densities and abnormal axonal pathfinding. In addition, we have shown that filopodia are shorter and reduced in number in dDAAM mutant primary neurons. the constitu-tively activated versions of ddaam promote filo-podia and neurite formation, i.e. cause phenotypes opposite to the loss-of-function analyses in all as-says we used. Because the fH2 domain of ddaam is a potent actin nucleation factor in vitro, these ob-servations together strongly suggest that ddaam plays a major role in the regulation of actin assembly during axonal growth. consistent with the fact that axonal growth regulation is an evolutionary highly conserved process, we have shown that ddaam promotes the formation of neurite-like protrusions when expressed in mouse P19 cells, whereas murine daam1 can functionally replace dDAAM in Dro-sophila. thus, our data strongly suggest that the regulation of actin assembly during axon growth is very likely to signify an evolutionary conserved daam function.

123

GEN

ETIC

S

József MIHáLY DRoSopHiLA DevelopmenTal bIoloGY

The localization of dDAAM in the Drosophila embryonic CNS (B,C) and in growth cones of cultured neurons (E-F).

our main goal in the future is to gain deeper in-sights into the molecular mechanism whereby daam family formins control axonal growth regulation. We want to understand how ddaam activity is regulated

and how it is connected to the axon guidance cues, and we aim to study the molecular mechanism of ddaam-dependent filopodia formation. Given that certain developmental disorders, accidental injuries and neurodegenerative diseases often result in severe axonal growth defects or axonal injuries, our studies on a key regulator of axonal growth may help the de-velopment of more efficient therapeutical tools.


124

GEN

ETIC

S

Left-right asymmetry has a fundamental role in the morphogenesis of internal organs. Disturbance of left-right signaling results in severe organ malformations. Male genitalia in Drosophila display a genetically determined directional rotation which can be used to study the molecular aspects of left-right asymmetry. Our research focuses on the identification and characterization of genes involved in this process.

Géza áDáM / Principal Investigator, Group Leader

János GAUSZ / senior scientistEdit GYáNYI / technician

Establishment of left-right asymmetry in Drosophila

Left-right (Lr) asymmetry arises as the conse-quence of genetically different regulation of the de-velopmental processes in the left and the right side of the body. In higher animals, Lr asymmetry is manifested predominantly in the asymmetric posi-tions and shapes of the internal organs. In humans, defects in laterality have a significant clinical impact causing birth defects, by which more than 1% of live births are affected. The heart, the most sensitive organ regarding Lr asymmetry signals, frequently responds with morphological malformations to mild defects in Lr asymmetry cues. In vertebrates, much of the mo-lecular details of the Lr patterning pathway has been uncovered. However, molecular aspects determin-ing situs choice and detailed cellular mechanisms of Lr asymmetric development remain to be explored. In vertebrates, the interpretation of the experimental data is difficult, because it is hard to perform quick and extensive genetic analyses for many genes. Drosophila offers a unique opportunity to overcome this problem, because it enables the performance of genome-wide genetic investigations.

certain organs of Drosophila (the gut, the male genitalia) undergo typical Lr asymmetric develop-

ment. The dextral rotation of the male genital plate is especially suitable for studying the Lr asymmetric processes in adults. during pupal development, the male genital plate undergoes a full dextral rotation as viewed from the posterior. The consequence of this ro-tation is that the spermiduct, which is tightly attached to the genital plate, performs a rightward looping around the hindgut. Investigating this phenotype, we isolated mutations of the Myo31DF gene which reverse the direction of the genital plate rotation as indicated by the sinistral looping of the spermiduct, causing a si-tus inversus phenotype. The Myo31DF gene encodes a type Id unconventional myosin protein. Loss of func-tion mutations of Myo31DF reverse not only the direc-tion of the genital plate rotation but also the looping of the gut.

lefT-RIGHT asYmmeTRIC DevelopemenT In DRoSopHiLA(DRoSopHiLA lefT-RIGHT asYmmeTRY GRoUp)

125

GEN

ETIC

S

Géza áDáM LEFT-RIGHT ASYMMETRIC DEVELOPEMENT IN DROSOPHILA

Myo31DF is one of the two sequenced genes that are known in the whole animal kingdom to cause a situs inversus phenotype with a 100% penetrance when mu-tated. The other one is inversin which is found exclu-sively in vertebrates. Though their primary molecular structures seem to be different, their size is approxi-mately the same, they contain 2 IQ motifs each, both can bind beta-catenin and they are expressed bilater-ally. for genitalia rotation, Myo31DF is required only in the abdominal segment 8 (a8) in the male genital disc. since the entire male genitalia develops from the abdominal segment 9 (a9), we concluded that a8 functions as a Lr signaling center for a9. to under-stand this function of a8, we are currently performing a genetic screen based on rna interference.

The Calpain B gene in Drosophila

calpains comprise a large family of calcium-acti-vated intracellular thiol proteases, which are involved in calcium-regulated events in the cell, catalyzing the proteolytic cleavage of numerous cytosolic, cytoskel-etal, and membrane-associated proteins. The activity of calpain proteases is regulated by the intracellular calci-um concentration and they are associated with multiple functions such as migration, adhesion and apoptosis.

Beside their physiological function, calpains are also involved in pathological processes. dysfunction of calpain3 in humans leads to limb girdle muscular dystrophy 2a, and mutations in the gene coding for calpain10 have been shown to correlate with an in-creased risk for type 2 diabetes. In contrast to mam-mals, where 15 calpain genes have been identified until now, the Drosophila genome contains only four genes encoding calpain-related sequences. However, out of these genes only two (CalpA and CalpB) encode canonical, active calpain enzymes. The CalpC gene product is an enzymatically inactive protein, whereas CalpD (sol) encodes an atypical calpain molecule. The structural characteristics of the Drosophila calpains are very similar to those of the mammalian enzymes, but they have a few distinguishing features: the calpa protein carries a 76 amino acid long hydrophobic in-sert in its c-terminal end, and the calpB protein has an unusually long n-terminal end of 224 amino acids. These extra sequences supposedly contribute to the compartmentalization of the calpain molecules.

to investigate its in vivo role, loss of function muta-tion of CalpB was generated via P element remobiliza-tion. The resulting mutants were fully viable and fertile displaying no morphological abnormalities. since the calpB protein is expressed at high levels in the border cells of the egg chambers, we investigated its possible role in the migration of these cells. In the CalpB mu-tants’ egg chambers, the migration of the border cells was significantly slower than in wild-type border cells. similar results were obtained when the expression of CalpB was blocked by expressing rna interference constructs in the border cells. These results indicate that CalpB is involved in the regulation of border cell migration.


126

GEN

ETIC

S

Two basic cell types exist in higher organisms: germ cells and somatic cells. Contrary to the somatic cells, which have adhesive properties, form tissues and organs, and are genetically inert, germ cells live as distinct en-tities within the sheath of the somatic cells. Germ cells are the only cells, which remain undifferentiated, staying genetically alive, allowing maintenance of the species. In most cases, the dichotomy between germ line and soma occurs during the early embryonic development. In our laboratory, we use genetic and genomic approaches to identify molecules and understand the mechanisms that underlie germ line determination in Drosophila mela-nogaster.

Miklós ERDÉLYI / Principal Investigator, Group Leader

Péter vILMOS / staff scientistFerenc JANKOvICS / staff scientistLászló HENN / staff scientistágnes vARGA / Phd studentMargit SZATHMáRI / technician

Maternal factors determine the germ cell fate

In most organisms a germ cell lineage is estab-lished early in embryogenesis. In the case of Dro-sophila, the primordial germ cells, or pole cells bud off from the posterior pole of the embryo just ninety minutes after egg laying, nearly two hours before cellularisation of the somatic cells. Injection experi-ments revealed that the posterior cytoplasm from late oocytes is able to induce ectopic germ cell fate. This demonstrates that the maternally produced pos-terior cytoplasm—also called pole plasm—contains all the factors necessary to instruct nuclei migrat-ing into it to take the germ cell fate. Thus, germline differentiation is under the control of genes active in the mother during oogenesis. a functional pole plasm, however, is not needed only for formation of the germ line, but also for proper development of the abdomen of the fly. mothers, which are homozygous mutants for pole plasm genes produce embryos that have both abdominal and germ line defects. The doubly mutant phenotype of embryos derived from mutant females lacking function of the pole plasm

genes reveals that the posterior cytoplasm contains morphogens for both the abdomen and the germline development.

Identification of pole plasm genes

most of the pole plasm genes have been identi-fied by their abdominal phenotypes (e.g. cappuccino, spire, staufen, oskar, vasa, valois, tudor, mago nashi) and some by their germ cell-less phenotypes (e.g. pip-squeak, germ cell-less, poirot, dmoesin). By detailed analysis of the pole plasm genes, a stepwise pathway of the pole plasm assembly has been established in which oskar plays the central role. assembly of the functional pole plasm requires several highly co-or-dinated cell biological processes such as mutual sign-aling between the oocyte and the follicular cell layer, microtubule reorientation of the oocyte, long-range kinesin-based oskar mrna transport along the mi-crotubules, translational control of the oskar mrna, actin-dependent anchoring of the oskar mrna and protein, and finally osk-mediated assembly of the pole plasm components.

GeRm Cell DevelopmenT In DRoSopHiLA MELAnoGASTER(DRoSopHiLA GeRm Cell DIffeRenTIaTIon GRoUp)

127

GEN

ETIC

S

Miklós ERDÉLYI GERM CELL DEVELOPMENT IN DROSOPHILA MELANOGASTER

Most of the genes of the oskar pathway are pleiotropic

With rare exceptions (osk, vasa, nanos), the identi-fied genes of the oskar pathway are not germline spe-cific, they also have other pleiotropic functions. How-ever, the unidentified signal or signals that specify the germ cell fate are most probably germline-specific. Thus, genetic analysis of the oskar pathway and identi-fication of the germline morphogen requires different genetic approaches. In order to identify new members of the oskar pathway we developed and used several genetic mutant isolation screens.

Interaction type of genetic screen

We have developed and used an interaction type of genetic screen in which a specific allele combina-tion of oskar, tropomyosin and staufen, three already known pole plasm genes provided a sensitized genetic background. In this sensitized background we identi-fied a lethal allele of Rab11, a new pleiotropic gene of the oskar pathway. We found that this small GtPase, known as a gene involved in vesicle targeting has a role in the oskar pathway as a regulator of the microtubule orientation of the developing oocyte.

Hobo-mediated germ cell-less mutant screen

making use of the relatively new hobo transposon mutagenesis system, we have identified poirot, a new germ cell-less gene, which encodes an sH3 domain binding protein. We found that poirot is a negative regulator of the Drosophila Bruton tyrosine kinase pro-tein, and this negative regulation is required for the proper anchoring of the oskar gene products.

Gene trap screen

We have developed and used a new P element based gene trap type transposon. making use of this mutator element, we isolated an isoform-specific, germ cell-less allele of the Dmoe gene. By this mutant, we showed that the Drosophila moesin has a role in

the anchoring of the oskar protein to the subcortical actin network. We also showed that Dmoe crosslinks the subcortical actin network and the cell membrane of the oocyte.

Microarray screen

to identify germ plasm-enriched, localized tran-scripts, we used a two-step method composed of cdna microarray and in situ rna hybridization techniques. We compared germ plasm deficient, normal and ec-topic germ plasm conditions in the cdna microarray experiments. rna species whose concentration in-creased when ectopic germ plasm was present and de-creased when the germ plasm was missing were select-ed. These candidates were then subjected to a second screen, which compared the distribution of the given rna in wild-type embryos and in eggs with ectopic germ plasm. We estimate that around one percent of the Drosophila genes encode for germ plasm enriched, localized transcripts.

Double-stranded RNA interference screen

double-stranded rna interference is one of the most powerful experimental systems of mod-ern Drosophila genetics. Based on the available se-quence information, gene-specific double-stranded rnas can be designed and introduced into living animals, which result in phenocopies equivalent with the hypomorphic classical mutant phenotypes. double-stranded rna species were synthesized, each of which was specific for one of the germ plasm enriched or germ cell specific transcripts. double-stranded rnas were injected into early Drosophila embryos. Germ cells of the injected embryos were labeled with the green fluorescent protein (GfP) tagged moesin, which allowed following the fate of the germ cells throughout the entire process of embryonic development. The dsrna-induced phe-nocopies were followed by automated in vivo video microscopy. The observed embryonic phenocopies were classified into the following categories: absence of germ cells or their decreased number, disoriented

128

GEN

ETIC

S

Miklós ERDÉLYI GERM CELL DEVELOPMENT IN DROSOPHILA MELANOGASTER

germ cells during migration, delayed germ cell mi-gration or gonad formation and failure of apoptosis of the disoriented germ cells.

A B

A. Embryonic germ cells (in-dicated in brown) are formed at the posterior pole of a wild-type embryo.

B. posterior pole of a germ cell-less embryo from a female mutant for a pole plasm gene


129

GEN

ETIC

S

István KISS / Principal Investigator, Group Leader

Erika vIRáGH / staff scientistBrigitta KISS / Phd studentMária KOPP / technician

neuropeptides/neurohormones are short peptidic fragments secreted by specific neurons in the cns. They are widespread in the animal kingdom from the worms up to the mammals. They exert hormonal effects and regulate many developmental and physi-ological functions e.g. development of the central and peripheral nervous system, production of the major hormones, fertility, water balance, cardiac rhythm, behaviour etc.

The genetic “dissection”, i.e. isolation of mutants and their analysis is the strategy which gives the high-est “resolution” in the analysis of gene function and interaction. Drosophila with its well-known genet-ics, molecular biology and fully annotated genome sequence offers unique advantages for such studies. as a holometabolous insect, it is especially suitable for studying the genetic regulation of insect develop-ment and physiology. The availability of complete in-sect (fruitfly, honey bee, Anopheles mosquito) genome sequences greatly accelerated the identification of neuropeptide genes and their receptors. Based on the Genome Project, the genes coding for these proteins are all known in Drosophila. most of the peptides have diverse effects depending on the availability of the dif-ferent receptors in the various tissues, their connec-tions to the different signalling pathways, etc. These issues are largely unknown even in the case of the bio-chemically well-known peptides. It is the elucidation of these diverse functions where the genetic approach in the fly can be very fruitful.

up to now, no systematic genetic analysis of neuro-hormone mechanism of action has been made in any system. We have started with the genetic analysis of a group of Drosophila genes producing the so-called fmrfamide-related (farP) neuropeptides (fmrfa-mides, myosuppressins and sulfakinin peptides) and their G-protein-coupled receptors (GPcrs). rnai (specific decay of mrna induced by double-stranded rna) mutants are available for all these genes and we have started with their analysis. The isolation of specific peptide and receptor mutants by targeted mutagenesis as well as transgenic constructs overex-pressing the gene products are also in progress. By analysing the mutant phenotypes and their interac-tions with representative signal transduction mu-tants, we establish the genetic network regulating the production, functions and interactions of these genes in neuropeptide signalling. such studies in the Dro-sophila model can make important contributions to the knowledge of the human and insect neurohor-mone systems, which is important with respect to their application in human pharmacology and insect control, respectively. This new project is based on in-ternational cooperation with czech and Belgian lab-oratories (dr. K. slama, Prague and Prof. dr. a. de Loof, Leuven, respectively)


moleCUlaR GeneTIC analYsIs of InseCT neURopepTIDes anD THeIR ReCepToRs In DRoSopHiLA MELAnoGASTER

130

GEN

ETIC

S

Our research focuses on cellular mechanisms of innate immunity using Drosophila melanogaster as a model organism. Studies on Drosophila contributed to our understanding of innate immunity in general, and uncovered similarities with the mammalian innate immune system. Although Drosophila possesses potent cellular immune responses, mediated by blood cells, the hemocytes, our knowledge on their heterogeneity and development is frag-mentary.

István ANDó / Principal Investigator, Group Leader

Éva KURUCZ / senior scientistRóbert MáRKUS / staff scientistviktor HONTI / staff scientistBarbara LAURINYECZ / staff scientistJános ZSáMBOKI / Phd studentGábor CSORDáS / Phd studentOlga KOvALCSIK / technicianSzilvia TáPAI / technician

The cellular immune response

microbes, parasites and abnormally developing tissues are invaded and eliminated by blood cells, the hemocytes. These cells are assumed to be developing in the central hematopoietic organ, the lymph gland, and serve as elements of rapid and effective cellular defence mechanisms, i.e. phagocytosis and encapsula-tion. morphologically different populations of hemo-cytes are responsible for these reactions, but due to the lack of molecular markers their origin, functions and lineage relationships are unclear.

Figure 1. GFP-labeled hemocytes in the Drosophila larva

to study heterogeneity, we identified immunologi-cal markers for blood cells. These markers, many of them identified by us recently as transmembrane re-

ceptors, help us study the heterogeneity, function and development of hemocytes using immunological and genetic approaches. using these markers we focus on the cellular events in the immune reactions, in par-ticular the encapsulation reaction.

research interests:

Definition of molecular markers for hemocyte subsets

Immunological epitopes of hemocyte-specific an-tigens can be used as markers for the identification of corresponding hemocytes or hemocyte subsets. We recently discovered immunological epitopes on hemo-cytes showing a cell-type specific expression pattern. These markers define subsets, i.e. the plasmatocytes, the lamellocytes and the crystal cells that have been identified so far by morphological and functional cri-teria. The newly discovered molecules, however, allow the dissection of morphologically similar populations to developmentally and functionally distinct subsets, e.g. stem cells and immediate precursors for the plas-matocytes and the lamellocytes. moreover, several an-tigens have been detected on lamellocytes, which are expressed sequentially during lamellocyte develop-ment and may mark distinct stages of differentiation.

ImmUnoloGY

131

GEN

ETIC

S

István ANDó IMMUNOLOGY

The expression pattern of these molecules in different hemocyte compartments is now under study.

Definition of hemocyte subsets and compartments

Three major subsets have been identified so far, namely the phagocytic plasmatocytes, the lamello-cytes responsible for the encapsulation reaction and the crystal cells involved in melanisation. The use of immunological markers allows the identification and isolation of hemocyte lineages and the different hemo-cyte compartments during development and following immune induction. recently we found that sessile he-mocytes form a functional compartment in the larva. The analysis of this compartment, as a niche for blood cell development, is in progress.

Figure 2. Lamellocytes encapsulating a parasitic egg. (Lamellocytes are labelled with Cy3-red).

Figure 3. GFP-labelled lamellocytes in the Drosophila larva

Hemocyte-specific molecules involved in the regulation of hemocyte development

In collaboration with dan Hultmark's lab in umea we have identified some of the genes for the cell-type specific molecules including the pan-hemocyte anti-gen Hemese, a new member of the glycophorin family and L1 lamellocyte marker. a plasmatocyte-antigen, nimrod was found to be involved phagocytosis. re-lated genes exist in other insect species and in verte-brates.


132

GEN

ETIC

S

One of the major limiting nutrients for plant growth is utilizable nitrogen in the environment. Acquisition and assimilation of nitrogen is therefore second in importance only to photosynthesis. Soil bacteria belonging to Rhizobiaceae are able to form a symbiotic relationship with leguminous plants, and in new plant organs, the root nodules, reduce the most abundant nitrogen source, the atmospheric nitrogen to ammonia. Host plants utilize the fixed nitrogen and in turn, provide carbon source and energy for nitrogen fixing bacteroids.

We investigate the presence and role of toxin-antitoxin (TA) systems in rhizobia under conditions of free-living state and during symbiosis with host plants. TA modules may participate in the adjustment of bacterial metabo-lism to varying environmental conditions. Moreover, the drastic physiological changes during the transition from free-living to symbiotic state may also require the active contribution of TA modules to metabolic regulation.

Ilona DUSHA / Principal Investigator, Group Leader

Gyöngyi CINEGE / staff scientistPaul Sebastian MICLEA / technician

toxin-antitoxin (ta) modules consisting of two partially overlapping genes are ubiquitous among bac-teria and archaea. ta systems encode proteins that form a complex acting as repressors for the ta oper-ons. The mechanism of action of ta modules is based on the different stabilities of toxin and antitoxin pro-teins. signals triggered by various stress factors lead to a decrease in the amount of labile antitoxin, thus the free stable toxin exerts its effect on various cellular tar-gets. The physiological function of chromosomally lo-cated toxins is still controversial. certain toxins, when activated by stress conditions induce significant loss of viability leading to programmed cell death. other observations demonstrated that ectopic expression of toxins resulted in bacteriostasis rather than bacteri-cidal effect. ta loci were also shown to participate in bacterial persistence and biofilm formation.

The proposed role of ta loci as general stress managers adjusting the metabolic rates under vary-ing environmental stimuli may be of special impor-tance during the adaptation of soil bacteria to oligo-

trophic conditions. In addition, symbiotic nitrogen fixing soil bacteria, which develop an intimate inter-action with leguminous plants also have the ability to adapt and function within the plant host cells during symbiosis.

our aim is to investigate the presence and role of ta systems in Sinorhizobium meliloti and Bradyrhizobium japonicum, the microsymbionts of two agriculturally important crops, alfalfa and soybean, respectively. We have shown that the ntrPR operon of Sinorhizobium meliloti represents a vapBC-type ta system, which is the most abundant group of the seven typical ta gene families. Insertion of transposon tn5 into the ntrR gene resulted in increased transcription of both nodu-lation genes (responsible for the production of bacte-rial nodulation signals the nod factors) and nitrogen fixation genes determining the enzyme nitrogenase. moreover, the dicarboxylate transport system provid-ing carbon source for bacteroids in nodules during symbiosis was also expressed at an increased level. as a result, alfalfa plants inoculated by this mutant strain

ToxIn-anTIToxIn moDUles In sYmbIoTIC nITRoGen-fIxInG soIl baCTeRIa(nITRoGen fIxaTIon GRoUp)

133

GEN

ETIC

S

Ilona DUSHA TOxIN-ANTITOxIN MODULES IN SYMBIOTIC NITROGEN-FIxING SOIL BACTERIA

had increased nitrogen content and biomass produc-tion as compared to those of the plants inoculated by the wild-type strain.

ta modules are surprisingly abundant in bacte-rial genomes. They are present in the chromosomes of almost all prokaryotes, often in very high numbers. Interestingly, obligate intracellular organisms have no functional ta loci, suggesting that these organisms which multiply under constant environmental con-ditions do not require ta modules as stress response mechanisms. Sinorhizobium meliloti also carries mul-tiple copies of ta modules representing different ta families. These loci are distributed on the chromo-some as well as on the megaplasmids of Sinorhizobium meliloti. some of them show high sequence homology to the ntrPR operon; however, our preliminary data

suggest that these additional copies are involved in the control of metabolic functions other than the regula-tion of nodulation and nitrogen fixation gene expres-sion. our aim is to determine the importance and role of ta copies, their possible cooperation, and their target sites in the cell. We investigate the molecular mechanism of the toxin molecules and the effect of ta systems on the stress tolerance of bacteria under free-living and symbiotic conditions. understanding their function may help to improve the efficiency of the symbiotic nitrogen fixation interaction with host plants as was already demonstrated for the ntrPR operon in Sinorhizobium meliloti.


134

GEN

ETIC

S

The Medicago Genetics Group in the Institute of Genetics has extensive experience in plant molecular biology, symbiotic nitrogen fixation, classical plant genetics and genetic mapping. The main trend of our work is to use genetics, genomics and molecular biology resources for structural, comparative and functional genomics of Medi-cago species and to study plant genes involved in symbiotic nodule development and nitrogen fixation. Besides the forward genetic approach, the phenotype-driven map-based cloning strategy we use modern genomic techniques to find candidate genes and verify them by reverse genetics. Genes identified are then subjected to detailed investiga-tion to reveal their structure, regulation as well as their location and the function of their product.

Gabriella ENDRE / Principal Investigator, Group Leader

Ernő KISS / senior scientistBoglárka OLáH / staff scientistAndrea BORBOLA / research assistantKatalin KONTáR / research assistantZoltán BOZSóKI / Phd student

Molecular background of the nitrogen fixing symbiosis

Legumes are essential sources of protein, carbohy-drates and minerals for animal and human nutrition. The high protein content of legume plants is made possible by the establishment of a root symbiosis with rhizobia to reduce atmospheric nitrogen. This symbi-otic capacity is indispensable for the global nitrogen cycle and allows farmers to reduce nitrogen (fertilizer) input and its associated cost and pollution and will be essential for the development of a sustainable agricul-ture. In addition, legumes contain unique bioactive compounds such as iso-flavonoids that can improve human and animal health (health promoting com-pounds). although legume crops are currently grown only on about 5% of cultivated land in europe, it is en-visaged that they will play an increasingly significant role due to their importance for sustainable agricul-ture, through reducing the use of costly and polluting nitrogenous fertilizers. Therefore this symbiosis is of high importance to agronomy and the environment, in which biotechnology is being used in environmen-tally friendly technologies to improve legume growth.

nitrogen-fixing symbiosis is initiated and main-tained by exchanges of signaling molecules between

the host plant and the microsymbionts—controlled by special genes in both organisms. The formation of nitrogen-fixing nodules on legumes’ roots requires an integration of infection by rhizobia at the root epider-mis and the initiation of cell division in the cortex, several cell layers away from the sites of infection. The main focus of our team has been the identification and function of plant symbiotic genes, especially using forward genetics or recently other genomic tools. We have identified the first symbiotic receptor gene from Medicago by map-based cloning approach (endre et al., nature, 2002). since then, several symbiotic genes have been identified and more are on the way in differ-ent laboratories all over the world.

our aim is to improve our knowledge about the genes and their protein products, as well as their inter-action during this important plant-bacterium symbio-sis for further possible exploitation of this biological

meDICaGo GeneTICs GRoUp

S. KALYANASUNDARAM / staff scientistErzsébet FEHÉRNÉ JUHáSZ / engineerSándor JENEI / engineerZsuzsanna LIPTAY / technician

135

GEN

ETIC

S

Gabriella ENDRE MEDICAGO GENETICS GROUP

process. to this end, besides classical and molecular genetic work our laboratory uses a wide variety of techniques of molecular biology, and our research ex-perience covers structural genomics, forward genetics, plant tissue culture, transformations, yeast two hybrid system, protein expression and purification.

Medicago genomics

model organisms proved to be useful in answer-ing biological questions that are more difficult to study directly in cultivated and often genetically re-calcitrant species because of their disadvantageous characteristics such as large genome, long life-cycle and lack of techniques for genetic manipulation. In recent years, Medicago truncatula has been rec-ognized as an excellent legume model in view of its advantageous characteristics (a small, diploid ge-nome, self-fertility and a short life cycle) and vari-ous genomic and genetic tools have been developed. different genomic and cdna libraries are available, and sequencing projects have resulted in large est datasets and long stretches of genomic sequences of the gene-rich parts of the genome. dna chips and diverse mutant and ecotype collections have also been developed. Thereby M. truncatula has become suitable for identifying genes in this plant and, sub-sequently, in other legumes when feasible.

our group has a long-standing research activity on different legume genetic systems such as alfalfa (Medi-cago sativa) and the model plant Medicago truncatula. after the pioneer work of the development of genetic maps, the group was involved in comparative genetic studies among different legume plants, and in the Bac end sequencing project in the structural genomics of M. truncatula. recently, functional genomic studies have also been initiated in our laboratory. ongoing projects include dna chip hybridizations and fur-ther investigation of the resulting candidate genes and their protein products. resources are also available for the ultimate validation of these candidate genes by a reverse genetic approach. a recently finished eu fP6 Grain Legumes Integrated Project has contributed significantly to this resource with a large-scale mu-tagenesis program of M. truncatula. We participated in this program and currently hold part of the seed collection of the insertional mutant lines. since the model legume M. truncatula is a close relative of sev-eral plants cultivated in europe (alfalfa, pea, clovers) and comparative genomic studies suggest good syn-teny with these species, we can also expect to be able to use the results obtained on the model plant in the improvement of the cultivated legume species.


136

GEN

ETIC

S

The stalling of DNA replication machinery that occurs as a consequence of encountering DNA damage is a challenging problem for cells. To rescue the stalled replication fork, different DNA damage bypass mechanisms have evolved that promote replication through DNA lesions. Increased error-prone bypass of DNA lesions causes increased mutagenesis, and as a consequence, a rise in the incidence of cancer. Error-free bypass processes, by contrast, keep mutagenesis low and reduce cancer frequencies. The goal of our project is to give insight into the mechanism and regulation of DNA damage bypass with special emphasis on the role of ubiquitin and SUMO modifications of DNA replication and repair proteins.

Lajos HARACSKA / Principal Investigator, Group Leader

András BLASTYáK / staff scientistIldikó HAJDÚ / staff scientistPéter BURKOvICS / staff scientistYathish J. ACHAR / staff scientistGali HIMABINDU / staff scientistZoltán GYURIS / Phd student

Genomic dna can be damaged by both external environmental agents and endogenous metabolic processes. to maintain the integrity of the genome, a variety of repair mechanisms have evolved that remove damaged bases from dna. However, as a result of mu-tations in repair systems, limited cellular repair capac-ity or timing, dna damage sometimes is not repaired before replication takes place. When the dna replica-tion machinery encounters an unrepaired dna lesion in the template strand, it faces a challenge, because the machinery is often unable to replicate past the lesion.

our research group is interested in the mechanisms that come into play when replication stalls at dna le-sions and that eventually lead to error-free or error-prone replication of damaged dna. error-prone rep-lication of damaged dna increases mutagenesis and leads to carcinogenesis, whereas error-free replication contributes to genetic stability.

The RAD6-RAD18-Dependent Pathway of Replication of Damaged DNA

Pathways for replication of damaged dna are highly conserved from yeast to human cells. In the yeast Saccharomyces cerevisiae, the pathways are well characterized genetically, and both error-free and mutagenic modes of damage bypass require the yeast genes RAD6 and RAD18. mutations in RAD6 and RAD18 confer a high degree of sensitivity to uv light and cause a defect in the replication of uv-damaged dna. furthermore, uv-induced mutagenesis does not occur in rad6 and rad18 mutants. rad6-rad18-mediated ubiquitin conjugation promotes replication through dna lesions via three different pathways:

ReplICaTIon of DamaGeD Dna In YeasT anD HUman Cells: ImplICaTIons foR mUTaGenesIs anD CaRCInoGenesIs

valéria SZUKACSOv / engineer Lajos PINTÉR / engineerKatalin ILLÉS / technicianIldikó KRAvJáR / technicianIlona NóTáRI / technician

137

GEN

ETIC

S

Lajos HARACSKA REPLICATION OF DAMAGED DNA IN YEAST AND HUMAN CELLS: IMPLICATIONS FOR MUTAGENESIS AND CARCINOGENESIS

the translesion synthesis pathways that depend on polymerase eta (Pol-eta) and Pol-zeta and the rad5-dependent postreplication repair pathway.

Translesion Synthesis by Specialized DNA Polymerases

Pol-eta is unique among eukaryotic dna polymer-ases in its proficiency at replicating through a uv-in-duced cis-syn thymine-thymine dimer. In humans, a defect in Pol-eta causes a variant form of xeroderma pigmentosum (XP-v). XP-v individuals are extremely sensitive to sunlight and they suffer from a high in-cidence of skin cancers. Pol-zeta promotes transle-sion synthesis by elongating the dna strand from the nucleotides that were inserted opposite the damaged base by another dna polymerase. By this mechanism, Pol-zeta promotes mutagenic translesion synthesis through certain dna lesions.

RAD5-Dependent Error-Free Replication of Damaged DNA

The RAD5-dependent pathway, an alternative pathway to those that depend on translesion synthesis polymerases plays a major role in error-free replica-tion of damaged dna and in reducing uv-induced mutagenesis in yeast. together with mms2 and ubc13,

rad5 forms a ubiquitin-conjugating enzyme complex that catalyzes the formation of polyubiquitin chains linked through the lysine-63 residue of ubiquitin. We have also shown that yeast rad5 has dna helicase ac-tivity that is specialized for replication fork regression. rad5 action promotes a “copy choice” type of synthe-sis through dna lesions, in which the newly synthe-sized daughter strand of the undamaged complemen-tary sequence is used as the template for bypassing the lesion.

Human Homologues of Yeast Rad5: HLTF and SHPRH Tumor Suppressors

recently, we have shown that HLtf and sHPrH are functional homologs of yeast rad5. HLtf is fre-quently inactivated in colorectal and gastric cancers and sHPrH is frequently mutated in a wide variety of cancers. a requirement for HLtf and sHPrH in error-free postreplication repair of damaged dna is in keeping with their cancer-suppression role.

our present aim is to keep identifying new players in the human rad6-rad18 dna damage bypass path-way which, similarly to hPoleta, sHPrH and HLtf tumor suppressors can be predicted to effect muta-genesis and carcinogenesis. We expect our research to provide greater insights into the areas of dna repair, mutagenesis and carcinogenesis.


138

GEN

ETIC

S

Ildikó UNK / Principal Investigator, Group Leader

Andrea DARABA / staff scientistMiklós HALMAI / Phd. studentvamsi KRISHNA / Phd studentSzilvia MINORITS / technician

cancer is one of the major causes of death in the present world. The seemingly unrelated nature of its different types has made it very difficult to find a com-mon cause that can trigger it. However, a growing body of evidence supports the idea that the roots of cancers lie in mutations in dna, the genetic material of cells. dna damages caused by extrinsic or intrinsic agents are usually removed from dna and repaired by one of the several dna repair systems of the cell preserv-ing the genetic information. However, high exposure to dna damaging agents can lead to the accumulation of unrepaired dna damages that can block the replication machinery leading to cell death. to ensure survival, cells have evolved mechanisms that can sustain dna replica-tion on damaged dna. These so-called damage toler-ance or dna damage bypass processes allow replication to continue on damaged dna without removing the damaged bases. In humans, increased error-prone by-pass of dna lesions causes increased mutagenesis and a rise in the incidence of cancers, whereas error-free repli-cation of damaged dna contributes to genetic stability.

In yeasts, the rad6-rad18 ubiquitin-conjugating complex governs three alternative pathways of rep-lication of uv-damaged dna: the rad5-dependent error-free, the dna polymerase η dependent error-free, and the dna polymerase ζ and rev1 dependent error-prone damage bypass.

The rad5-dependent pathway operates through a template switching mechanism, but specialized, so-called translesion synthesis dna polymerases work in the Polη and also in the Polζ and rev1 dependent pathways.

to activate any of the above three sub-pathways, first Pcna, the processivity factor of the replicative polymerase has to be ubiquitinated by rad6-rad18. monoubiquitination of Pcna activates the Polη and

Dna DamaGe ToleRanCe In eUkaRYoTes(Dna RepaIR GRoUp)

Many factors participate in the regulation of peripheral immune tolerance and maintenance of the immune homeostasis by controlling the inflammatory and autoimmune processes in mammals. A powerful mechanism is the modulation of the pattern of cell surface glycosylation and the expression of soluble or cell-bound lectins inter-acting with their ligands. Among these lectins, galectin-1 is one of the master immunoregulators.

139

GEN

ETIC

S

Ildikó UNK DNA DAMAGE TOLERANCE IN EUKARYOTES

the Polζ pathways, dependent on translesion synthesis polymerases. Polyubiquitination of that same residue of Pcna activates the rad5 pathway.

The most important questions about the rad6-rad18 dependent dna lesion bypass concern its regu-lation: How can ubiquitination of Pcna govern all three pathways? What are the downstream steps?

our main goal is to investigate the regulatory mechanisms of dna lesion bypass by rad6-rad18.

our hypothesis: to maintain the accurate duplica-tion of the genetic material, the access of low fidelity translesion synthesis dna polymerases, such as Polη and rev1, to the replication fork must be restricted to dna lesion sites. also, a mechanism should exist which provides preference to error-free lesion bypass pathways over the error-prone lesion bypass pathway. The regulation most probably involves specific protein interactions and posttranslational modifications, as strongly suggested by genetic data from yeast. Proteins

that interact with translesion synthesis polymerases are most probably involved in the regulation of dna damage bypass reactions.

We will identify proteins that mediate the rev1 de-pendent lesion bypass pathway and characterize these proteins by genetic and biochemical means. We will investigate whether the replicative polymerase Polδ is subject to posttranslational modification upon en-countering a dna lesion. also, we will examine the role of Pcna in mediating protein interactions neces-sary for activating the switch between the three sub-pathways. our results will provide insight into the mechanism of the regulation of lesion bypass path-ways and the polymerase exchange reactions between the replicative polymerase and translesion synthesis polymerases occurring at dna lesion sites.


140

GEN

ETIC

S

Éva MONOSTORI / Principal Investigator, Group Leader

Roberta FAJKA-BOJA / staff scientistágnes CZIBULA / staff scientistAndrea BLASKó / Phd studentGábor J. SZEBENI / Phd studentJulianna NOváK / Phd studentÉva KRISTON-PáL / Phd studentLea vÉGH / Phd studentAndrea GERCSó / technician

lYmpHoCYTe sIGnal TRansDUCTIon laboRaToRY

Many factors participate in the regulation of peripheral immune tolerance and maintenance of the immune homeostasis by controlling the inflammatory and autoimmune processes in mammals. A powerful mechanism is the modulation of the pattern of cell surface glycosylation and the expression of soluble or cell-bound lectins inter-acting with their ligands. Among these lectins, galectin-1 is one of the master immunoregulators.

Control of T cell viability by extracellular galectin-1

although galectin-1 is widely expressed in a large number of tissues and fulfills pleiotropic extracellular functions, it specifically acts on the immune response by preventing autoimmune and inflammatory proc-esses. Its inhibitory effects derive from reducing the production of inflammatory cytokines and inducing apoptosis of activated t lymphocytes. These functions encourage us to think about galectin-1 as a potential therapeutic drug in autoimmune and inflammatory diseases. on the other hand, galectin-1 contributes to tumor progression, partly by participating in gen-eration of tumor immunoprivilege. The molecular mechanism of t cell apoptosis induced by galectin-1 has been recently described in our laboratory (fig. 1). The apoptotic pathway involves the obligatory ac-tivation of tyrosine kinases, p56lck and ZaP 70 fol-lowed by tyrosine phosphorylation of intracellular substrates. after this, acid sphingomyelinase converts sphingomyelin into apoptotic second messenger, cera-mide and as a consequence, depolarization of the mi-tochondrial membrane occurs. final steps include the activation of caspase 9 and caspase 3 resulting in mas-sive proteolysis of proteins and breakdown of nuclear dna. In these studies apoptosis has been induced by

recombinant galectin-1; however, we have proved that cell-derived galectin-1 reduces t-cell viability via an identical pathway (fig. 2). Galectin-1 secreted by the tumor cells does not appear as a soluble factor, rather it couples to its glycoconjugate ligands on the tumor cell surface. Hence t-cell apoptosis induced by the tu-mor cells in co-culture system requires direct cell-cell interaction.

Figure 1. Galectin-1 induced apoptotic pathway. Inhibition of p56lck and ZAP 70 tyrosine kinases, activity of acid sphingomyelinase and re-lease of ceramide, the decrease of the mitochondrial membrane poten-tial or activation of caspases result in failure of T-cell apoptosis induced by recombinant, soluble galectin-1.

141

GEN

ETIC

S

Éva MONOSTORI LYMPHOCYTE SIGNAL TRANSDUCTION LABORATORY

Figure 2. Tumor cell-derived galectin-1 induces T-cell apoptosis on identical pathway as recombinant soluble galectin-1. HeLa: galectin-1 non-producing tumor cells, U87: galectin-1 producing tumor cells.


142

GEN

ETIC

S

István RASKó / Principal Investigator, Group Leader

Mónika MóROCZ / staff scientistBernadett CSáNYI / staff scientistKatalin PRISKIN / staff scientistAnita SZÉCSÉNYI-KáITY / staff scientistMária RADó / technician

During the last several years it has become clear that ancient DNA (aDNA) is preserved in archeological speci-mens, but usually in tiny amounts, seriously damaged, often contaminated with inhibitory substances from the soil or with fungal or bacterial DNA. Ancient DNA offers applications in genetic studies of ancient human populations and in reconstructing human population history.

Archeological genetics

mitochondrial dna (mtdna) is used in adna studies to trace maternal lineages, on account of its high copy number and the lack of recombination. Y chromosomal non-recombining regional polymor-phisms, on the other hand, are useful in following paternal inheritance. These two tools are routinely used in human phylogeographic studies. Human mtdna d-loop and Y chromosomal mutations have accumulated sequentially along radiating maternal and paternal lineages during and after the process of human colonization of different geographical re-gions of the world. Their analysis enables the study of the genetic traces of migration and the admixtures of different human communities and helps in estimat-ing the degree of relationships within and between populations. an efficient method was developed in our laboratory to isolate Pcr-amplifiable dna from ancient bones.

The mitochondrial and Y chromosomal archeo-genetic map of the carpatian basin at the time of the arrival of the Hungarian tribes in the 10th – 11th cen-turies has been established. Pcr-amplifiable adna was isolated from bones originating from archeologi-cally characterized burial sites in Hungary. after se-

quencing, the mtdna and Y chromosome haplotypes and the monophyletic clades (haplogroups) which the samples belong to were determined. These data and those from modern Hungarian and secler samples were used to assign the relationships between the sam-ples and the sequences of 153 worldwide populations in our database.

recently lactose tolerance, a trait which has evolved as a result of cultural selection is being studied in re-cent and ancient Hungarian populations.

In compliance with ancient Hungarian burial cus-toms, noble Hungarians in the 10th – 11th centuries were buried with their horses. The determination of the genetic relationships of these animals by means of mitochondrial dna analysis is in progress.

Figure 1. From bone to mitochondrial sequences

GenomIC appRoaCHes In sTUDIes of HUman DIsease anD popUlaTIon oRIGIns(moleCUlaR HUman GeneTICs GRoUp)

143

GEN

ETIC

S

István RASKó GENOMIC APPROACHES IN STUDIES OF HUMAN DISEASE AND POPULATION ORIGINS

Interaction of genetic pathways in nerve specific differentiation

We have previously established that a gene, which belongs to the formin gene family is participating in the induction of nerve specific differentiation in mouse teratocarcinoma P19 cell line. studies are in progress to understand the underlying genetic inter-actions.


144

GEN

ETIC

S

Gyula HADLACZKY / Principal Investigator, Group Leader

Imre CSERPáN / senior scientistKatalin FODOR / senior scientistRóbert KATONA / senior scientistTünde PRAZNOvSZKY / senior scientistErika CSONKA / staff scientistIldikó SINKó / staff scientist

Péter BLAZSó / Phd studentMária DEáK / technicianGyöngyi HOLLó / technicianJudit KERESŐ / technicianKinga SZÉKELY-SZŰCS / technician

Artificial chromosomes are attractive tools for the better understanding of chromosome structure and function with additional potential as gene delivery vectors in various fields of gene technology. In the last decade, we have developed a methodology for the in vivo generation of mammalian satellite DNA based artificial chromosomes (SATACs) with defined genetic content. This is based on the induction of de novo chromosome formations via large-scale amplification, which can be initiated by targeted integration of any exogenous DNA sequence into the satellite/rDNA region of certain host chromosomes of mammalian cells. Co-amplification of sequences at the inte-gration site results in de novo formed chromosome arms and new chromosomes composed of the exogenous DNA provided as well as endogenous satellite/rDNA sequences.

Mammalian artificial chromosomes

While satacs are heterochromatic, they never-theless provide a suitable chromosomal environment for stable, persisting expression of the integrated ex-ogenous genetic material. satacs can be engineered in different mammalian species including humans, and can be purified and transferred into many types of recipient somatic cells and zygotes. transgenic ani-mals have successfully been generated with purified satacs, and the transmission of the artificial chromo-some through several generations has been achieved. moreover, tissue-specific expression of a therapeutic gene in transgenic offspring has been demonstrated.

The feasibility of exploiting satellite dna-based artificial chromosome has been established in differ-ent fields of biotechnology. satacs represent a novel protein production platform both for cellular protein production and for production of therapeutic mol-ecules in body fluids of transgenic animals. stable and heritable satacs with large carrying capacity may serve as potential vectors for animal breeding and in production of humanized cells, tissues and organs for xenotransplantation.

Figure 1. Generation of satellite DNA-based artificial chromosomes (A-E), mouse SATACs (F)

Human satacs composed of rdna and non-coding satellite dna sequences that lack transcrip-tion units for undesired and unknown genes can be regarded as genetically "neutral" and hence are proto-types of safe or low-risk artificial chromosome vectors for gene therapy.

eight years of collaborative r&d with chromos molecular systems Inc., a canadian company that was built on the basic science of the chromosome Group resulted in the artificial chromosome expression system (ace system). The ace system comprises:

CHRomosome sTRUCTURe anD fUnCTIon(laboRaToRIes of HUman moleCUlaR GeneTICs II)

145

GEN

ETIC

S

Gyula HADLACZKY CHROMOSOME STRUCTURE AND FUNCTION

(i) a pre-engineered platform mac with multiple ac-ceptor sites (Platform ace), which is capable of harboring a number of different genes, it has large carrying capacity, and represents a non-integrating safe vector.

(ii) the ace Integrase, a lambda integrase enzyme, which has been modified to render the integrase function-ally independent of bacterial host cell factors and ca-pable of operating in a mammalian context.

(iii) the ace targeting vector (atv) is a plasmid-based shuttle vector that conveys a gene(s) of in-terest onto Platform ace by means of targeted recombination between the recombination ac-ceptor attP sites present on Platform ace and the recombination donor attB site of the atv, catalyzed by the ace Integrase. The ace system is a platform technology for protein production, transgenesis and gene therapy.

Figure 2. Human satellite DNA-based artificial chromosome (*).

Figure 3. The ACE System

In 2007, GlaxosmithKline acquired the technol-ogy for pharmaceutical protein production; chromos and its successor calyx Bioventures Inc. retained the technology for the field of gene therapy and transgen-ics. at the same time, the Brc obtained an exclusive right to use the technology in the field of gene therapy restricted geographically to Hungary. Brc is the own-er of 49 artificial chromosome patents (28 pending, 21 issued, granted).

exploration of the potential of artificial chromo-somes in gene therapy is still a challenge for basic sci-ence. The major areas of our current research are related to those basic science tasks that may establish the feasi-bility for safe use of artificial chromosomes as vectors in the medicine of the 21st century. These include: i. construction and engineering of therapeutic artifi-

cial chromosomes, preferably with appropriate con-trol of the expression of therapeutic gene(s).

ii. Improved and efficient delivery of artificial chro-mosomes, preferably in a cell-specific manner

iii. demonstration of therapeutic effect(s) in appro-priate cellular and/or animal model systems.

recently, the first successful combined artificial chromosome-stem cell therapy was completed on the mouse model of an incurable genetic disease. (sup-ported by the ministry of economy and transport of the Hungarian republic, and the eu, GvoP 3.1.1-aKf0082). This result demonstrated the feasibility of the use of artificial chromosome technology in human gene therapy.

future directions include:i. establishment of a preclinical artificial chromo-

some-stem cell unit for human gene therapy suit-able for animal model experiments.

ii. artificial chromosome-mediated generation and therapeutic modification of stem cells for gene therapy.for many years, we have been working in close col-

laboration with andor udvardy's group at the Insti-tute of Biochemistry, Brc.


146

GEN

ETIC

S

Zsolt PÉNZES / Principal Investigator, Group Leader

Miklós BOZSó / staff scientistPéter BIHARI / Phd studentBotond SIPOS / Phd student

of the various forms of insect herbivory, the most intimate and complex form is gall induction. Galls are plant tissues but their development is largely controlled by insect genes. They represent discrete microhabitats that support relatively closed com-munities of specialist inhabitants. This property has made galls important model systems in many sub-ject areas ranging from population dynamics to the evolution of communities. In spite of the utility of insect galls in these topics, the high diversity of gall-ing taxa, processes shaping their distribution and evolution are poorly understood. recent advances of molecular approaches may provide insight into these processes.

Phylogenetics of gall wasps

With around 1400 described species, gall wasps constitute the second largest group of galling insects, and they induce some of the most structurally com-plex and diverse galls known. They are obligate plant parasites; the most important host plants are oaks. oak gall wasps (tribe Cynipini) are cyclically parthe-nogenetic insects including many well-known species distributed mainly in Holarctic. a number of species within Cyinipidae have lost the ability to induce galls themselves but instead develop inside the galls of other cynipids: these are called inquilines (tribe Synergini). Their host specificity varies from species to species. Little is known about this group.

despite recent advances in Cynipidae phyloge-netics, details are to be explored. early taxonomy

was based on wasp morphology and gall structure. for now, the utility of dna sequence markers in reconstructing phylogenetic relationship at various levels of divergence is well established. theoreti-cal advances also provide a standard tool to com-bine datasets collected on levels of morphology and dna sequences. We revised relationships in Chilas-pis – Dryocosmus (Cynipini) and Synophrus (Syn-ergini) genera. Work on Synergus (Synergini) is in progress.

This work is done in collaboration with G. melika (Pest diagnostic Laboratory, PPscd of county vas, Hungary), G. stone (univ. edinburgh, uK) and J. Pu-jade-villar (univ. Barcelona, spain).

Genetic diversity of an inquiline gall wasp

Previous work on gall wasps has revealed sub-stantial intra-specific variation, particularly be-tween regions known to represent discrete Pleis-tocene glacial refuges. asia minor is known to be a centre of diversity. It is a potential source for west-ward range expansion into europe. By projecting genetic diversity of populations into a geographic map, colonization routes can be reconstructed by the aid of molecular markers in different scales of space and time.

Parasitism may result in accelerated rates of mo-lecular evolution and it may engender bursts of spe-ciation and diversification. signs of these events can be detected by studying genetic differentiation. The evolution of host-races appears to represent one of

InseCT pHYloGeneTICs anD pHYloGeoGRapHY(moleCUlaR bIoDIveRsITY UnIT)

147

GEN

ETIC

S

Zsolt PÉNZES INSECT PHYLOGENETICS AND PHYLOGEOGRAPHY

the main routes to diversification in phytophagous insects. closed communities of galls provide unique systems to test this theory.

Synergus umbraculus is a common inquiline species living predominantly in galls of different Andricus spe-cies. our work shows that it is characterized by high

genetic diversity. different wasp hosts are used simul-taneously. multiple colonization events may account for the pattern detected in the carpathian basin.


148

GEN

ETIC

S

Henrik GyurkovicsIsh-Horowicz, d., Pinchin, s.m., Ingham, P.W. and Gyurko-vics, H. (1989). autocatalytic ftz activation and metameric in-stability induced by ectopic ftz expression. Cell 57(2): 223-232.

Gyurkovics, H., Gausz, J., Kummer, J., Karch, f. (1990). a new homeotic mutation in the Drosophila bithorax complex re-moves a boundary separating two domains of regulation. EMBO J. 9(8): 2579-2585.

Galloni, m., Gyurkovics, H., schedl, P., Karch, f. (1993). The bluetail transposon: evidence for independent cis-regulatory domains and domain boundaries in the bithorax complex. EMBO J. 12(3): 1087-1097.

farkas, G., Gausz, J., Galloni, m., reuter, G., Gyurkovics, H. and Karch, f. (1994). The trithorax-like gene encodes the Dro-sophila GaGa factor. Nature 371(6500): 806-808.

von allmen, G., Hogga, I., spierer, a., Karch, f., Bender, W., Gyurkovics, H. and Lewis, e. (1996). splits in fruitfly Hox gene complexes. Nature 380(6570): 116.

mihály, J., Hogga, I., Gausz, J., Gyurkovics, H. and Karch, f. (1997). In situ dissection of the fab-7 region of the bithorax complex into a chromatin domain boundary and a Polycomb response element. Development 124(9): 1809-1820.

sipos, L., mihály, J., Karch, f., schedl, P., Gausz, J. and Gy-urkovics, H. (1998). transvection in the Drosophila abd-B do-main: extensive upstream sequences are involved in anchoring distant cis-regulatory regions to the promoter. Genetics 149(2): 1031-1050.

Barges, s., mihály, J., Galloni, m., Hagstrom, K., muller, m., shanower, G., schedl, P., Gyurkovics, H. and Karch, f. (2000). The fab-8 boundary defines the distal limit of the bithorax complex iab-7 domain and insulates iab-7 from initiation ele-ments and a Pre in the adjacent iab-8 domain. Development 127(4): 779-790.

mihály, J., Barges, s., sipos, L., maeda, r., cleard, f., Hogga, I., Bender, W., Gyurkovics, H. and Karch, f. (2006). dissecting the regulatory landscape of the abd- B gene of the bithorax complex. Development 133: 2983-2993.

Iampietro, c., cleard, f., Gyurkovics, H., maeda, r.K. and Karch, f. (2008). Boundary swapping in the Drosophila Bitho-rax complex. Development 135: 3983-3987.

László Sipossipos, L., mihály, J., Karch, f., schedl, P., Gausz, J. and Gy-urkovics, H. (1998). transvection in the Drosophila abd-B do-main: extensive upstream sequences are involved in anchoring distant cis-regulatory regions to the promoter. Genetics 149(2): 1031-1050.

mollaaghababa, r., sipos, L., tiong, s.Y., Papoulas, o., arm-strong, J.a., tamkun, J.W. and Bender, W. (2001). mutations in Drosophila heat shock cognate 4 are enhancers of Polycomb. Proc. Natl. Acad. Sci. U.S.A. 98(7): 3958-3963.

Bajusz, I., sipos, L., Györgypál, Z., carrington, e., Jones, r., Gausz, J. and Gyurkovics, H. (2001). The trithorax-mimic al-lele of enhancer of zeste renders active domains of target genes accessible to Polycomb group dependent silencing in Drosophi-la melanogaster. Genetics 159: 1135-1150.

sipos, L. and Gyurkovics, H. (2005). Long-distance interac-tions between enhancers and promoters - The case of the abd-B domain of the Drosophila bithorax complex. FEBS J. 272: 3253-3259.


sipos, L., Kozma, G., molnár, e. and Bender, W. (2007). In situ dissection of a Polycomb response element in Drosophila mel-anogaster. Proc. Natl. Acad. Sci. U.S.A. 104(30): 12416-12421.

Kozma, G., Bender, W. and sipos, L. (2008). replacement of a Drosophila Polycomb response element core, and in situ anal-ysis of its dna motifs. Mol. Genet. Genomics 279(6): 595-603.

József Mihálymihály, J., Hogga, I., Gausz, J., Gyurkovics, H. and Karch, f. (1997). In situ dissection of the Fab-7 region of the bithorax complex into a chromatin domain boundary and a Polycomb-response element. Development 124: 1809-1820.

mihály, J., mishra, r.K. and Karch, f. (1998). a conserved se-quence motif in Polycomb-response elements. Molecular Cell 1: 1065-1066.

Barges, s., mihály, J., Galloni, m., Hagstrom, K., muller, m., shanower, G., schedl, P., Gyurkovics, H. and Karch, f. (2000). The Fab-8 boundary defines the distal limit of the bithorax complex iab-7 domain and insulates iab-7 from initiation ele-ments and a Pre in the adjacent iab-8 domain. Development 127: 779-790.

Boutros, m., mihály, J., Bouwmeester, t. and mlodzik, m (2000). signaling specificity by frizzled receptors in drosophi-la. Science 288, 1825-1828.

mishra, m., mihály, J., Hagstrom, K., schweinsberg, s., Barges, s., spierer, a., Karch, f. and schedl, P (2001). The iab-7 Poly-comb response element maps to a nucleosome free region of chromatin and requires both GaGa and Pleiohomeotic for si-lencing activity. Molecular and Cellular Biology 21: 1311-1318.


matusek, t., djiane, a., Jankovics, f., Brunner, d., mlodzik, m. and mihály, J (2006). The Drosophila formin daam regu-lates the tracheal cuticle pattern through organizing the actin cytoskeleton. Development 133: 957-966.

matusek, t., Gombos, r., szécsényi, a., sánchez-soriano, n., czibula, a., Pataki, c., Gedai, a., Prokop, a., raskó, I. and mi-hály, J. (2008). formin proteins of the daam subfamily play a role during axon growth. J. Neurosci. 28: 13310-13319.

Géza ÁdámGorjanácz, m., Ádám, G., török, I., mechler, B.m., szlanka, t. and Kiss, I. (2002). Importin- alpha2 is critically required for the assembly of ring canals during Drosophila oogenesis. Dev. Biol. 251: 271–282.

InsTITUTe of GeneTICsseleCTeD pUblICaTIons:

149

GEN

ETIC

S

Llano, e., Ádám, G., Pendás, a.m., Quesada, v., sánchez, L.m., santamaría, I., noselli, s. and López-otín, c. (2002). structur-al and enzymatic characterization of Drosophila dm2-mmP, a membrane-bound matrix metalloproteinase with tissue-spe-cific expression. J. Biol. Chem. 277: 23321-23329.

Ádám, G., Perrimon, n. and noselli, s. (2003). The retinoic-like juvenile hormone controls the looping of left-right asym-metric organs in Drosophila. Development 130: 2397-2406.

Ádám, G., Gausz, J., noselli, s., Kurucz, É., andó, I. and ud-vardy, a. (2004). tissue- and developmental stage-specific changes in the subcellular localization of the 26s proteasome in the ovary of Drosophila melanogaster. Gene Expression Pat-terns 4: 329-333.

spéder, P., Ádám, G. and noselli, s. (2006). type Id uncon-ventional myosin controls left–right asymmetry in Drosophila. Nature 440: 803-807.

Kókai, e., szuperák, m., alphey, L., Gausz, J., Ádám, G. and dombrádi, v. (2006). Germ line specific expression of a pro-tein phosphatase Y interacting protein (PPYr1) in Drosophila. Gene Expression Patterns 6: 724-729.

Miklós ErdélyiJankovics, f., sinka, r. and erdélyi, m. (2001). an interaction type of genetic screen reveals a role of the rab11 gene in oskar mrna localization in the developing Drosophila melanogaster oocyte. Genetics 158(3): 1177-1188.

Jankovics, f., sinka, r., Lukacsovich, t. and erdélyi, m. (2002). moesIn crosslinks actin and cell membrane in Drosophila oocytes and Is required for osKar anchoring. Curr Biol. 12(23): 2060-2065.

sinka, r., Jankovics, f., somogyi, K., szlanka, t., Lukacsovich, t. and erdélyi, m. (2002). Poirot, a new regulatory gene of Dro-sophila oskar acts at the level of the short oskar protein iso-form. Development 129(14): 3469-3478.

szuperák, m., Zvara, Á. and erdélyi, m. (2005). Identification of germ plasm-enriched mrnas in Drosophila melanogaster by the cdna microarray technique. Gene Expr.Patterns 5: 717-723.

István Kisstörök, t., tick, G., alvarado, m. and Kiss, I. (1993). PlacW in-sertional mutagenesis on the second chromosome of Drosophi-la melanogaster: Isolation of lethals with different overgrowth phenotypes. Genetics 135: 71-80.

török, I., strand, d., schmitt, r., tick, G., török, t., Kiss, I. and mechler, B.m. (1995). The overgrown hematopietic or-gans31 tumor suppressor gene of Drosophila encodes an Im-portin-like protein accumulating in the nucleus at the onset of mitosis. J. Cell Biol. 129: 1473-1489.

spradling, a.c., stern, d., Kiss, I., roote, J. and rubin, G.m. (1995). Gene disruption using P transposable elements: an in-tegral component of the Drosophila genome project. Proc. Nat. Acad. Sci. U.S.A. 92: 10824-10830.

ohkura, H., török, t., tick, G., Hoheisel, J., Kiss, I. and Glover, d.m. (1997). mutation of a gene for a Drosophila kinesin-like protein, Klp 38B, leads to failure of cytokinesis. J. Cell Sci. 110: 945-954.

török, I., Herrmann-Horle, d., Kiss, I., tick, G., speer, G., schmitt, r. and mechler, B.m. (1999). down-regulation of rps21, a putative translation initiation factor interacting with P40, produces viable minute imagos and larval lethality with overgrown haematopoi-etic organs and imaginal discs. Molec. Cell. Biol. 19: 2308-2321.

török, t., Gorjánácz, m., Bryant, P.J. and Kiss, I. (2000). Prod is a novel dna-binding protein that binds to the 1.686 g/cm3 10 bp satellite repeat of Drosophila melanogaster. Nucl. Acids Res. 28: 3551-3557.

chen, J., Godt, d., Gunsalus, K., Kiss, I., Goldberg, m. and Laski, f.a. (2001). cofilin/adf is required for cell motility during Drosophila ovary development and oogenesis. Nature Cell Biol. 3: 204-209.

Gorjánácz, m., Ádám, G., török, I., mechler, B.m., szlanka, t. and Kiss, I. (2002). Importin-2 is critically required for the assembly of ring canals during Drosophila oogenesis. Dev. Biol. 251: 271-282.

Page, a.r., Kovacs, a., deak, P., torok, t., Kiss, I., dario, P., Bastos, c., Batista, P., Gomes, r., ohkura, H., russell, s. and Glover, d.m. (2005). spotted-dick, a zinc-finger protein of Drosophila required for expression of orc4 and s phase. EMBO J. 24: 4304-4315.

Gorjánácz, m., török, I., Pomozi, I., Garab, G., szlanka, t., Kiss, I. and mechler, B.m. (2006). domains of Importin-alpha2 required for ring canal assembly during Drosophila oogenesis. J. Struct Biol. 154: 27-41.

chen, d., ahlford, a., schnorrer, f., Kalchhauser, I., fellner, m., viràgh, e., Kiss, I., syvänen, a.c. and dickson, B.J. (2008). High-resolution, high-throughput snP mapping in Drosophila melanogaster. Nat. Methods 5: 323-329.

István AndóKurucz, É., Zettervall, c.-J., sinka, r., vilmos, P., Pivarcsi, a., ekengren, s., Hegedűs, Z., andó, I. and Hultmark, d. (2003). Hemese, a hemocyte-specific transmembrane protein affects the cellular immune response in Drosophila. Proc. Natl. Acad. Sci. U.S.A. 100: 2622-2627.

vilmos, P., nagy, I., Kurucz, É., Hultmark, d., Gateff, e., andó, I. (2004). a rapid rosetting method for separation of hemocyte sub-populartions in Drosophila melanogaster. Dev. Comp. Im-munol, 28: 555-563.

sinenko, s.s., Kim, e.K., Wynn, r., manfruelli, P., andó, I., Wharton, K., Perrimon, n. and mathey-Prevot, B. (2004). Yantar, a conserved arginine-rich protein is involved in Dro-sophila hemocyte development. Dev. Biol. 273: 48-62.

Zettervall, c.-J., anderl, I., Williams, m.J., Palmer, r., Kurucz, É., andó, I. and Hultmark, d. (2004). a direct screen for genes involved in Drosophila blood cell activation. Proc. Natl. Acad. Sci. U.S.A. 101: 14192-14197.

márkus, r., Kurucz, É., rus, f. and andó, I. (2005). sterile wound-ing is a minimal and sufficient trigger for a cellular immune re-sponse in Drosophila melanogaster. Immunol Lett. 101(1): 108-111.

Williams, m.J., andó, I. and Hultmark, d. (2005). Drosophila melanogaster rac2 is necessary for a proper cellular immune response. Genes Cells. 10(8): 813-823.

rus, f., Kurucz, É., márkus, r., Inenko, s.a., Laurinyecz, B., Pataki, c., Gausz, J., Hegedűs, Z., udvardy, a., Hultmark, d. and andó, I. (2006). expression pattern of filamin-240 in Dro-sophila blood cells. Gene Expr. Patterns. 6(8): 928-934.


150

GEN

ETIC

S

Kurucz, É., váczi, B., márkus, r., Laurinyecz, B., vilmos, P., Zsámboki, J., csorba, K., Gateff, e., Hultmark, d. and andó, I. (2007). definition of Drosophila hemocyte subsets by cell-type specific antigens. Acta Biol. Hung. 58: 95-111.

Kurucz, É., márkus, r., Zsámboki, J., medzihradszky, K.f., darula, Zs., vilmos, P., udvardy, a., Krausz, I., Lukacsovich, t., Gateff, e., Zettervall, c-J., Hultmark, d. and andó, I. (2007). nimrod, a Putative phagocytosis receptor with eGf repeats in Drosophila Plasmatocytes. Current Biology, 17(7): 649-654.

sipos, B., somogyi, K., andó, I. and Pénzes, Zs. (2008). 2prhd: a tool to study the patterns of repeat evolution. BMC Bioinfor-matics. 18: 9:27.

somogyi, K., sipos, B., Pénzes, Zs., Kurucz, É., Zsámboki, J., Hultmark, d. and andó, I. (2008). evolution of genes and repeats in the nimrod superfamily. Mol. Biol. Evol. 25(11): 2337–2347.

márkus, r., Laurinyecz, B., Kurucz, É., Honti, v., Bajusz, I., si-pos, B., somogyi, K., Kronhamn, J., Hultmark, d. and andó, I. (2009). sessile hemocytes as a hematopoietic compartment in Drosophila melanogaster. Proc. Natl. Acad. Sci. U.S.A. 106(12): 4805-4809.

Ilona Dushadusha, I. and Kondorosi, Á. (1993). Genes at different regula-tory levels are required for the ammonia control of nodulation in Rhizobium meliloti. Mol. Gen. Genet. 240: 435-444.

dusha, I., oláh, B., szegletes, Zs., erdei, L. and Kondorosi, Á. (1999). syrM is involved in the determination of the amount and ratio of the two forms of the acidic exopolysaccharide ePsI in Rhizobium meliloti. Mol. Plant-Microbe Interact. 12: 755-765.

dusha, I., austin, s., dixon, r. (1999). The upstream region of the nodD3 gene of Sinorhizobium meliloti carries enhancer sequences for the transcriptional activator ntrc. FEMS Micro-biol. Lett. 179(2): 491-499.

oláh, B., Kiss, e., Györgypál, Z., Borzi, J., cinege, Gy., csanádi, Gy., Batut, J., Kondorosi, Á. and dusha, I. (2001). mutation in the ntrR gene, a member of the vap gene family, increases the symbiotic efficiency of Sinorhizobium meliloti. Mol. Plant-Mi-crobe Interact. 14: 887-894.

Puskás, L., nagy, Z., Kelemen, J., rüberg, s., Bodogai, m., Becker, a. and dusha, I. (2004). Wide-range transcriptional modulating effect of ntrR under microaerobiosis in Sinorhizo-bium meliloti. Mol. Gen. Genomics, 272: 275-289.

Bodogai, m., ferenczi, s., Bashtovyy, d., miclea, P., Papp, P. and dusha, I. (2006). The ntrPr operon of Sinorhizobium me-liloti is organized and functions as a toxin-antitoxin module. Mol. Plant-Microbe Interact. 19: 811-822.

Gabriella EndreKaló, P., endre, G., Zimányi, L., csanádi, G. and Kiss, G.B. (2000). construction of an improved linkage map of diploid alfalfa (Medicago sativa). Theor. Appl. Genet. 100: 641-657.

endre, G., Kereszt, a., Kevei, Z., mihacea, s., Kaló, P. and Kiss, G.B. (2002). a receptor kinase gene regulating symbiotic nod-ule development. Nature 417: 962-966.

endre, G., Kaló, P., Kevei, Z., Kiss, P., mihacea, s., szakál, B., Kereszt, a. and Kiss, G.B. (2002). Genetic mapping of the non-nodulation phenotype of the mutant mn-1008 in tetraploid alfalfa (Medicago sativa). Mol. Gen. Genomics 266: 1012-1019.

Liu, J., Blaylock, L.a., endre, G., cho, J., town, c.d., vanden-Bosch, K.a. and Harrison, m.J. (2003). transcript profiling coupled with spatial expression analyses reveals genes involved in distinct developmental stages of the arbuscular mycorrhizal symbiosis. Plant Cell 15: 2106-2123.

Kuppusamy, K.t., endre, G., Prabhu, r., Penmetsa, r.v., veereshlingam, H., cook, d.r., dickstein, r. and vanden-Bosch, K.a. (2004). LIN, a Medicago truncatula gene required for nodule differentiation and persistence of rhizobial infec-tions. Plant Physiol. 136: 3682–3691.

Kevei, Z., seres, a., Kereszt, a., Kaló, P., Kiss, P., tóth, G., en-dre, G. and Kiss, G.B. (2005). significant microsynteny with new evolutionary highlights is detected between Arabidopsis and legume model plants despite the lack of macrosynteny. Mol. Gen. Genomics 274: 644-657.

mun, J.H., Kim, d.J., choi, H.K., Gish, J., debelle, f., mudge, J., denny, r., endre, G., saurat, o., dudez, a.m., Kiss, G.B., roe, B., Young, n.d. and cook, d.r. (2006). distribution of mic-rosatellites in the genome of Medicago truncatula: a resource of genetic markers that integrate genetic and physical maps. Genetics 172: 2541-2555.

Kevei, Z., Lougnon, G., mergaert, P., Horváth, G.v., Kereszt, a., Jayaraman, d., Zaman, n., marcel, f., regulski, K., Kiss, G.B., Kondorosi, Á., endre, G., Kondorosi, É. and ané, J-m. (2007). a 3-hydroxy-3-methylglutaryl coenzyme a reductase interacts with norK in the nodulation signaling pathway. Plant Cell 19: 3974-3989.

tadege, m., Wen, J., He, J., tu, H., Kwak, Y., eschstruth, a., cayrel, a., endre, G., Zhao, P.X., chabaud, m., ratet, P. and mysore, K.s. (2008). Large scale insertional mutagenesis using Tnt1 retrotransposon in the model legume Medicago trunca-tula. Plant J. 54: 335-347.

Kiss, e., muñoz, a., oláh, B., Kaló, P., morales, m., Heckmann, a.B., Borbola, a., Lózsa, a., Kontár, K., middleton, P., downie, J.a., oldroyd, G.e.d. and endre, G. (2009). LIn, a novel type of u-box/Wd40 protein, controls early infection by rhizobia in legumes, submitted.

Lajos HaracskaHaracska, L., Yu, s.L., Johnson, r.e., Prakash, L. and Prakash, s. (2000). efficient and accurate replication in the presence of 7,8-dihydro-8-oxoguanine by dna polymerase η. Nat. Genet. 25: 458-461.

Johnson, r.e., Washington, m.t., Haracska, L., Prakash, s. and Prakash, L. (2000). eukaryotic polymerases ι and ζ act sequen-tially to bypass dna lesions. Nature 406: 1015-1019.

Haracska, L., unk, I., Johnson, r.e., Johansson, e., Burgers, P.m., Prakash, s. and Prakash, L. (2001). roles of yeast dna polymerases δ and ζ and of rev1 in the bypass of abasic sites. Genes & Dev. 15: 945-954.

Haracska, L., Johnson, r.e., unk, I., Phillips, B., Hurwitz, J., Prakash, s. and Prakash, L. (2001). targeting of human dna polymerase ι to the replication machinery via interaction with Pcna. Proc. Natl. Acad. Sci. U.S.A. 98: 14256-14261.


151

GEN

ETIC

S

Haracska, L., Kondratick, c.m., unk, I., Prakash, s. and Pra-kash, L. (2001). Interaction with Pcna is essential for yeast dna polymerase η function. Mol. Cell 8: 407-415.

Haracska, L., Prakash, L. and Prakash, s. (2003). a mecha-nism for the exclusion of low-fidelity human Y-family dna polymerases from base excision repair. Genes & Dev. 17: 2777-2785.

Haracska, L., torres-ramos, c.a., Johnson, r.e., Prakash, s. and Prakash, L. (2004). opposing effects of ubiquitin conjuga-tion and sumo modification of Pcna on replicational bypass of dna lesions in yeast. Mol. Cell. Biol. 24: 4267-4274.

Burkovics, P., szukacsov, v., unk, I. and Haracska, L. (2006). Human ape2 protein has a 3 '-5 ' exonuclease activity that acts preferentially on mismatched base pairs. Nucleic Acids Res. 34: 2508-2515.

unk, I., Hajdú, I., fátyol, K., szakál, B., Blastyák, a., Bermu-dez, v., Hurwitz, J., Prakash, L., Prakash, s. and Haracska, L. (2006). Human sHPrH is a ubiquitin ligase for mms2-ubc13-dependent polyubiquitylation of proliferating cell nuclear an-tigen. Proc. Natl. Acad. Sci. U.S.A. 103: 18107-18112.

Blastyák, a., Pintér, L., unk, I., Prakash, L., Prakash, s. and Haracska, L. (2007). Yeast rad5 protein required for postrep-lication repair has a dna helicase activity specific for replica-tion fork regression. Mol. Cell 28(1): 167-175.

unk, I., Hajdú, I., fátyol, K., Hurwitz, J., Yoon, J.H., Prakash, L., Prakash, s. and Haracska L. (2008). Human HLtf func-tions as a ubiquitin ligase for proliferating cell nuclear antigen polyubiquitination. Proc. Natl. Acad. Sci. U.S.A. 105(10): 3768-3773.

Ildikó Unkunk, I., Kiss-tóth, e. and Boros, I. (1994). transcription factor aP-4 participates in activation of Bovine Leukemia virus long terminal repeat by p34 tax. Nucleic Acids Res. 22: 4872-4875.

unk, I., Haracska, L., Johnson, r.e., Prakash, s. and Prakash, L. (2000). apurinic endonuclease activity of yeast apn2 pro-tein. J. Biol. Chem. 275: 22427-22434.

Haracska, L., Kondratick, c.m., unk, I., Prakash, s. and Pra-kash, L. (2001). Interaction with Pcna is essential for yeast dna polymerase η function. Mol. Cell 8: 407-415.

Haracska, L., unk, I., Johnson, r.e., Johansson, e., Burgers, P.m., Prakash, s. and Prakash, L. (2001). roles of yeast dna polymerases δ and ζ and of rev1 in the bypass of abasic sites. Genes Dev. 15: 945-954.

unk, I., Haracska, L., Prakash, s. and Prakash, L. (2001). 3’-Phosphodiesterase and 3’→5’ exonuclease activities of Yeast apn2 Protein and requirement of These activities for repair of oxidative dna damage. Mol. Cell. Biol. 21: 1656-1661.

unk, I., Haracska, L., Gomes, X.v., Burgers, P.m.J., Prakash, L. and Prakash, s. (2002). stimulation of 3’→5’ exonuclease and 3’-phosphodiesterase activities of yeast apn2 by Pcna. Mol. Cell. Biol. 22: 6480-6486.

unk, I., Hajdú, I., fátyol, K., szakál, B., Blastyák, a., Bermu-dez, v., Hurwitz, J., Prakash, L., Prakash, s. and Haracska, L. (2006). Human sHPrH is a ubiquitin ligase for mms2-ubc13-dependent polyubiquitylation of proliferating cell nuclear an-tigen. Proc. Natl. Acad. Sci. U.S.A. 103: 18107-18112.

unk, I., Hajdú, I., fátyol, K., Hurwitz, J., Yoon, J.H., Praksh, L., Prakash, s. and Haracska, L. (2008). Human HLtf functions as a ubiquitin ligase for proliferating cell nuclear antigen poly-ubiquitination. Proc. Natl. Acad. Sci. U.S.A. 105: 3768-3773.

Éva Monostorimonostori, É., desai, d., Brown, m.H., cantrell, d.a. and crumpton, m.J. (1991). activation of human t lymphocytes via cd2antigen results in tyrosine phosphorylation of t cell antigen receptors -chains. J. Immunol. 144: 1010.

fajka-Boja, r., szemes, m., Ion, G., Légrádi, Á., caron, m. and monostori, É. (2002). receptor tyrosine phosphatase, cd45 binds Galectin-1 but does not mediate its apoptotic signal in t cell lines. Immunol. Lett. 82/1-2: 149-154.

Légrádi, a., chitu, v., szukacsov, v., fajka-Boja, r., szűcs, K.s. and monostori, É. (2004). Lysophosphatidylcholine is a regula-tor of tyrosine kinase activity and intracellular ca2+ level in Jurkat t cell line. Immunol. Lett. 91: 17-21.

vas, v., fajka-Boja, r., Ion, G., dudics, v., monostori, É. and uher, f. (2005). Biphasic effect of recombinant galectin-1 on the growth and death of early hematopoietic cells. Stem Cells 23: 279-287.

Ion, G., fajka-Boja, r., tóth, G.K., caron, m. and monostori, É. (2005). role of p56 lck and ZaP 70 mediated tyrosine phos-phorylation in galectin-1 induced cell death. Cell Death Differ. 12(8): 1145-1147.

Ion, G., fajka-Boja, r., Kovács, f., szebeni, G., Gombos, I., czi-bula, Á., matkó, J. and monostori, É. (2006). acid sphingomy-elinase mediated release of ceramide is essential to trigger the mitochondrial pathway of apoptosis by galectin-1. Cell Signal. 18: 1887-1896.

Kiss, J., Kunstár, a., fajka-Boja, r., dudics, v., tóvári, f., Légrádi, Á., monostori, É. and uher, f. (2007). a novel anti-inflammatory function of human galectin-1: inhibition of he-matopoietic progenitor cell mobilization. Exp. Hematol. 35: 305-313.

urbán, v.s., Kiss, J., Kovács, J., Gócza, e., vas, v., monostori, É. and uher, f. (2008). mesenchymal stem cells cooperate with bone marrow cells in therapy of diabetes. Stem Cells 26: 244-253.

fajka-Boja, r., Blaskó, a., Kovács-sólyom, f., szebeni, G.J., tóth, G.K. and monostori, É. (2008). co-localization of galectin-1 with Gm1 ganglioside in the course of its clathrin- and raft-de-pendent endocytosis. Cell. Mol. Life Sci. 65(16): 2586-2593.

István Raskóraskó I. and downes, c.s. (1994). Genes in medicine. molec-ular biology and human genetic disorders. Chapman & Hall London.

Kalmár, t., Bachrati, c., marcsik, a. and raskó, I. (2000). a simple and efficient method for Pcr amplifiable dna extrac-tion from ancient bones. Nucl. Acids Res. 28: e67.

Bogácsi-szabó, e., Kalmár, t., csányi, B., tömöry,Gy., czibula, Á., Priskin, K., Horváth, f., downes,c.s. and raskó, I. (2005). mito-chondrial dna of ancient cumanians: culturally asian steppe nomadic Immigrants with substantially more Western eurasian mitochondrial dna Lineages. Human Biology 77: 639-662.


152

GEN

ETIC

S

tömöry, G., csányi, B., Bogácsi-szabó, e., Kalmár, t., czibula, Á., csősz a., Priskin, K., mende, B., Lango, P., downes, c.s. and rasko, I. (2007). comparison of maternal lineage and bio-geographic analyses of ancient and modern Hungarian popu-lations. Am. J. Phys. Anthropol. 134: 354-368.

Priskin, K., tömöry, G., Bogácsi-szabó, e., csányi, B., vörös I. and raskó, I. (2007). mitochondrial dna control region analysis of a late neolithic aurochs (Bos primigenius Boj. 1827.) from the carpathian Basin. Acta Biol. Hung. 58: 131-137.

csányi, B., Bogácsi-szabó, e., tömöry, G., czibula, Á., Priskin, csősz, a., mende, B., Lango, P., csete, K., Zsolnai, a., conant, e.K., downes,c.s. and raskó, I. (2008). Y-chromosome analy-sis of ancient Hungarian and two modern Hungarian-speak-ing populations from the carpathian Basin. Ann. Hum. Genet. 72: 519-534.

matusek, t., Gombos, r., szécsényi a., sánchez-soriano, n., czibula, Á., Pataki c., Gedai, a., Prokop, a., raskó I. and mi-hály, J. (2008). formin proteins of the daam subfamily play a role during axon growth. J. Neurosci. 28: 13310-13319.

nagy, d., Bogácsi szabó, e., várkonyi, Á., csányi, B., czibula, Á., Bede, o., tari, B. and raskó, I. (2009). Prevalence of adult-type hypolactasia as diagnosed with genetic and lactose hydro-gen breath tests in Hungarians. Eur. J. Clin. Nutr. 63: 909–912.

Gyula HadlaczkyHadlaczky, Gy., Praznovszky, t., cserpán, I., Kereső, J., Péterfy, m., Kelemen, I., atalay, e., szeles, a., szelei, J., tubak, v. and Burg, K. (1991). centromere formation in mouse cells cotrans-formed with human dna and a dominant marker gene. Proc. Natl. Acad. Sci. U.S.A. 88: 8106-8110.

Praznovszky, t., Kereső, J., tubak, v., cserpán I., fátyol, K. and Hadlaczky, Gy. (1991). De novo chromosome formation in rodent cells. Proc. Natl. Acad. Sci. U.S.A. 88: 11042-11046.

fátyol, K., cserpán, I., Praznovszky, t., Kereső, J. and Had-laczky, Gy. (1994). cloning and molecular characterization of a novel chromosome specific centromere sequence of chinese hamster. Nucl. Acids Res. 22: 3728-3736.

Kereső, J., Praznovszky, t., cserpán, I., fodor, K., Katona, r., csonka, e., fátyol, K., Holló, Gy., szeles, a., ross, a.r., sum-ner, a.t., szalay, a.a. and Hadlaczky, Gy. (1996). De novo chromosome formations by large-scale amplification of the centromeric region of mouse chromosomes. Chromosome Res. 4: 226-239.

Holló, Gy., Kereső, J., Praznovszky, t., cserpán, I., fodor, K., Katona, r., csonka, e., fátyol, K., szeles, a., szalay, a.a. and Hadlaczky, Gy. (1996). evidence for a megareplicon covering megabases of centromeric chromosome segments. Chromo-some Res. 4: 240-247.

deJong, G., telenius, a.H., telenius, H., Perez, c.f., drayer, J.I. and Hadlaczky, Gy. (1999). mammalian artificial chromosome pilot facility: Large-scale isolation of functional satellite dna-based artificial chromosomes. Cytometry 35: 129-133.

telenius, H., szeles, a., Kereső, J., csonka, e., Praznovszky, t., Imreh, s., maxwell, a., Perez, c.f., drayer, J.I. and Hadlac-zky Gy. (1999). stability of a functional murine satellite dna-based artificial chromosome across mammalian species. Chro-mosome Res. 7(1): 3-7.

csonka, e., cserpán, I., fodor, K., Holló, Gy., Katona, r., Kereső, J., Praznovszky, t., szakál, B., telenius, a., deJong, G., udvardy, a. and Hadlaczky, Gy. (2000). novel Generation of Human satellite dna-based artificial chromosomes in mammalian cells. J. Cell Sci. 113(18): 3207-3216.

Hadlaczky, Gy. (2001). satellite dna-bases artificial chromosomes for use in gene therapy. Curr. Opin. Mol. Ther. 3(2): 125-132.

cserpán, I., Katona, r., Praznovszky, t., novák, e., rózsavölgyi, m., csonka, e., mórocz, m., fodor, K. and Hadlaczky, Gy. (2002). The chaB4 and nf1-related long-range multisequence dna families are contiguous in the centromeric heterochro-matin of several human chromosomes. Nucl. Acids Res. 30(13): 2899-2905.

monteith, d.P., Leung, J.d., Borowski, a.H., co, d.o., Praznovski, t., Jiric, f.r., Hadlaczky, Gy. and Perez, c.f. (2003). Pronuclear microinjection of purified artificial chro-mosomes for generation of transgenic mice: Pick-and-Inject technique. In mammalian artificial chromosomes: Methods and Protocols. eds.: sgaramella, v. and eridani, s. Methods in Mol. Biol. 240: 227-242.

Lindenbaum, m., Perkins, e., csonka, e., Greene, a., flem-ing, e., Hadlaczky, Gy., macdonald, n., maxwell, a., Perez, c. and Ledebur, H. Jr. (2004). The ace system: engineering ar-tificial chromosomes to rapidly generate high-expressing cell lines for manufacture of recombinant proteins. Nucleic Acids Res 32(21): e172.

duncan, a. and Hadlaczky, Gy. (2007). chromosomal engi-neering. Curr. Opin. Biotech. 18(5): 420-424.

Katona, r.L., sinkó, I., Holló, Gy., székely-szűcs, K., Praznovsz-ky, t., Kereső, J., csonka, e., fodor, K., cserpán, I., szakál, B., Blazsó, P., udvardy, a. and Hadlaczky, Gy. (2008). a combined artificial chromosome-stem cell therapy method in a model experiment aimed at the treatment of Krabbe disease in the twitcher mouse. Cell. Mol. Life Sci. 65: 3830-3838.

Zsolt PénzesÁcs, Z., melika, G., Pénzes, Zs., Pujade-villar, J. and stone, G.n. (2007). The phylogenetic relationships between Dryocos-mus - Chilaspis and allied genera of oak gallwasps (Hymenop-tera, cynipidae: cynipini) Systematic Entomology 32: 70-80.

somogyi, K., sipos, B., Pénzes, Zs., Kurucz, É., Zsámboki, J., Hultmark, d. and andó, I. (2008). evolution of genes and re-peats in the nimrod superfamily. Molecular Biology and Evolu-tion 25: 2337-2347.

sipos, B., somogyi, K., andó, I. and Pénzes, Zs. (2008). t2prhd: a tool to study the patterns of repeat evolution. BMC Bioinfor-matics 9: 27.

tavakoli, m., melika, G., sadeghi, s.e., Pénzes, Zs., assareh, m.a., atkinson, r., Bechtold, m., mikó, I., Zargaran, m.r., aligolizade, d., Barimani, H., Bihari, P., Pirozi, f., fülöp, d., somogyi, K., challis, r., Preuss, s., nicholls, J. and stone, G.n. (2008). new species of oak gallwaps from Iran (Hymenoptera: cynipidae: cynipini). Zootaxa 1699: 1-64.

Pénzes, Zs., melika, G., Bozsóki, Z., Bihari, P., mikó, I., tava-koli, m., Pujade-villar, J., fehér, B., fülöp, d., szabó, K., Bozsó, m., sipos, B., somogyi, K. and stone, G.n. (2009). a taxonomic and phylogenetic re-appraisal of the inquiline wasps of the ge-nus Synophrus Hartig, 1843 (Hymenoptera: cynipidae: syn-ergini). Systematic Entomology, in press.


153

PLA

NT

BIO

LOG

Y

Institute of Plant BiologyH-6726 Szeged, Temesvári krt. 62.H-6701 Szeged, P.O. Box 521, Hungary

Brc

154

PLA

NT

BIO

LOG

Y

The development of plants depends on reliable signaling mechanisms providing information on the actual state of the outer environment. The multilevel interaction of these mechanisms (e.g. light, clock and hormonal regulation) forms a regulatory network, which allows plants to respond adequately to changes in the ambient environment.

Our research focuses on (i) the signal transduction pathways induced by ultraviolet light, (ii) the molecular mecha-nism by which the red/far-red absorbing photoreceptors convert light into a biochemical signal, (iii) the structure and function of the plant circadian clock and (iv) the transcriptional regulation of genes involved in the biosynthesis of steroi-dal phytohormones (brassinosteroids, BRs).

moleCUlaR meCHanIsms of lIGHT anD CloCk sIGnalInG paTHWaYs In ARABiDopSiS

Ferenc NAGY / Principal Investigator, Group Leader

Éva áDáM / staff scientist Erzsébet FEJES / staff scientist László KOZMA-BOGNáR / senior scientistMiklós SZEKERES / senior scientist Péter GYULA / staff scientistAndrás vICZIáN / staff scientist László MENCZEL / research assistant Balázs FEHÉR / staff scientistAnita KAPUS / staff scientist vladyslava SOKOLOvA / staff scientist

Ultraviolet light induced signaling cascades

ultraviolet light is a biologically important and dy-namic component of sunlight. There is accumulating evidence that ultraviolet irradiation induces not only stress responses but, similarly to the visible part of the spectrum, it also functions as an environmental cue to regulate plant growth and development. We showed by microarray analysis that short, non-damaging pulses of uv induce a characteristic change in the ex-pression profile of hundreds of genes and subsequently identified a number of key regulatory factors orches-trating this specific plant response. In collaboration with roman ulm’s laboratory in freiburg, Germany we demonstrated that the bZIP transcription factor HY5, the multifunctional e3 ligase coP1 (constitu-tively photomorphogenic 1) and the uvr8 (ultraviolet resistant 8) protein play important roles in mediat-ing uv-induced signaling. It has been demonstrated that uv light induces specific and rapid interaction of uvr8 and coP1 in planta and that this step is a very early and critical step in uv-induced signaling. We are interested in defining additional components of the uv-induced signaling network and in identifying

specific cis-acting regulatory elements mediating uv-induced gene expression. uv and visible light induced signaling cascades share a number of key regulatory components, thus we intend to characterise how uv irradiation modifies phytochrome and circadian clock controlled signaling and ultimately plant growth and development.

Phytochrome-mediated signaling

Arabidopsis contains a small gene family encoding the phytochromes PHYa-PHYe. The photosensory function of the phytochrome molecule is based on its capacity to undergo reversible interconversion be-tween the red light-absorbing Pr form and the far-red light-absorbing Pfr form. It has been shown that these conformational changes activate/deactivate signaling pathways, thereby leading to changes in the expression of about 2500–3000 genes that underlie developmental responses to light. We have demonstrated that light in-duces import of all phytochrome species into the nuclei in a wavelength and intensity dependent fashion and that phytochromes of nuclear localisation form nucle-ar bodies, which presumably represent multi-protein

Kata TERECSKEI / staff scientistAndrea PALáGYI / staff scientistJános BINDICS / Phd studentBlanka GODZA / Phd studentLídia HATEGAN / Phd studentMárk SIPOS / Phd studentRóbertné HAJó / technicianKatalin JóSZAI / technicianHedvig MAJZIK / technicianGabriella vERES / technician

155

PLA

NT

BIO

LOG

Y

Ferenc NAGY MOLECULAR MECHANISMS OF LIGHT AND CLOCK-SIGNALING PATHWAYS IN ARABIDOPSIS

complexes (figure 1). The biological function of these nuclear bodies is poorly understood. our laboratory uses a number of experimental approaches to define the precise role of these phytochrome-containing nu-clear bodies in light-induced signaling.

Figure 1. Nucleocytoplasmic distribution and formation of PHYA to PHYE-GFP-containing speckles in 7-day-old dark-adapted seedlings.

(A) to (J) Epifluorescence images of hypocotyl cells of transgenic Arabi-dopsis seedlings expressing the PHYA-GFP ([A] and [B]), PHYB-GFP ([C] and [D]), PHYC-GFP ([E] and [F]), PHYD-GFP ([G] and [H]), and PHYE-GFP ([I] and [J]) fusion proteins. Epifluorescence images of nuclei of dark-adapted seedlings ([A], [C], [E], [G], and [I] and of seedlings transferred to white light for 10 min (B) or 6 h ([D], [F], [H], and [J]) are shown. Positions of selected nuclei (nu) are indicated.

We have demonstrated that fHY1 (far-red elongat-ed hypocotyl 1 and its close homolog fHL1 (fhy1-like) are necessary and sufficient to regulate light-induced nuclear import of PHYa in plants and in a cell-free system. Based on these observations and using fHY1 and PHYa as building blocks, we aim to create a syn-thetic light-regulated nuclear import/gene expres-sion system, which can be used in plant, yeast or even mammalian cells.

Photoactivated PHYa is an extremely labile mole-cule with a half-life of ~20 min. The factors controlling this rapid degradation process are largely unknown. We have carried out a genetic screen and isolated mu-tants showing increased stability of an ectopically ex-pressed PHYa-luciferase (PHYa-Luc) fusion protein in response to light (figure 2). We have confirmed that light-induced degradation of the endogenous PHYa receptor is also inhibited in these mutants. further characterization of the mutants and mapping of the mutations will reveal novel components/factors of light-controlled protein degradation in plants.

Figure 2. Measuring PHYA levels using the PHYA-LUC fusion protein in two isolated mutants

Panel A: 4-day-old etiolated (cD) seedlings expressing the PHYA-LUC fusion protein under the control of the PHYA promoter were treated with 6 hours of 20 μmol/m2/s red light (R) before the presented lumi-nescent images were taken by a sensitive CCD camera. The lumines-cence, proportional to the actual PHYA level of two chosen PHYA deg-radation mutants (225/1 and 005-7A) can be compared to the wild-type (WT) plantlets.

Panel B: 4-day-old etiolated seedlings (the same lines as on Panel a), bearing the PHYa:PHYa-Luc transgene were illuminated with 20 µmol/m2/s red light. The emitted luminescence was perceived by a ccd camera. The signals from about 10-15 seedlings were measured at every indicated time point. The measured values were averaged and normalized to the corresponding dark control value.


The circadian system in Arabidopsis thaliana

circadian clocks are biological timing mechanisms; they generate a 24h basic oscillation and rhythmically regulate a wide range of processes in many organisms. In eukaryotes, the circadian oscillator relies on tran-scriptional/translational feedback loops operated by the clock genes and clock proteins. circadian clocks are synchronized to the periodic environmental changes (e.g. day/night cycles) by specific stimuli; among these the most important is light. Phytochrome photore-ceptors are involved in setting the clock by transduc-ing the light signal to the central oscillator. We have showed that transcription of phytochrome genes and nuclear import of phytochrome receptor proteins are both regulated by the clock, indicating a two-way re-

156

PLA

NT

BIO

LOG

Y

Ferenc NAGY MOLECULAR MECHANISMS OF LIGHT AND CLOCK-SIGNALING PATHWAYS IN ARABIDOPSIS

lationship between the components of the light input pathway and the oscillator.

In order to identify new components of the plant circadian clock, we have initiated a large-scale mutant screening program and isolated several new circadian mutants in Arabidopsis. among them, the lip1-1 (light insensitive period 1) mutant displays novel circadian phenotypes arising from specific defects in the light input pathway to the oscillator. The LIP1 gene encodes a functional, plant-specific atypical small GtPase and therefore we postulate that it acts at the posttranscrip-tional level. our current work is focused on elucidat-ing the molecular mode of action of LIP1, which will allow us to integrate LIP1 in the model of the plant clock.

characterization of additional mutants from the screen is in progress and is expected to help determine the number and hierarchy of circadian circuits exist-ing in higher plants.


Developmental regulation of brassinosteroid biosynthesis

In higher plants brassinosteroids (Brs) control essential functions, such as elongation, photomor-

phogenesis, germination, f lowering time and fer-tility. the steroid hormone environment is largely determined by the activity of Br biosynthetic genes, which are regulated primarily at the transcrip-tional level. In Arabidopsis, the transcript levels of all known Br biosynthetic genes are coordinately feedback-regulated by the biologically active end product of the synthesis pathway. In addition, at least two key genes of Br biosynthesis, CPD and the Br c-6 oxidase-encoding CYP85A2 are also under developmental, organ-specific, light-responsive and circadian control. since Brs are not transported within the plant, these regulatory mechanisms are crucial for the homeostasis and morphogenic effects of the hormone.

We found that, in addition to the hormone gra-dient, light-dependent changes of susceptibility also influence Br responses. our goal is to elucidate how, and to what extent Br accumulation and physiologi-cal sensitization contribute to the activation of Br signaling. to this end, we develop reporter lines al-lowing in vivo detection of Br distribution, and char-acterize the dependence of the hormone response on the expression level and functioning of Br signaling components.


157

PLA

NT

BIO

LOG

Y

Environmental factors which impose adverse effects on photosynthetic organisms include light, drought, heavy metals and oxidative agents. By using a multidisciplinary approach, we investigate the mechanisms by which abiotic stress fac-tors and inducers of reactive oxygen species damage plants and cyanobacteria. We also study the defense and acclima-tion responses which are induced by these stimuli.

lIGHT- anD oxIDaTIve sTRess In planTs anD CYanobaCTeRIa(laboRaToRY of moleCUlaR sTRess- anD pHoTobIoloGY)

Imre vASS / Principal Investigator, Group Leader

Éva HIDEG / senior scientistPéter KóS / staff scientist Zsuzsanna DEáK / staff scientistMárta DOROGI / staff scientistÉva KISS / staff scientist Loredana PECA / staff scientist

Electron transport in the Photosystem II complex of photosynthesis

Photosystem II (PsII) of the photosynthetic appara-tus is a multisubunit pigment-protein complex in the thylakoid membrane, which performs light-induced oxidation of water and reduction of plastoquinone. We study the mechanisms of charge separation and charge recombination by thermoluminescence and chlorophyll fluorescence measurements. our results show that the dominating charge recombination route is the indirect non-radiative pathway via the primary radical pair (P680

+Phe-). By using site-directed mutants we have shown the participation of several amino acid residues (d1-ala344, cP43-Glu354) of the d1 protein in the ligation of the mn cluster of water oxidation.

Damage of the photosynthetic apparatus by visible and ultraviolet light

visible light is not only the basic driving force of photosynthesis, but also an important damaging fac-tor. We have demonstrated that photodamage involves the 3[P680

+Phe-] → 3P680 process via the main route of charge recombination, which leads to the production of highly damaging singlet oxygen through the inter-

action of 3P680 and molecular o2. We have also shown that the efficiency of 1o2 formation is influenced by the redox potential of Phe, which plays an important role in protection against photodamage.

Figure 1. Electron transport components of the PSII complex.

The flux of uv-B (290–320 nm) radiation is increas-ing at the surface of earth due to the recent depletion of the stratospheric ozone layer, which is potentially harmful for all forms of life. In plant cells, an impor-tant target of uv-B is the photosynthetic apparatus. We have demonstrated that the primary site of dam-age by uv-B (and uv-a) radiation is the mn cluster of the water-oxidizing complex in PsII. This effect is correlated with direct uv- absorption by mn(III) and

Leyla ABASOvA / staff scientistIstván Zoltán vASS / Phd studentCsaba István NAGY / Phd studentLászló SASS / research assistantGabriella FLEIT / technician

158

PLA

NT

BIO

LOG

Y

Imre vASS LIGHT- AND OxIDATIVE STRESS IN PLANTS AND CYANOBACTERIA

mn(Iv) ions. uv-stress induced damage of PsII can be repaired via de-novo synthesis of the d1 and d2 re-action center subunits. We have also shown that this repair process involves the ftsH protease, cyclic nu-cleotides, especially cGmP, as well as the HsP16 heat shock protein.

Stress-induced defense and acclimation responses in cyanobacteria

We investigate the molecular background of vari-ous stress responses (high light, uv radiation, heavy metals, oxidative agents, oxygen availability etc.). We have studied the light-dependent regulation of the psbA genes, encoding the d1 reaction center protein, in several cyanobacteria using quantitative rt Pcr and have shown differential responses within the psbA gene family. our recent interest includes the mecha-nism of heavy-metal tolerance and heavy-metal in-duced expression of Synechocystis 6803 genes. We have studied gene expression patterns in response to co2+, ni2+, Zn2+, cd2+, cr3+, cr6+, as3+, as5+, and constructed bioluminescent Synechocystis 6803 strains, which spe-cifically respond to co2+, ni2+ or Zn2+.

Complex plant stress monitoring

Quantitative assessment of plant responses to vari-ous stress conditions can be achieved by combined application of various remote sensing methods (dig-

ital photography, thermal imaging, chlorophyll fluo-rescence imaging) together with highly controlled growth environment. Based on these principles we are developing a complex plant stress diagnostic system, which is used for quantitative testing of drought toler-ance as well as leaf rust infection in different wheat varieties.

Detection of active oxygen species in plant systems

reactive oxygen species (ros) are associated with stress responses acting as primary elicitors, propaga-tors of oxidative damage, by-products as well as sig-nalling molecules. The involvement of ros in plant stress is generally concluded from detecting products of oxidative damage. In collaboration with university of Pécs (Pte, Hungary), we have developed direct de-tection techniques based on fluorescence quenching of reporter molecules upon combining with ros.

a combination of two imaging techniques: one based on detecting spatial and intensity distribution of the ros sensors and another based on variable chl-a fluorescence provides valuable information on light-driven ros generation and its detrimental effects on the investigation of the role of ros production in leaves.


Photo Chl fluorescence DanePy fluorescence

Figure 2. The effect of leaf rust on photosynthetic activity and ROS production in wheat leafs.

159

PLA

NT

BIO

LOG

Y

Our laboratory focuses on light energy converting biological systems and similar artificial assemblies. The main goal of our research is to understand the structure and function of photosynthetic membranes; furthermore we participate in the research and development of environmentally friendly self-assembling systems for solar energy conversion, and we also design and construct scientific instruments.

Photosynthesis is the energetic basis of life on Earth. The atmospheric oxygen, and thus also the ozone shield, are of photosynthetic origin. The fossil energy carriers are ‘solar-energy deposits’ of the past, and the greenhouse gas CO2 that is released during their combustion can only be recycled by photosynthetic organisms. Hence, a better understanding of photosynthesis is of paramount importance, which can also help in designing artificial solar energy utilizing systems.

In recent years, we have (i) revealed the three dimensional ultrastructure of the photosynthetic membrane system of plants – the most abundant and one of the most complex membrane systems of the biosphere, (ii) offered an explana-tion for the (enigmatic) role of non-bilayer lipids in the organization and dynamics of the bilayer biological membrane, (iii) have shown that pigment-protein complexes are assembled into highly organized but structurally flexible macro-domains, and (iv) discovered a novel, biological thermo-optic mechanism, which appears to play important roles in the regulation of physiologically relevant processes. We also design and construct innovative instruments that provide unique physical information on the molecular architecture of microscopic samples.

lIGHT eneRGY ConveRTInG bIoloGICal sTRUCTURes, pHYsIoloGICal pRoCesses, pHYsICal meCHanIsms(laboRaToRY of pHoTosYnTHeTIC membRanes)

Győző GARAB / Principal Investigator, Group Leader

László MUSTáRDY / emeritus senior scientistLászló KOváCS / senior scientist Szilvia Z. TóTH / staff scientistZsuzsanna váRKONYI / staff scientistOttó ZSIROS / research associateGábor STEINBACH / staff scientistYuliya MILOSLAvINA / staff scientist

The structure and flexibility of granal thylakoid membranes; light harvesting systems

during photosynthesis the absorbed light energy is converted into chemical energy. since sunlight is quite ‘dilute’ (the photon flux density is low), photons must be harvested by an ‘antenna’ system. The high efficiency of this system, which captures photons and transfers light (excitation) energy into the photochemical reaction centers, can only be ensured by a highly organized mo-lecular assembly, the (macro-)organization of which is still largely unknown. The main goal of our research is to reveal the structure and function of these self-assem-bling antenna and membrane systems, and to construct similar artificial molecular devices.

our electron tomographic investigations and earlier data obtained with ‘conventional’ electron microscopy, supervised in part by László mustárdy, revealed the 3-di-mensional ultrastructure of the granum-stroma thylakoid membrane system, a quasi-helical assembly (figure 1).

Figure 1. Idealized 3D model of granum-stroma thylakoid membrane system

Petar H. LAMBREv / staff scientistMilán SZABó / staff scientistágnes BUZA / Phd studentvaléria NAGY / Phd studentTünde TóTH / Phd studentágnes RÉDAI / technician

160

PLA

NT

BIO

LOG

Y

Győző GARAB LIGHT ENERGY CONVERTING BIOLOGICAL STRUCTURES, PHYSIOLOGICAL PROCESSES, PHYSICAL MECHANISMS

Granal chloroplasts are a rather late but immensely successful product of evolution. They appeared ‘only’ about half a billion years ago, but have ‘conquered’ all green plants and thus have become the most abundant membrane system in nature. concerning the structure and dynamics of this multilamellar membrane system under physiologically relevant conditions, non-invasive neutron and X-ray scattering measurements are performed, mostly on european large scale facility instruments. spectroscopic techniques are used to reveal the self-assembly, molecular organization and structural flexibility of this hierarchically organized complex membrane system.

By using nmr and optical spectroscopic tech-niques, we have recently shown that thylakoid mem-branes contain significant amounts of non-bilayer li-pid phase(s), which appear to be associated with the bilayer membrane. The importance of this question is signified by the fact that in energy-converting mem-branes non-bilayer lipids constitute half or more of the total lipid content; these lipids, their interactions with proteins, and non-bilayer phases might play impor-tant roles in determining the structure and dynamics of membranes.

Polarization spectroscopic investigations revealed that these membranes contain highly ordered, but structurally flexible units, the chiral macrodomains. We would like to understand the roles of different light harvesting components and different lipid classes in macro-organization, a project co-supervised by László Kovács. Granal thylakoid membranes and lamellar ag-gregates of isolated light-harvesting antenna complexes display very interesting spectroscopic features; they are non-recomposable from their constituents („the whole is more than the sum of its parts”). Their investigation as models is justified by the fact that similar features are exhibited by more complex entities, such as nuclei, chromosomes, protein aggregates, and by observations suggesting that most regulatory processes occur at this level of complexity, rather than at lower levels of the structural hierarchy.

While studying the structural flexibility of chiral macrodomains, we have discovered a novel, biological thermo-optic mechanisms. during thermo-optical re-

organizations ultrafast local heat-transients, originat-ing from the dissipation of excess photon energy, cause elementary structural changes due to the “built-in” thermally instable structural units in the close vicinity of the dissipation (figure 2). In this way, the excess en-ergy, which cannot be used in photosynthesis, induces structural changes. according to other data, these re-organizations play important roles in the regulation of photosynthesis, especially in high-intensity light and at elevated temperatures. similar reorganizations have also been observed in diatoms. (These algae deserve special attention for their ability to remove the atmos-pheric co2 from the fast carbon cycle, and sequester the fixed co2 into the sediments of deep oceans.)

Figure 2. Scheme of the thermo-optic mechanism. Ultrafast (10–20 ps), local (~1 nm radius) heat jumps facilitate the thermally inducible elemen-tary structural transitions

self-assembling light harvesting natural and recon-stituted multilamellar systems are of great potential interest with regard to solar energy converting sys-tems; they are ideally suited to supply energy for pho-tochemical reactions located on catalytic surfaces to produce hydrogen or other fuel. We also participate in national and international collaborations in this field.

Heat stress—replacing the oxygen evolving complex in vivo

Plants, algae and cyanobacteria are capable of water oxidation. during this process, molecular oxygen is released to the atmosphere and the electrons are used in a multistep electron transport process to produce carbohydrates from co2. This process also depends on the activity of the water-oxidizing complex (Woc), which supplies electrons for carbon fixation. However,

161

PLA

NT

BIO

LOG

Y

Győző GARAB LIGHT ENERGY CONVERTING BIOLOGICAL STRUCTURES, PHYSIOLOGICAL PROCESSES, PHYSICAL MECHANISMS

Woc is very sensitive to environmental stresses, espe-cially to heat stress, which can lead to its full inactiva-tion. our studies on different plant species and algae have shown that Woc can be replaced by ascorbate (vitamin c). By this means, ascorbate can transiently maintain photosynthetic activity, and thus protect the machinery (figure 3). The aim of our studies—led by szilvia Z. tóth—is to explore the physiological signifi-cance and the potential biotechnological applications of this mechanism.

Figure 3. Ascorbate, as an alternative electron donor to photosystem II

Order or disorder in biological samples? DP-LSM: a novel tool for mapping anisotropy

Highly organized molecular macroassemblies are found in many hierarchically organized biological systems, e.g. in nuclei, chromosomes, viruses, stacked membranes, tissues, actin-based structures. However, our understanding of the self-assembly, molecular organization, structural dynamics and physiologi-cal functions of these complex molecular assemblies is still rudimentary, mainly because in most samples these features can hardly be discerned macroscopi-cally. In recent years we have elaborated new imag-ing methods to measure and map the anisotropy in

microscopic samples. In collaboration with spin-off companies, we have equipped our laser scanning con-focal microscope (Lsm) with differential polarization (dP) attachments. With the dP-Lsm, 8 additional independent physical parameters can be mapped, which all carry unique information on the anisotropic molecular architecture of the samples. dP images re-vealing important novel features have been recorded on different biological systems, such as granal chlo-roplasts, plant cell walls (figure 4), cell membranes, amyloid filaments, actin-based structures, and also on intelligent materials, such as liquid crystals and pig-ment nanorods. The dP-Lsm has been awarded gold and silver medals at major international exhibitions.

Figure 4. Fluorescence intensity (A) and fluorescence detected linear dichroism images (B) of plant cell walls


A

B

162

PLA

NT

BIO

LOG

Y

The lipid composition of photosynthetic membranes is essential in the construction of the photosynthetic ma-chinery and in developing resistance to various stress conditions. We use a combination of physiological, biophysical and molecular biological approaches to investigate the structural and functional importance of phosphatidylglyc-erol in the functions and the formation of the photosynthetic apparatus, the role of lipid-protein, lipid-carotenoid and carotenoid-protein interaction in stress responses.

fUnCTIonal anD sTRUCTURal ImpoRTanCe of lIpIDs In pHoTosYnTHeTIC membRanes(laboRaToRY of planT lIpID fUnCTIon anD sTRUCTURe)

Zoltán GOMBOS / Principal Investigator, Group Leader

Mihály KIS / senior scientistIldikó DOMONKOS / staff scienctistBettina UGHY / staff scienctistHajnalka LACZKó-DOBOS / staff scienctistÖzge SÖZER / Phd studentAnna SALLAI / technician

The role of phosphatidylglycerol in photosynthetic electron transport processes

among glycoglycerolipids, dominant lipids of pho-tosynthetic membranes, there is only one phospholipid species, phosphatidylglycerol (PG). PG is an indispens-able lipid in cyanobacteria and higher plants, which are able to carry out oxygenic photosynthesis. crystallo-graphic studies of protein complexes involved in photo-synthesis have revealed the presence of PG molecules in the reaction centre (rc) of PsI and PsII complexes.

Figure 1. Electron micrographs of Synechocystis PCC6803 phosphatidyl-glycerol (PG) deficient mutant cells. Swollen, non-dividing cells are visible in the culture grown in the absence of PG. After re-addition of PG to the PG-depleted cells, a complete recovery of the cells could be observed.

We generated several PG-deficient mutants of vari-ous cyanobacterial strains. These PG mutants need-ed an exogenous supply of PG for their growth. The growth rate of the mutant cells in the presence of PG was hardly distinguishable from that of the wild type; however, that of the mutant cells was gradually sup-pressed during PG depletion. re-addition of PG mol-ecules restored cell division (fig. 1).

short-term depletion of PG hardly influenced the activity of PsI; however, that of PsII was severely af-fected. PG depletion suppressed the electron transport in PsII around primary and secondary quinones, in-hibited the assembly of the cP43 subunit to PsII rc and disturbed the formation of the functionally active dimers of PsII. Long-term PG depletion suppressed the oligomerization of PsI rcs (fig. 2) and decreased the PsI activity of the PG-deficient cells.

Figure 2. Top view of a PSI trimer, and disintegration of a trimer to mono-mers occurring after PG depletion.

163

PLA

NT

BIO

LOG

Y

Zoltán GOMBOS FUNCTIONAL AND STRUCTURAL IMPORTANCE OF LIPIDS IN PHOTOSYNTHETIC MEMBRANES

We concluded that PG depletion affects cyanobacte-rial cell division and PG is necessary for the oligomeriza-tion and functioning of photosynthetic reaction centres.

We investigated the remodeling of PG molecules in PG-deficient mutant cells of Synechocystis Pcc6803 without manipulation of the enzymes involved in this remodeling process. The exogenously added ar-tificial dioleoyl-PG was transformed into photosyn-thetically more essential natural PG derivatives. our experiments demonstrated remodeling of lipids in a prokaryotic photosynthetic bacterium.

Carotenoids, indispensable components for assembly and functions of photosystems

a gradual elimination of PG from the photosynthet-ic membranes of the mutant cells resulted in increased sensitivity to light, which resulted in photobleaching of photosynthetic pigments and ultimately to cell death. The induction of photobleaching can be explained by the formation of a triplet state of chlorophyll, which initiates the formation of reactive oxygen species that in turn in-duce the degradation of photosynthetic pigments in the PG-depleted cells. We demonstrated that in PG-deficient mutant Synechocystis Pcc6803 cells PG depletion induc-es an accumulation of myxoxanthophyll and echinenone in both thylakoid and cytoplasmic membranes. an in-crease in myxoxanthophyll and echinenone content upon PG depletion suggests that PG depletion regulates the biosynthetic pathway of specific carotenoids.

In order to get a better understanding of carotenoid involvement in photosynthetic functions, we construct-ed the first oxygenic photosynthetic prokaryotic mutant completely deficient in carotenoid (car) synthesis.

Figure 3. HPLC chromatograms of the pigments of wild-type and caro-tenoidless mutant Synechocystis PCC6803 cells.

complete elimination of cars (fig. 3) did not sig-nificantly affect the assembly of PsI and cytochrome b6f

complexes. The carotenoid-deficient mutant strain of Synechocystis Pcc6803 did not have any oxygen-evolving activity. The cells became light sensitive. The lack of cars blocked the construction of PsII rcs and the functions related to PsII protein complexes. elimination of cars does not affect severely the transcription of the studied photosynthetic rc genes, although the translation of protein subunits of PsII was remarkably suppressed.

We concluded that cars are indispensable constitu-ents of the photosynthetic apparatus and essential for protection of chlorophylls against photooxidation. furthermore, they are also required for the synthesis and assembly of PsII subunits.

The role of membrane lipids in low temperature stress processes in wheat

cell membranes are dynamic structures, which are constantly formed and degraded. changes in the lipid composition of membranes play an essential role in the adaptation of living organisms to various environmental effects. freezing temperature is one of the major abiotic stresses living organisms, especially plants, have to face. for example, the growing season and the geographical diversity of plant species are highly influenced by frost damage. The major food grain crop consumed by hu-mans is bread wheat. appropriate frost tolerance of win-ter wheat is essential for its safe production in countries under temperate climate. The primary sites of freezing injury in plants are cell membranes, especially the plas-ma membranes. Because of the importance of the plasma membrane in plant adaptation against the deleterious effects of low temperature, detailed analysis of the al-terations of their lipids will substantially help our under-standing of the role of the structural modifications of the membrane and the signals generated in the membrane.

Thus the major objective of this project is comprehen-sive lipid species profiling using special wheat genetic stocks such as substitution and recombinant lines. We hope to lo-calize and map genes involved in the metabolism of lipids. This may enable us to provide useful molecular markers linked to the mapped genes, thus helping mas.


164

PLA

NT

BIO

LOG

Y

Under extreme environmental conditions the reduction in growth of plant organs and productivity is highly de-pendent on the functionality of basic cellular mechanisms including cell division or defence reactions. The primary aims of ongoing research projects are the identification and characterization of genes, protein complexes involved in the control of cell cycle progression and sensing hormonal and stress signals. Comparison of stress sensitive and resistant genotypes from wheat and barley is expected to reveal novel mechanisms in adaptation to oxidative stresses or drought. Brachypodium distachion, a new model for temperate grasses is also used as experimental system in studies on linkage between drought adaptation and root development. In our candidate gene approach much attention is devoted to the analysis of the stress-protective function of rice and wheat aldo-keto reductases.

sTRess Responses anD aDapTaTIon meCHanIsms In planTs: ReGUlaTIon of Cell DIvIsIon CYCle anD pRoTeCTIve paTHWaYs(planT Cell DIvIsIon CYCle anD sTRess aDapTaTIon GRoUp)

Gábor v. HORváTH / Principal Investigator, Group Leader

Dénes DUDITS / research Professor Sándor BOTTKA / senior scientist János GYÖRGYEY / senior scientist Edit áBRAHáM / staff scientist László BAKó / staff scientist Pál MISKOLCZI / staff scientist Aladár PETTKó-SZANDTNER / staff scientistMátyás CSERHáTI / staff scientist Bettina NAGY / staff scientist Mária SZÉCSÉNYI / staff scientist Zoltán TURóCZY / staff scientist

Plant retinoblastoma-related proteins as key regulators of cell division and growth

cell division is one of the most fundamental cellular events that contributes to the realization of the develop-mental program and organization of plant structure. In plants as in other eukaryotic organizations, the cell divi-sion cycle includes the replication of dna molecules in s-phase and subsequent organization of chromosomes, which are equally distributed to the daughter cells dur-ing mitosis (m-phase). two major checkpoints, the first before entering s-phase at the end of G1-phase and the second before mitosis at the end of G2-phase control the progression through the cycle. figure 1 proposes a cen-tral role for the retinoblastoma-related proteins (rBrs) in the regulation of the G1-s transition through inter-action with cyclin–cyclin-dependent kinase (cdK) and phosphatase (PP2a) complexes. The regulatory

function of rBr depends on its phosphorylation status and interaction with e2f/dP transcriptional factors. Based on our research data the described model sug-gests alternative ways for the stress and hormone-acti-vated ca2+ signals in blocking G1-s transition in alfalfa (Medicago sativa, ms) cells. The ca2+-activated PP2a phosphatase complex can ensure rBr dephosphoryla-tion resulting in the block in the binding of the e2f/dP transcriptional factors, consequently the induction of s-phase genes is inhibited. We also showed that cdK complexes can phosphorylate rBr protein from alfalfa. This modification is expected to release the e2f/dP fac-tors for activation of s-phase genes. The cdK function is under the control of the inhibitor protein (cKIms) that is also a phosphoprotein and substrate of the ca2+-dependent protein kinase (cdPKms). The cellular ca2+ level can be increased under hormonal or stress effects. We showed that the stress hormone abscisic acid (aBa)

Zoltán ZOMBORI / staff scientistEmine DINC / Phd studentAndrás CSERI / Phd studentPetra MAJER / Phd studentPing YU / Phd studentIldikó BORKA / technicianKatalin LáSZLó / technicianGyörgyi SáNDOR / technicianKatalin TÖRÖK / technicianKrisztina TALPAS / technician

165

PLA

NT

BIO

LOG

Y

Gábor v. HORváTH STRESS RESPONSES AND ADAPTATION MECHANISMS IN PLANTS: REGULATION OF CELL DIVISION CYCLE AND PROTECTIVE PATHWAYS

reduced cdK activity in cultured alfalfa cells. In con-trast, auxin and cytokinins activate this kinase prima-rily responsible for rBr phosphorylation and activation of genes in s-phase.

Figure 1: Phosphorylation of retinoblastoma-related protein (RBRMs) by cyclin-dependent kinases or dephosphorylation by phosphatase (PP2A) are under the control of Ca2+-dependent protein kinase (CDPKMs) and cyclin-dependent kinase inhibitor (CKIMs)

cloning of the rice rBr cdnas (OsRBR1 and OsRBR2) allowed the production of transgenic cells or plants with overproduction or down-regulation in the synthesis of the corresponding mrnas (figure 2). The reduced synthesis of osrBr1 protein caused significant increase in the frequency of s-phase cells, whereas over-production decreased the number of dna synthesizing cells, as shown by figure 2. cultured cells of antisense line exhibited higher cell mass production in suspen-sion culture. We consider retinoblastoma-related pro-teins to be central regulators of plant cell division cycle and our present research is focused on the role of phos-phorylation, degradation of rBrs in the control of plant growth, development and stress adaptation.

Figure 2: Influence of RBR mRNA levels on the frequency of DNA syn-thesizing cells labelled with 5-ethynyl-2’-deoxyuridine (green or yellow-coloured) and biomass production in suspension culture.


Drought responses in cereals: a genomic approach

Plants have developed a wide range of defence strate-gies to maintain the functional integrity of cells and the whole organism under drought stress. efficient root sys-tem, effective ros eliminating pathways both play im-portant role in surviving periods of water shortage. We aim to reveal key molecular components of long term drought adaptation of the root system in cereals. remote wheat genotypes having different root growth charac-teristics (figure 3) and upland rice cultivars are studied in genome wide transcript profiling experiments.

Figure 3: Root development of wheat genotypes having different drought adaptation strategies under control (80%) and limited (40%) water supply.

comparative transcriptome analysis of a spring vari-ety, Kobomugi with a winter variety Plainsman that fol-lows avoidance strategy revealed that stress and defence related genes are more frequently up-regulated in the first one, while cell wall, growth and signal transduction-related transcripts are more abundant in the second one.

transcriptome analysis of upland rice cultivars highlighted genes exhibiting daily fluctuation in in-duction during drought. a found gene is not only induced proportionally to the extent of stress, but strictly root specific as well. Its promoter was fused to reporter gene construct and transformed into cereals to evaluate practical usability. Groups of co-ordinately regulated rice genes allow us to search for common

166

PLA

NT

BIO

LOG

Y

Gábor v. HORváTH STRESS RESPONSES AND ADAPTATION MECHANISMS IN PLANTS: REGULATION OF CELL DIVISION CYCLE AND PROTECTIVE PATHWAYS

regulatory elements in the whole promoterome using our computer algorithm that finds new common ele-ments in drought induced genes.

ros eliminating machinery of wheat plays impor-tant role in aerial part of plant as well. comparing re-sponses of two wheat genotypes, we found differences in ascorbate metabolism: both ascorbate oxidation and transcription levels of enzymes processing ascor-bate were changed. relative transcript levels of ascor-bate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase isoenzymes showed different transcriptional changes in the two genotypes.

Brachypodium distachion, a new model for temper-ate grasses is also used to implement our experimental system and we will study linkage of drought adapta-tion and root development.


Reactive carbonyl detoxification and stress tolerance in plants

The productivity of plants is greatly affected by en-vironmental stresses such as drought, increased soil salinity, high or low temperature. The production of reactive oxygen species (ros) and their toxic products, the reactive carbonyl compounds (rcc) are common feature of most environmental stresses, their reaction with proteins can significantly inhibit the normal ac-tivity of basic enzymes such as phosphoribulo kinase (figure 4/a).

Figure 4/A: Inhibition of the activity of recombinant phosphoribulo ki-nase enzyme by different chloroplastic reactive carbonyl compounds.

Improvement of scavenging capacity of cells can lead to increased stress tolerance. Plant aldose/alde-hyde reductases are important factors for such function since they have a wide range of enzymatic activity on lipid peroxidation and glycolysis derived reactive car-bonyls (like 4-hydroxy non-2-enal and methylglyoxal) and their detoxification capacity can limit the harmful effects of such stress-derived compounds. our main goals are: i, analysis of the molecular background of en-zyme modifications by reactive carbonyl compounds; ii, searching for candidate genes that can be used in the detoxification process; iii, based on the results improve stress tolerance of important crop plants. according to our previous results transgenic tobacco plants ectopi-cally expressing the alfalfa aldose reductase, the MsALR cdna under the control of the camv 35s promoter were more tolerant to dehydration stress and recovered better from damages caused by water deficit than the untransformed wild type plants and were more tolerant to stress caused by high temperature and light intensity or uv-B irradiation. analysis of transgenic wheat plants over-producing the alfalfa enzyme by the complex diag-nostic system developed in collaboration with the cereal research non-Profit co. revealed significant improve-ments in agronomical parameters during drought stress (figure 4/B). This result supported the start of a conven-tional breeding program that aims the improvement of stress tolerance of Hungarian wheat varieties.

Figure 4/B: Over-production of MsALR enzyme can increase drought stress tolerance of transgenic wheat plants (Experiment of J. Pauk, Cereal Research Non-Profit Co.).


CY-45 control wheat

AKR 284 transgenic wheat

167

PLA

NT

BIO

LOG

Y

Plants and animals have different developmental strategies for growth, yet individuals of both attain charac-teristic species-specific sizes constrained by their developmental genetic programmes. Additionally, growth can be significantly influenced by environmental factors, specifically so in plants. We are seeking to understand how plant growth is regulated at the molecular level and through which molecular mechanisms environmental signals are able to influence growth.

moleCUlaR ReGUlaToRs of planT GRoWTH

Zoltán MAGYAR / Principal Investigator

Gyöngyi Angéla BATTANCS / technician

The key factor in growth is the duration of cell proliferation and the timing of the exit from prolifer-ation to cell expansion and differentiation (figure 1). In plants, cell proliferation is largely concentrated in specialised regions known as meristems, which con-tain the stem cells. In meristems udifferentiated cells are produced by cell proliferation, and when these cells stop dividing, as they leave the meristematic region they differentiate into specific tissues. during differentiation, plant cells frequently increase their dna content by a modified mitotic cycle called en-doreduplication, a process of continous dna syn-thesis without intervening mitosis. We are interested in the molecular mechanisms which maintain stem cell activity in the meristems; control the balance be-tween cell division and differentiation and regulate the switch from mitotic cell cycle to endoreduplica-tion in a parallel fashion during organ development (figure 1). our main interest is in genes involved in the regulatory mechanisms which make the deci-sion to enter or leave the division cycle. We cloned a family of genes from the model plant Arabidop-sis thaliana called e2f transcription factors, which are related to genes that control the same process in animals. The canonical role for e2f transcription factors is to regulate cell cycle entry, but it is becom-ing apparent in many sytems that e2fs have broader functions and that, besides the regulation of cell

cycle transitions, they also coordinate cell prolifera-tion with cell growth and differentiation. according to the current model, e2fs can work both as positive and negative regulators of transcription, depending on their structure and on the function of the retino-blastoma (rB) tumour suppressor protein.

auxin is a plant growth hormone that regulates cell division in a concentration dependent manner; elevated auxin levels activate cell division in the meristems, whereas reduced amounts repress mi-tosis as cells leave the meristematic regions, and in parallel it enhances cell growth. We discovered that auxin increases the stability of the e2fB protein, and co-expression of e2fB with its dimerization partner dPa in plant cells could maintain cell pro-liferation in the absence of auxin. cytokinin, an-other plant hormone works opposite to auxin and the antagonistic functions of these two hormones appear to be a key mechanism which regulates mer-istematic functions. our recent work indicates that cytokinin can change the activity of e2fB from a transcriptional activator to a transcriptional repres-sor. our aim is to understand the molecular mecha-nisms leading to this switch in e2f activity during hormone signalling, and to identify the downstream targets of e2fs by using the chromatin immunopre-cipitation (chIP) method.

168

PLA

NT

BIO

LOG

Y

Figure 1: Mechanisms for organ size control. (a) Organ formation, exem-plified here by leaf development, consist of two stages. The first phase is underpinned by cell proliferation, characterized by intense macromo-lecular/cytoplasmic synthesis and rapid cell division. The second phase is characterized by cell expansion and differentiation. Differentiation takes place along a basipetal gradient (that is, from leaf tip to leaf base), as indicated here by the gradient in cell size and cell greening. The red arrow summarizes proliferative inputs, and the black arrow indicates the arrest of proliferation and the initiation of differentiation. (b,c) The two principal mechanisms for controlling organ size. Enlargement of organs can be produced by either (b) increasing proliferation signals or (c) de-laying the transition between proliferation and differentiation. In both cases the number of cells available for organ formation at the end of the proliferative phase is increased, but the underlying mechanisms are dif-ferent (Bögre et al., Genome Biology 2008)

The maintenance of stem cells in the plant meristems is crucial for the growing plant. Previous studies demon-strated that the retinoblastoma-related protein 1 (rBr1) is a stem cell regulator in plants. recently we have found that ectopic co-expression of e2fB and dPa het-erodimeric transcription factors increases the amount of stem cells in Arabidopsis roots (figure 2), which further supports the involvement of the plant rBr-e2f pathway in the regulation of stem cell maintenance.

Figure 2: Ectopic co-expression of E2FB with DPA increases the amount of stem cells in the root meristem of Arabidopsis as indicated by red ar-row. Position of the quiescent centre (QC) is indicated.

to unravel the molecular pathway controlling the switch from proliferation to differentiation, we use the first developing leaf pair of Arabidopsis thaliana as a model system. In this model system, cells gradually exit the mitotic cell cycle and engage into an endoredu-plication cycle as they start to differentiate (figure 1). our results indicate that different e2f proteins enter into complex with the single rBr1 protein at different developmental stages. We suggest that two e2fs from Arabidopsis, e2fa and e2fB have antagonistic func-tions when they form complexes with rBr1; e2fa-rBr1 keeps cells in the mitotic cycle, whereas binding of rBr1 to e2fB stimulates cell cycle exit during leaf development (figure 3). our major aim is to identify the downstream targets of these e2f transcriptional complexes. stress such as drought could change the activity of e2f complexes, which might lead to growth repression. We seek to understand how and why under stress conditions plants stop growing, and what hap-pens at the cellular level. molecular insights into this process and how it is signalled may lead to opportuni-ties to engineer crops with increased stress tolerance and, consequently, with higher yields.

Figure 3: Proposed working model for E2FA and E2FB function when they form complexes with RBR1.


Zoltán MAGYAR MOLECULAR REGULATORS OF PLANT GROWTH

169

PLA

NT

BIO

LOG

Y

Plants exhibit a remarkable developmental plasticity as a consequence of their sessile way of life. During plant ontogenesis, exogenous (environmental) and endogenous (developmental) signals converge on interlinked signal-ing pathways having dynamic impacts on plant form and function. We use molecular, biochemical and cellular approaches to reveal and characterize plant-specific pathways of cellular signaling that underlie this developmen-tal plasticity.

lInkInG CellUlaR sIGnalInG To planT DevelopmenT(fUnCTIonal Cell bIoloGY GRoUp)

Attila FEHÉR / Principal Investigator, Group Leader

Krisztina ÖTvÖS / staff scientistMónika DOMOKI / staff scientistJudit BÍRó / staff scientistCsilla FODOR-DUNAI / staff scientistManuela E. JURCA / staff scientistRóza NAGY / technicianGyöngyvér KATONA / technician

Signaling through ROP GTPases

rHo GtPases constitute a major branch of the ras superfamily of small GtP-binding proteins and func-tion as GdP/GtP switches that are activated by diverse extracellular stimuli. once activated, each rHo GtPase interacts with a wide spectrum of functionally diverse downstream effectors to initiate signaling pathways. In plants, a specific class of rHo GtPases exists forming the roP subfamily. Their involvement in various cellu-lar processes (e.g. cell elongation, tip growth, pathogen defence, hormone signaling etc.) has been demonstrated, but the signaling cascades they are involved in are hardly explored. It is more and more obvious that plants have evolved specific rHo (roP) GtPase effectors as com-pared to other eukaryotes. especially their links to up-stream receptor and downstream effector kinases seem to be unique and largely unknown (figure 1).

In our laboratory we identified a specific group of plant kinases exhibiting GtP-bound roP GtPase-dependent in vitro activity (figure 2). These kinases can be considered therefore the first potential roP GtPase effector kinases in plants. our aim is to identify further elements of this signaling pathway and to verify its biological significance using transgenic and mutant

plants. moreover, the structure of the plant roP-interacting kinases (rrKs) predicts a novel, specific way for their functional interaction with roP GtPases, as they do not have any GtPase-binding motif. Therefore we also aim to reveal the structural and biochemical background of this molecular interaction. furthermore, we are interested in the characterization of the link of upstream receptor kinases to roP GtPase signaling. In this field we collaborate with the group of dr. antje Berken (Germany).

Figure 1. Potential ways for the regulation of ROP GTPase activity by phosphorylation. The three indicated pathways are still hypothetical as supported only by in direct or in vitro data. Further regulatory steps based on the phosphorylation of ROP regulators and effectors can also be hypothesized but are presently unknown. Red arrows indicate activa-tion, blue arrows inhibition/inactivation, violet arrows both. ROP – „Rho of plants” GTPase; GEF – guanine nucleotide exchange factor; GAP – GTPase-activating protein; GDI – guanine nucleotide dissociation inhibitor;

170

PLA

NT

BIO

LOG

Y

Figure 2. In vitro activation of the Medicago RRK1 kinase by the consti-tutive active (CA) Rop6 GTPase. (For details: Dorjgotov et al. (2009) FEBS Letters 583:1175)

Nucleosome assembly protein (NAP)-related proteins (NRPs) and plant development

nucleosome assembly proteins (naPs) and their close relatives, the naP-related proteins (nrPs) are multifunctional proteins having roles, in addition to nucleosome assembly, in gene transcription, cell divi-sion, apoptosis and in various signaling pathways. We demonstrated that Arabidopsis nrPs have in vitro and in vivo histone phosphatase inhibitor activity. We also identified nrP-containing protein complexes associ-ated with cell cycle and transcriptional regulation. fur-thermore, it was also established, in collaboration with cropdesign nv, Ghent, Belgium, that nrP-overex-pressing rice plants gain a considerable yield increase. our further aim is to identify the components of the nrP-related protein complexes and to define their bio-logical functions in association with plant development and cell cycle regulation.

Figure 3. Comparison of the gene exprerssion pattern of young seeds from drought (D) or drought and heat (DH) treated Capelle Desprez (CA) and Plainsman V (PL) wheat cultivars using a custome microarray. (blue decreased, red increased relative expression)

Genomic approaches to reveal gene expression networks associated with plant developmental and stress tolerance pathways

We use microarray technology, est sequencing and real-time quantitative Pcr (rt-QPcr) to identify gene expression patterns associated with plant-specific developmental pathways such as somatic embryogen-esis, fertilization and seed development. related to these studies, a custom oligonucleotide microarray representing 15,000 wheat genes related to seed-de-velopment has been set up based on previous in silico gene expression analysis. The microarray was success-fully used to identify the effect of drought and heat on early seed development (figure 3). This work is in collaboration with dr. Beáta Barnabás (agricultural research Institute, Has, martonvásár, Hungary).


Attila FEHÉR LINKING CELLULAR SIGNALING TO PLANT DEVELOPMENT

171

PLA

NT

BIO

LOG

Y

Extreme environments such as high soil salinity, drought, high temperature or cold require special adapta-tion of plants. Moreover, environmental stress represents serious limitations for agricultural productivity. Osmotic stress develops in plants during drought or when the soil has high salt content. Our group studies the regulation of osmotic stress responses in the model organism Arabidopsis thaliana. Using genetic approaches such as T-DNA insertion mutagenesis and random cDNA transfer, we have identified several Arabidopsis genes, which have an influence on responses to salt, drought and oxidative stress or ABA signalling. Proline metabolism is used to study metabolic responses to such environmental constrains.

GeneTIC anD moleCUlaR DIsseCTIon of osmoTIC anD aba sIGnals (aRabIDopsIs moleCUlaR GeneTICs GRoUp)

László SZABADOS / Principal Investigator, Group Leader

Csaba KONCZ / scientific advisorágnes CSÉPLŐ / staff scientistLaura ZSIGMOND / staff scientistGábor RIGó / staff scientistCsaba PAPDI / staff scientist

Development of genetic tools to identify and study novel regulatory genes

t-dna insertion mutagenesis has been developed in Arabidopsis as an important tool for the identifi-cation and functional characterization of regulatory genes which can control osmotic stress tolerance. We have established a tagged mutant collection with an-notation of more than thousand insertion sites. using a promoter trap technique which employed the firefly luciferase as reporter gene, numerous in situ gene fu-sions have been identified where the t-dna insert was inserted in stress-responsive genes. a stress-responsive aBc transporter has been characterized, which was activated by high salinity, osmotic pressure and aBa.

screening for altered stress sensitivity, several tagged mutants have been identified with reduced stress toler-ance. In the case of the ppr40 mutant, germination and growth were hypersensitive to salt, high osmotics, ab-scisic acid (aBa) and oxidative agents. The insertion in-activated the PPR40 gene which codes for a previously unknown PPr domain protein, involved in the regula-tion of mitochondrial electron transport. recently we have developed an efficient way to identify and clone

novel regulatory genes through the construction and employment of the controlled overexpression system, which is based on a transformation competent cdna library. The utility of the system has been confirmed in three genetic screens. stress regulatory genes have been identified which control salt tolerance, aBa sensitivity and expression of stress-induced genes.

Figure 1. Use of the COS cDNA transfer system to identify Arabidopsis genes controlling stress responses. A) Screening for salt tolerance: iden-tification of salt tolerant plants under controlled in vitro conditions. B) Use of bioluminescence imaging to identify regulatory genes with the capacity to enhance the activity of the ADH1-LUC reporter gene. Arrow shows the position of a seedling with enhanced luciferase activity. C) Segregation of T2 generation seedlings with luciferase activity.

Mary Prathiba JOSEPH / Phd studentImma PEREZ SALAMó / Phd studentAnnamária KIRáLY / technicianIldikó vALKAI / technician

172

PLA

NT

BIO

LOG

Y

Proline metabolism and stress regulation

Proline accumulation during drought or salt stress is a well-known phenomenon in plants. In order to characterize regulation of proline accumulation, the Arabidopsis P5CS1 and P5CS2 genes, which encode the rate-limiting enzyme of proline biosynthesis, the pyrroline-5-carboxylate synthase, have been cloned and characterized in the last decade. We showed that functional divergence of the two P5cs genes is manifested in differential transcriptional regulation of these genes during plant development and in re-sponses to biotic and abiotic stress. moreover, differ-

ences in intracellular localization of the GfP-tagged P5cs1 and P5cs2 proteins suggested that intracellular compartmentalization is important to regulate house-keeping and stress-induced proline accumulation. The importance of proline accumulation in osmotic stress tolerance has been confirmed with loss-of-function insertion p5cs1 mutants, which showed enhanced salt sensitivity and oxidative damage. The p5cs2 mutations lead to embryo lethality, and therefore confirmed the essential housekeeping function of the P5cs2 gene.


László SZABADOS GENETIC AND MOLECULAR DISSECTION OF OSMOTIC AND ABA SIGNALS

173

PLA

NT

BIO

LOG

Y

Ferenc Nagytóth, r., Kevei, e., Hall, a., millar, a.J., nagy, f. and Kozma-Bognár, L. (2001). circadian clock-regulated expression of phytochrome and cryptochrome genes in Arabidopsis. Plant Physiol. 127: 1607-1616.

Kircher, s., Gil, P., Kozma-Bognár, L., fejes, e., speth, v., Bau-er, d., Ádám, É., schäfer, e. and nagy, f. (2002). nucleo-cyto-plasmic partitioning of the plant photoreceptors phytochrome a, B, c, d and e is differentially regulated by light and exhibits a diurnal rhythm. Plant Cell 14: 1541-1555.

Bancos, s., nomura, t., sato, t., molnár, G., Bishop, G.J., Kon-cz, c., Yokota, t., nagy, f. and szekeres, m. (2002). regulation of transcript levels of the Arabidopsis cytochrome P450 genes involved in brassinosteroid biosynthesis. Plant Physiol. 130: 504-513.

ulm, r., Baumann, a., oravecz, a., máté, Z., Ádám, É., oake-ley, J., schäfer, e. and nagy, f. (2004). Genome-wide analysis of gene expression reveals HY5 function in the uv-B response of Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 101: 1397-1402.

Hiltbrunner, a., viczián, a., Bury, e., tscheuschler, a., Kirch-er, s., tóth, r., Honsberger, a., nagy, f., fankhauser, c. and schäfer, e. (2005). nuclear accumulation of the phytochrome a photoreceptor requires fHY1. Curr. Biol. 15: 2125-2130.

dodd, a.n., salathia, n., Hall, a., Kevei, É., tóth, r., nagy, f., Hibberd, J.m., millar, a.J. and Webb, a.a. (2005). Plant cir-cadian clocks increase photosynthesis, growth, survival, and competitive advantage. Science 309: 630-633.

oravecz, a., Baumann, a., máté, Z., Brzezinska, a., molinier, J., oakeley, e.J., Ádám, É., schäfer, e., nagy, f. and ulm, r. (2006). constItutIveLY PHotomorPHoGenIc 1 is required for the uv-B response in Arabidopsis. The Plant Cell 18:1975-1990.

Locke, J.c., Kozma-Bognár, L., Gould, P.d., fehér, B., Kevei. É., nagy, f., turner, m.s., Hall, a. and millar, a.J. (2006). experimental validation of an expected feedback loop in the multi-oscillator clock of Arabidopsis thaliana. Mol. Syst. Biol. 2: 59.

Bancos, s., szatmári, a.-m., castle, J., Kozma-Bognár, L., shi-bata, K., Yokota, t., Bishop, G.J., nagy, f. and szekeres, m. (2006). diurnal regulation of the brassinosteroid-biosynthetic CPD gene in Arabidopsis. Plant Physiol. 141: 299-309.

ohnishi, t., szatmári, a.-m., Watanabe, B., fujita, s., Bancos, s., Koncz, c., Lafos, m., shibata, K., Yokota, t., sakata, K., szekeres, m. and mizutani, m. (2006). c-23 hydroxylation by Arabidopsis cYP90c1 and cYP90d1 reveals a novel shortcut in brassinosteroid biosynthesis. Plant Cell 18: 3275-3288.

Kevei, É., schäfer, e. and nagy, f. (2007). Light-regulated nu-cleo-cytoplasmic partitioning of phytochromes, J. Exp. Bot. 58: 3113-3124.

Kevei, É., Gyula, P., fehér, B., tóth, r., viczián, a., Kircher, s., rea, d., dorjgotov, d., schäfer, e., millar, a.J., Kozma-Bognár, L. and nagy, f. (2007). Arabidopsis circadian clock is regulated by the small GtPase LIP1. Curr. Biol. 17: 1456-64.

sáfrány, J., Haasz, v., máté, Z., ciolfi, a., fehér, B., oravecz, a., stec, a., dallmann, G., morelli, G., ulm, r. and nagy, f. (2008). Identification of a novel cis-regulatory element for uv-B induced transcription in Arabidopsis. Plant J. 54: 402-414.

Kerényi, Z., mérai, Z., Hiripi, L., Benkovics, a., Gyula, P., Lacomme, c., Barta, e., nagy, f. and silhavy, d. (2008). Inter-kingdom conservation of mechanism of nonsense-mediated mrna decay. EMBO J. 27: 1585-1595.

Pfeiffer, a., Kunkel, t., Hiltbrunner, a., neuhaus, G., Wolf, I., speth, v., Ádám, É., nagy, f. and schäfer, e. (2008). a cell free system for light dependent nuclear import of phytochrome. Plant J. 57: 680-689.

Imre Vasscser, K. and vass, I. (2007). radiative and non-radiative charge recombination pathways in Photosystem II studied by thermo-luminescence and chlorophyll fluorescence in the cyanobacte-rium Synechocystis 6803. BBA-Bioenergetics 1767: 233-243.

cheregi, o., sicora, c., Kós. P.B., Barker, m., nixon, P. and vass, I. (2007). The role of the ftsH and deg proteases in the repair of uv-B radiation-damaged Photosystem II in the cy-anobacterium Synechocystis Pcc 6803. BBA-Bioenergetics 1767: 820-828.

szilárd, a., sass, L., deák, Z. and vass, I. (2007). The sensitiv-ity of Photosystem II to damage by uv-B radiation depends on the oxidation state of the water-splitting complex. BBA-Bioen-ergetics 1767: 876-882.

vass, I., cser, K. and cheregi, o. (2007). molecular mecha-nisms of light-stress of photosynthesis. Ann. N.Y. Acad. Sci. 1113: 114-122.

Hideg, É., Kós. P.B. and vass, I. (2007). Photosystem II dam-age induced by chemically generated singlet oxygen in tobacco leaves. Physiol. Plant. 131: 33-40.

Hideg, É., Kós, B.P and schreiber, u. (2008). Imaging of nPQ and ros formation in tobacco leaves: heat inactivation of the water-water cycle prevents down-regulation of Ps II. Plant Cell Physiol. 42: 1879–1886.

Peca, L., Kós, P.B., máté, Z., farsang, a. and vass, I. (2008). construction of bioluminescent cyanobacterial reporter strains for detection of nickel, cobalt, and zinc. FEMS Micro-biol. Lett. 289: 258-264.

Kós, P.B., deák, Z., cheregi, o. vass, I. (2008). differential regulation of psbA and psbD gene expression, and the role of the different d1 protein copies in the cyanobacterium Ther-mosynechococcus elongatus BP-1. BBA-Bioenergetics 1777: 74-83.

Šnyrychová, I., ayaydin, f. and Hideg, É. (2009). detecting hy-drogen peroxide in leaves in vivo – a comparison of methods. Physiol. Plant. 135: 1–18.

vass, I. and cser, K. (2009). Janus-faced charge recombina-tions in photosystem II photoinhibition. Trends Plant Sci. 14: 20-205.

Győző GarabBarzda, v., Istokovits, a., sidimijev, I. and Garab, G. (1996). structural flexibility of chiral macroaggregates of light-har-vesting chlorophyll a/b pigment-protein complexes. Light-induced reversible structural changes associated with energy dissipation. Biochemistry-US 35: 8981-8985.

InsTITUTe of planT bIoloGYseleCTeD pUblICaTIons:

174

PLA

NT

BIO

LOG

Y

simidjiev, I., stoylova, s.s., amenitsch, H., Jávorfi, t., mus-tárdy, L., Laggner, P., Holzenburg, a. and Garab, G. (2000). self-assembly of large, ordered lamellae from non-bilayer lip-ids and integral membrane proteins in vitro. Proc. Natl. Acad. Sci. U.S.A. 97: 1473-1476.

Garab, G., Lohner, K., Laggner, P. and farkas, t. (2000). self-regulation of the lipid content of membranes by non-bilayer lipids. Trends Plant Sci. 5: 489-494.

Garab, G., cseh, Z., Kovács, L., rajagopal, s., várkonyi, Z., Wentworth, m., mustárdy, L., dér, a., ruban, a.v., Papp, e., Holzenburg, a. and Horton, P. (2002). Light-induced trimer to monomer transition in the main light-harvesting antenna complex of plants: Thermo-optic mechanism. Biochemistry-US 41: 15121-15129.

dobrikova, a.G., várkonyi, Z., Krumova, s.B., Kovács, L., Kostov, G.K., todinova, s.J., Busheva, m.c., taneva, s.G. and Garab, G. (2003). structural rearrangements in chloroplast thylakoid membranes revealed by differential scanning calor-imetry and circular dichroism spectroscopy. Thermo-optic ef-fect. Biochemistry-US 42: 11272-11280.

mustárdy, L. and Garab, G. (2003). Granum revisited. a three-dimensional model - where things fall into place. Trends Plant Sci. 8: 117-122.

Kovács, L., damkjaer, J., Kereiche, s., Ilioaia, c., ruban, a.v., Boekema, e.J., Jansson, s. and Horton, P. (2006). Lack of the light-harvesting complex cP24 affects the structure and func-tion of the grana membranes of higher plant chloroplasts. Plant Cell 18: 3106-3120.

Lambrev, P.H., várkonyi, Z., Krumova, s., Kovács, L., milo-slavina, Y., Holzwarth, a.r. and Garab, G. (2007). Importance of timer-trimer interactions for the native state of the plant light-harvesting complex II. Biochim. Biophys. Acta – Bioen-erg. 1767: 847-853.

mustárdy, L., Buttle, K., steinbach, G. and Garab, G. (2008). The three-dimensional network of the tylakoid membranes in pants: Quasihelical model of the granum-stroma assembly. Plant Cell 20: 2552-2557.

Krumova, s.B., dijkema, c., de Waard, P., van as, H., Garab, G. and van amerongen, H. (2008). Phase behaviour of phos-phatidylglycerol in spinach thylakoid membranes as revealed by P-31-nmr. BB A –Biomembranes 1778: 997-1003.

szabó, m., Lepetit, B., Goss, r., Wilhelm, c., mustárdy, L. and Garab, G. (2008). structurally flexible macro-organization of the pigment-protein complexes of the diatom Phaeodactylum tricornutum. Photosynth Res. 95: 237–245.

Yang, c., Lambrev, P., chen, Z., Jávorfi, t., Kiss, a.Z., Paulsen, H. and Garab, G. (2008). the negatively charged amino acids in the lumenal loop inf luence the pigment binding and conformation of the major light-harvesting chlorophyll a/b complex. Biochim. Biophys. Acta – Bioenerg. 1777: 1463–1470.

steinbach, G., Pomozi, I., Zsiros, o., Páy, a., Horváth, G.v. and Garab, G. (2008). Imaging fluorescence detected linear dichr-oism of plant cell walls in laser scanning confocal microscope. Cytometry 73a: 202-208.

tóth, sZ., Puthur, J.t., nagy, v. and Garab, G. (2009). experi-mental evidence for ascorbate-dependent electron transport in leaves with inactive oxygen-evolving complexes. Plant Physiol. 149: 1568–1578.

Garab, G. and van amerongen, H. (2009). Linear dichroism and circular dichroism in photosynthesis research. Photosynth Res. doI 10.1007/s11120-009-9424-4.

Zoltán GombosGombos, Z., Kis, m., Páli, t. and vígh, L. (1987). nitrate star-vation induces homeoviscous regulation of lipids in the cell envelope of the blue-green alga Anacystis nidulans. Eur. J. Bio-chem. 165: 461-465.

Wada, H., Gombos, Z. and murata, n. (1990). enhancement of chilling tolerance of a cyanobacterium by genetic manipula-tion of fatty acid desaturation. Nature (London) 347: 200-203.

Gombos, Z., Wada, H. and murata, n. (1992). unsaturation of fatty acids in membrane lipids enhances the tolerance of the cyanobacterium Synechocystis Pcc6803 to low-temperature photoinhibition. Proc. Natl. Acad. Sci. U.S.A. 89: 9959-9963.

Gombos, Z., Wada, H. and murata, n. (1994). The recovery of photosynthesis from low-temperature photoinhibition. Proc. Natl. Acad. Sci. U.S.A. 91: 8787-8791.

Gombos, Z., Kanervo, e., tsvetkova, n., sakamoto, t., aro, e.m. and murata, n. (1997). Genetic enhancement of the ability to tolerate photoinhibition by introduction of unsaturated bonds into membrane glycerolipids. Plant Physiol. 115: 551-559.

Kis, m., Zsiros, o., farkas, t., Wada, H., nagy, f. and Gombos, Z. (1998). Light-induced expression of fatty acid desaturase genes. Proc. Natl. Acad. Sci. U.S.A. 95: 4209-4214.

Hagio, m., Gombos, Z., vákonyi, Zs., masamoto, K., sato, n., tsuzuki, m. and Wada, H. (2000). direct evidence for require-ment of phosphatidylglycerol in photosystem II of photosyn-thesis. Plant Physiol. 124: 795-804.

Gombos, Z., várkonyi, Zs., Hagio, m., Iwaki, m., Kovács, L., masamoto, K., Itoh, s. and Wada, H. (2002). Phosphatidylglyc-erol requirement for the function of elektron acceptor plasto-quinone QB in the photosystem II reaction center. Biochemistry 41: 3796-3802.

várkonyi, Zs., masamoto, K., debreceny, m., Zsiros, o., ughy, B., Gombos, Z., domonkos, I., farkas, t., Wada, H. and szal-ontai, B. (2002). Low-temperature-induced accumulation of xanthophylls and its structural consequences in the photosyn-thetic membranes of the cyanobacterium Cylindrospermopsis raciborskii. an ftIr spectroscopic study. Proc. Natl. Acad. Sci. U.S.A. 99: 2410-2415.

domonkos, I., malec, P., sallai, a., Kovács, L., Itoh, K., shen, G., ughy, B., Bogos, B., sakurai, I., Kis, m., strzalka, K., Wada, H., Itoh, s., farkas, t. and Gombos, Z. (2004). Phosphatidylg-lycerol is essential for oligomerization of Photosystem I reac-tion center. Plant Physiol. 134: 1471-1478.

domonkos, I., Laczkó-dobos, H. and Gombos, Z. (2008). Lipid-assisted protein-protein interactions that support pho-tosynthetic and other cellular activities. Prog. Lipid Res. 47: 422-435.

Laczkó-dobos, H., ughy, B., tóth, s.Z., Komenda, J., Zsiros, o., domonkos, I., Párducz, Á., Bogos, B., Komura, m., Itoh, s. and Gombos, Z. (2008). role of phosphatidylglycerol in the function and assembly of Photosystem II reaction center, stud-ied in a cdsA-inactivated PaL mutant strain of Synechocystis sp. Pcc6803 that lacks phycobilisomes. BBA-Bioenergetics 1777: 1184-1194.


175

PLA

NT

BIO

LOG

Y

domonkos, I., malec, P., Laczkó-dobos, H., sözer, o., Klodaw-ska, K., Wada, H., strzalka, K. and Gombos, Z. (2009). Phos-phatidylglycerol depletion induces an increase in myxoxan-thophyll biosynthetic activity in Synechocystis Pcc6803 cells. Plant Cell Physiol. 50: 374-382.

Gábor V. Horváthoberschall, a., deák, m., török, K., sass, L., vass, I. Kovács, I., fehér, a., dudits, d., Horváth, G.v. (2000). a novel aldose/aldehyde reductase protects transgenic plants against lipid peroxidation under chemical and drought stresses. Plant J. 24: 437-446.

Hideg, É., nagy, t., oberschall, a., dudits, d., vass, I. (2003). detoxification function of aldose/aldehyde reductase during drought and ultraviolet-B (280-320 nm) stresses. Plant Cell Env. 26: 513-522.

Hegedűs a, erdei s, Janda t, tóth e, Horváth G, dudits d. (2004). transgenic tobacco plants overproducing alfalfa al-dose/aldehyde reductase show higher tolerance to low temper-ature and cadmium stress. Plant Sci. 166: 1329-1333.

Pettkó-szandtner, a., mészáros, t., Horváth, G.v., Bakó, L., csordás-tóth, É., Blastyák, a., Zhiponova, m., miskolczi, P. and dudits, d (2006). activation of an alfalfa cyclin-depend-ent kinase inhibitor by calmodulin-like domain protein ki-nase, Plant J. 46: 111-123.

dudits, d., cserháti, m., miskolczi, P., Horváth, v.G. (2007). The growing family of plant cyclin-dependent kinases with multiple functions in cellular and developmental regulation. In: Cell cycle control and development Ed.: Dirk Inze. Black-well Publishing, Oxford. (annual Plant rewievs, volume 32, pp. 1-30.)

Lendvai a., Pettkó-szandtner, a., csordás-tóth. É., miskolczi, P., Horváth, G.v., Györgyey, J. and dudits d. (2007). dicot and monocot plants differ in retinoblastoma-related protein sub-families. J. Exp. Bot. 58: 1663-1675.

miskolczi, P., Lendvai, Á., Horváth, G.v., Pettkó-szandtner, a. and dudits, d. (2007). conserved functions of retinoblastoma proteins: from purple retina to green plant cell. Plant Sci. 172: 671-683.

Zombori, Z., Jancsó, m., Zvara, Á., Pauk, J., Györgyey. J. (2008). Investigation of the effect of drought stress on the rice transcriptome. Acta Biol. Szeged. 52(1):143-145.

Kotogány, e., dudits, d., Horváth, G.v., ayaydin, f. (2009). a rapid and robust assay for detection of dna replication phase in plant cells and tissues by using ethynyldeoxyuridine. sub-mitted

secenji, m., Hideg, É., Bebes, a., Györgyey, J. (2009). transcrip-tional differences in gene families of the ascorbate-glutathione cycle in wheat during mild water-deficit. Plant Cell Reports (in press)

Gallé, a., csiszár, J., secenji, m., Guóth, a., cseuz, L., tari, I., Györgyey, J., erdei, L. (2009). Glutathione transferase activity and expression patterns during grain filling in flag leaves of wheat genotypes differing in drought tolerance: response to water deficit. J. Plant Physiol. doi:10.1016/j.jplph.2009.05.016 (in press)

Zoltán Magyarmagyar, Z., atanassova, a., de veylder, L., rombauts, s. and Inze, d. (2000). characterization of two distinct dP-related genes from Arabidopsis thaliana. FEBS Letters 486(1): 79-87.

vlieghe, K., Boudolf, v., Beemster, G.t., maes, s., magyar, Z., atanassova, a., de almeida engler, J., de Groodt, r., Inze, d. and de veylder, L. (2005). The dP-e2f-like gene deL1 con-trols the endocycle in Arabidopsis thaliana. Current Biology 15(1): 59-63.

magyar, Z., de veylder, L., atanassova, a., Bakó, L., Inzé, d. and Bögre, L. (2005). The role of the Arabidopsis e2fB tran-scription factor in regulating auxin-dependent cell division. Plant Cell 17(9): 2527-2541.

mészáros, t., Helfer, a., Hatzimasura, e., magyar, Z., serazet-dinova, L., rios, G., Bardoczy, v., teige, m., Koncz, c., Peck, s. and Bögre, L. (2006). The Arabidopsis maP kinase kinase mKK1 participates in defence responses to the bacterial elici-tor flagellin. Plant Journal. 48(4): 485-498.

Horváth, B.m., magyar, Z., Zhang, Y., Hamburger, a.W., Bakó, L., visser, r.G., Bachem, c.W. and Bögre, L. (2006). eBP1 regulates organ size through cell growth and proliferation in plants. EMBO Journal 25(40): 4909-4920.

Lopez, e., dillon, e., magyar, Z., Khan, s., Hazeldine, s., de Jager, s., murray, J., Beemster, G., Bögre, L. and shanahan, H. (2008). distinct light-mediated gene expression and cell cycle program in the shoot apex and cotyledons. Plant Cell 20(4): 947-968.

magyar, Z. (2008). Keeping the balance between prolifera-tion and differentiation by the e2f transcriptional regulatory network is central to plant growth and development. In: Plant Growth signaling ed. by László Bögre and Gerrit Beemster; springer; Plant cell monographs. vol. 10. pp 89-105.

Bögre, L., magyar, Z. and Lopez-Juez, e. (2008). new clues to organ size control in plants. Genome Biology 9(7): 226.

Attila FehérPasternak, t. P., Prinsen, e., ayaydin, f., Potters, G., asard, H., onckelen, H. a. and fehér, a. (2002). The role of auxin, pH, and stress in the activation of embryogenic cell division in leaf protoplast-derived cells of alfalfa. Plant Physiology 129: 1807-1819.

fehér, a., Pasternak, t.P. and dudits, d. (2003). transition of somatic plant cells to an embryogenic state. Plant Cell, Tissue, and Organ Culture 74:201-228.

Ötvös, K., Pasternak, t. P., miskolczi, P., domoki, m., dorjgo-tov, d., szűcs, a., Bottka, s., dudits, d. and fehér, a. (2005). nitric oxide is required for, and promotes auxin-mediated ac-tivation of, cell division and embryogenic cell formation but does not influence cell cycle progression in alfalfa cell cultures. The Plant Journal 43: 849-860.

domoki, m., Györgyey, J., Biro, J., Pasternak, t.P., Zvara, a., Bottka, s., Puskás., L.G., dudits, d. and fehér, a. (2006). Identification and characterization of genes associated with the induction of embryogenic competence in leaf-protoplast-derived alfalfa cells. BBA-Gene Structure and Expression 1759: 543-551.


176

PLA

NT

BIO

LOG

Y

Zhiponova, m.K., Pettkó-szandtner, a., stelkovics, É., neer, Z., Bottka, s., Krenács, t., dudits, d., fehér, a. and szilák, L. (2006). mitosis-specific promoter of the alfalfa cyclin-depend-ent kinase gene (medsa;cdKB2;1) is activated by wounding and ethylene in a non-cell division-dependent manner. Plant Physiology 140: 693-703.

szűcs, a., dorjgotov, d., Ötvös, K., fodor, c., domoki, m., Györgyey, J., Kaló, P., Kiss, G.B., dudits, d. and fehér, a. (2006). characterization of three rop GtPase genes of alfalfa (Medicago sativa L.). BBA Gene Structure and Expression 1759: 108-115.

fehér, a. (2006). Why somatic plant cells start to form em-bryos? In: a. mujib and J. samaj eds., somatic embryogenesis, Plant cell monographs, vol. 2., springer-verlag, Berlin Heidel-berg, pp. 85-101.

Pasternak, t., Ötvös, K., domoki, m. and fehér, a. (2007). Linked activation of cell division and oxidative stress defense in alfalfa leaf protoplast-derived cells is dependent on exog-enous auxin. Plant Growth Regulation 51: 109-117.

fehér, a., Ötvös, K., Pasternak, t. and Pettkó-szandtner, a. (2008). The involvement of reactive oxygen species (ros) in the cell cycle activation (G0-to-G1 transition) of plant cells. Plant Signaling & Behavior 3: 823-826.

fehér, a., Jurca, m.e., fodor-dunai, c. and dorjgotov, d. (2008). regulation of roP GtPase signaling at the gene ex-pression level. The Open Plant Science Journal 2: 21-30.

Barnabás, B., Jäger, K. and fehér, a. (2008). The effect of drought and heat stress on reproductive processes in cereals. Plant, Cell & Environment 31: 11-38.

Jurca, m.e., Bottka, s. and fehér, a. (2008). characterization of a family of Arabidopsis receptor-like cytoplasmic kinases (rLcK class vI). Plant Cell Reports 27: 739-748.

dorjgotov, d., Jurca, m.e., fodor-dunai, c., szűcs, a., Ötvös, K., Klement, É., Bíró, J. and fehér, a. (2009). Plant rho-type (rop) GtPase-dependent activation of receptor-like cytoplas-mic kinases in vitro. FEBS Letters 583: 1175-1182.

László Szabadosszabados, L., Kovács, I., oberschall, a., Ábrahám, e., Kerekes, I., Zsigmond, L., nagy, r., alvarado, m., Krasovskaja, I., Gál, m., Berente, a., rédei, G.P., Ben-Haim, a. and Koncz, c. (2002). distibution of 1000 sequenced t-dna tags in the Ara-bidopsis genome. Plant J. 32: 233-242.

Ábrahám, e., rigó, G., székely, G., nagy, r., Koncz, cs. and szabados, L. (2003). Light-dependent induction of proline bio-synthesis by abscisic acid and salt stress is inhibited by brassi-nosteroid in Arabidopsis. Plant Mol. Biol. 51: 363-372.

alvarado, m., Zsigmond, L., Kovács, I., cséplö, Á., Koncz, cs. and szabados, L. (2004). Luciferase gene trapping in Arabidop-sis: tagging of stress-responsive genes. Plant Physiol. 134: 1-10.

fabro, G., Kovács, I., Pavet, v., szabados, L. and alvarez, m.e. (2004). Proline accumulation and atP5cs2 gene activation are induced by plant-pathogen incompatible interactions in Arabi-dopsis. Mol. Plant Micr. Interaction 17: 343-350.

farkas, I., dombrádi, v., miskei, m., szabados, L. and Koncz, cs. (2007). Arabidopsis PPP family of serine/threonine phos-phatases. Trends Plant Sci. 12: 169-176.

székely, Gy., Ábrahám, e., cséplő, Á., rigó, G., Zsigmond, L., csiszár, J., ayaydin, f., strizhov, n., Jásik, J., schmelzer, e., Koncz, cs. and szabados, L. (2008). duplicated P5cs genes of Arabidopsis play distinct roles in stress regulation and developmental control of proline biosynthessis. Plant J. 53: 11-28.

Zsigmond, L., rigó, G., székely, Gy., Ötvös, K., szarka, a., darula, Zs., medzihradszky, K.f., Koncz, cs., Koncz, Zs. and szabados, L. (2008). Arabidopsis PPr40 connects abiotic stress responses to mitochondrial electron transport. Plant Physiol. 146: 1721-1737.

Papdi, cs., Ábrahám, e., Joseph, m.P., Popescu, c., Koncz, cs. and szabados, L. (2008). functional identification of Arabidop-sis stress regulatory genes using the controlled cdna overex-pression system, cos. Plant Physiol. 147: 528–542.


177

Laboratories of Core FacilitiesH-6726 Szeged, Temesvári krt. 62.H-6701 Szeged, P. O. Box 521, Hungary

Brc

LABO

RATO

RIES

OF

CORE

FA

CILI

TIES

178

LABO

RATO

RIES

OF

CORE

FA

CILI

TIES

sCIenTIfIC seCReTaRIaT

Anikó PáYScientific Secretary, Biological Research Center, Hungarian Academy of Sciences

H-6726 szeged, temesvári krt. 62.H-6701 szeged, P.o. Box 521., Hungary

Phone: 36-62-599-763, 36-62-599-774 fax: 36-62-432-576e-mail: [email protected]

Edina GONDA LEHMANNSecretary

Phone: 36-62-599-763e-mail: [email protected]

László SIKLóSManager of the International Training Course (ITC)

Phone: 36-62-599-611, 36-62-599-772e-mail: [email protected]

Tímea vARGASecretary for Foreign Relations, ITC Secretary


Grants Office:

Péter HEFFNERConsultant


Anita KISS and Klára REMÉNYIAssistants


179

LABO

RATO

RIES

OF

CORE

FA

CILI

TIES

Dna seqUenCInG laboRaToRY

Anikó PáY / Principal Investigator

Anikó BIRó / technician

Plasmid and fragment dnas have been sequenced using fluorescent dye-labelled (Bigdye) terminators and cycle sequencing method in this laboratory for several years.

from the beginning of 2003, the runs and the anal-yses of the sequencing reactions are done on an aBI Prism 3100 Genetic analyzer capillary sequencer. us-ing the 50 cm capillaries, the obtained dna sequences are reliable up to 600–700 nucleotides.

results can be transferred to a paper form (printed electropherograms) or to an electronic platform.

This laboratory operates as a custom-service facility and charges a fee for dna sequence determinations.


Edit KOTOGáNY / technician

The facscalibur system (Becton dickinson) can be used for many diagnostic and research applications including multicolour analysis of fluorescently labelled cells or cell particles. The facscalibur can measure several parameters: forward light scatter (fsc), side light scatter (ssc) and 3 or 4 different fluorescence pa-rameters.

our 2 flow-cytometers are used by several research groups of the Brc for many purposes, like dna con-tent analysis (monitoring cell cycle and apoptosis), studies on antibodies and cell-surface markers, in-vestigations of intracellular calcium level, research of transformation efficiency etc.

The facscalibur system also functions as a cell sorter.


floW-CYTomeTeR (faCsCalibur) laboRaToRY

180

LABO

RATO

RIES

OF

CORE

FA

CILI

TIES

nUCleIC aCID sYnTHesIs laboRaToRY

The aim of the Nucleic Acid Synthesis Laboratory is to deliver high-quality DNA- and RNA oligomers quickly and directly for the ongoing BRC projects. We produce a great variety of specifically modified nucleic acid se-quences on demand by chemical synthesis. We are also committed to the research of new applications of chemically modified oligonucleotides as structural elements and genomic tools in various living organisms. In order to realize this goal, we also take part in various scientific collaboration projects with a number of research laboratories.

In 1967, when a large research group of chemists headed by H. G. Khorana announced their project to produce the first chemically synthesized gene, there were a lot of scientists who expressed their doubts whether such a synthetic product would ever find real biological use. When we take a look at the develop-ment of molecular biology, we can firmly state that to-day’s achievements could not be reached without the results of synthetic nucleic acid chemistry.

Figure. Small and high-throughput automated nucleic acid synthesiz-ers capable of 2 and 48 simultaneous syntheses, respectively. The small plastic synthesis columns (blue arrow) and the reagent flasks (red ar-rows) can be observed.

The elements of modern nucleic acid synthesis

In the nucleic acid synthesis Laboratory we use the most up-to-date chemistry for synthesizing piec-es of dna with defined base-sequences. The chain length of these molecules ranges from 6 to 200 nucle-otides. Key elements of modern synthesis are the care-fully optimized chemistry called phosphoramidite method, the solid supported procedure which means that the growing chain is attached to a suitable solid particle throughout the procedure, and finally the full automation of the process. for this purpose, we use

Sándor BOTTKA / Principal Investigator

Mónika PUMMER / staff scientist

181

LABO

RATO

RIES

OF

CORE

FA

CILI

TIES

a high-throughput nucleic acid synthesizer for large scale production and a smaller instrument with high flexibility for building oligonucleotides bearing vari-ous chemical modifications.

Natural and modified sequences

oligonucleotides with natural structure, the work-ing horses of molecular biology are involved in many known procedures as primers, probes and even total synthetic genes. on the other hand, chemically modi-fied structures enable new applications – labeling, visualization, stabilization, conjugation, enzyme- and structural studies. as a collaboration partner, we syn-thesize almost all types of oligonucleotide derivatives, elaborate new compounds and actively participate in developing novel methods in diverse fields of molecu-lar biology.

Targeted inhibition of gene expression

Perhaps this application is the most exciting area of oligonucleotide research. These types of compounds are able to act directly on living cells and organisms as active compounds or drugs. oligonucleotides bind to the nucleic acids of the cells in a sequence-selective fashion, and thereby cause specific inhibition of gene expression. our own research project is devoted to the research of various applications of this phenomenon. The so-called antisense oligonucleotide and small in-hibitory rna projects are focused mainly to plant sys-tems, and the final goal is to develop a novel genomic research tool.


Sándor BOTTKA NUCLEIC ACID SYNTHESIS LABORATORY

182

LABO

RATO

RIES

OF

CORE

FA

CILI

TIES

laboRaToRY of fUnCTIonal GenomICs

László G. PUSKáS / Principal Investigator, Group Leader

Klára KITAJKA / staff scientistágnes ZvARA / staff scientist László HACKLER / staff scientistDalma MÉNESI / staff scientistZoltán KIS / Phd studentNóra FARAGó / Phd studentRozália CSAPó / technician

Microarrays are chemically activated glass slides with a large number of oligonucleotides, cdnas, pro-teins or drug-like compounds spotted on their surfaces in high density. They are novel and extraordinary tools for functional molecular biology providing a rapid and comprehensive approach to simultaneously monitor the different mutations in the genome, the expression levels of thousands of known and uncharacterized genes, and protein expression differences between diverse biologi-cal samples in a comparative way at different system levels (genome, transcriptome, proteome). since 2000, the Laboratory of functional Genomics in Brc has suc-cessfully applied the microarray technique for genomic, transcriptomic and proteomic research. The laboratory is equipped with all the high-tech instruments, hard-ware and software background necessary for microar-ray printing, reading and data analysis and validation: arrayer robot (microGrid II tas) that enables spotting high density microarrays (up to 25.000 spots/slide), a fully automated hybridization station (ventana) with precisely regulated hybridization conditions and a con-focal laser scanner (agilent technologies) which can read the arrays with high sensitivity and resolution, for data validation Quantitative real time Pcr machine (corbett research). We are currently building a local database containing all the information about the ex-periments using sun workstation (sun microsystem) as hardware and GenePix (spot analysis) and omniviz

(cluster analysis) as software background. This labora-tory works partly as a custom-service and has several scientific cooperations with other laboratories and in-stitutes all over Hungary and abroad.

Genome level: changes within the chromosome: deletion or amplification is quite frequent in vari-ous diseases. specific rearrangements, in many cases, are characteristic of the individual diseases and states. comparative genomic hybridization (cGH) is a rapid, high throughput, dna microarray based method that provides a lot of information about the genomic bal-ance of cells, mono- or trisomies, amplifications and deletions in a simple experimental procedure. cGH of-fers a new solution for amplification/deletion analysis applied so far, because it is extremely well applicable and high throughput way for the overall analysis of the whole genome. using this method we analyzed a clini-cal case. merkel cell carcinoma (mcc) was diagnosed in a woman’s upper lip. after a long tumour-free period, an anaplastic carcinoma with neuroendocrine features developed in her palatine tonsil, raising the possibility of a late haematogenous metastasis, a second field tu-mour, or a second primary tumour. The regional lymph nodes were devoid of metastasis. our aim was to reveal whether the tumours have a common origin. using ar-ray-based cGH and an improved doP-Pcr technique we could demonstrate that our protocol preserves the original copy number of different chromosomal regions

Using approaches of functional genomics, we are focusing on gene activity profiling, gene copy number altera-tions, methylation pattern analysis and protein expression profiling in different organisms, including human, rat, mouse, wheat, rice, carp, dog, wild dear. Global gene expression changes can be followed in diverse physiologic and pathologic states. This makes possible to reveal changes at the genome level to better understand diseases, stress resistance in plants and to follow the effects of drug treatments.

183

LABO

RATO

RIES

OF

CORE

FA

CILI

TIES

in amplified genomic dna more accurately than stand-ard doP-Pcr techniques. In the case of mcc the partly similar and partly different molecular patterns indicated a genetic relationship between the tumours, and exclud-ed the possibility that the tonsillar tumor was a metasta-sis. The common origin was further confirmed, namely out of 3 early markers (9p, 3p and 17p), two (9p and 17p) were common in both cancer samples. These findings suggest that a genetically altered field was the reason for the development of the tonsillar cancer; thus, it can be regarded pathogenetically as a second field tumour.

Transcriptome level: The most important and most informative application of dna microarrays is the parallel study of gene expression from different biological samples that focuses on the functionally active parts of the genome. The method has enabled large numbers of genes, from specific cell populations, to be studied in a single experiment. a primary goal of expression profiling studies is to characterize genes that are expressed abnormally. Global gene expression changes can be followed in diverse physiological and pathological states. dna microarrays with sets of syn-thetic oligonucleotides on their surface can be used to obtain a molecular fingerprint of the gene expression of cells. We have successfully applied the microar-ray technique to analyze transcriptome changes due to many different environmental effects. to study the dietary effects of essential fatty acids on gene expres-sion, we have monitored expression profiles of tissues obtained from transgenic animals, osteoporosis and inflammation animal models, bacteria and plants

under different stress conditions, human cell lines treated with different drugs, and human tissues of dif-ferent pathological states. We selected many different stress-response genes to create a special microarray for analyzing different toxicological stresses in differ-ent organisms. using this very specialized microarray a toxicological profile of many small molecules, drug-like compounds or any other biological or non-biolog-ical sample can be analyzed.

Proteome level: although transcript profiling offers a good opportunity to identify genes that play a role in diseases, even the complete mrna fingerprints have their limitations, since proteins carry out most of the cellular functions. numerous protein modifications, such as rna splicing and posttranslational modifica-tion (e.g. phosphorylations, glycosylations) are known that protein functions are dependent on. The genomic or the transcript sequence does not give full informa-tion about the different protein-protein interactions, how and where these interaction occur inside the cells under various conditions. to obtain detailed informa-tion about a complex biological sample, information about many proteins and protein-protein interactions is required. Protein chips are also used in our laboratory (commercially available – sigma, or in-house made) for screening protein expression and protein modifications in a high throughput manner. focused protein micro-arrays are planned to be developed in the future.


László G. PUSKáS LABORATORY OF FUNCTIONAL GENOMICS

Figure 1. DNA microarrays A: hybridized with Cy5 labeled cDNA probe obtained from human lymphocyte total RNA B: hybridized with Cy5 labeled cDNA (obtained from human healthy tissue total RNA) and Cy3 labeled cDNA (obtained from human tumor tissue total RNA).

A B

184

LABO

RATO

RIES

OF

CORE

FA

CILI

TIES

pRoTeomICs ReseaRCH GRoUp

Katalin F. MEDZIHRADSZKY / Principal Investigator, Group Leader

Éva HUNYADI-GULYáS / staff scientistZsuzsanna DARULA / staff scientistÉva KLEMENT / staff scientistHajnalka GYŐRI-DÜRGŐ / technicianágnes áRvA / technician

Proteomics research has become one of the most dynamic research areas. Well equipped Proteomics centers have been established all over the world. In-vestigating the proteome provides answers to exciting questions, such as, which genes are transcribed and eventually translated; in what form are these proteins biologically active; which other proteins they inter-act with; how are these processes controlled etc. mass spectrometry has become the method of choice for pro-teomics research. It is equally well suitable for protein identification, de novo sequencing, for the characteriza-tion of post-translational modifications, or other cova-lent labeling. It also can be utilized for investigating the 3d structure of proteins as well as the spatial organiza-tion of protein complexes. Last but not least, besides the qualitative characterization of the proteome mass spectrometry can also deliver quantitative results.

We have extensive collaborations within the Bio-logical research center and with academic organiza-tions in Hungary and abroad. Biological samples are provided by our collaborative partners. our tasks are the analytical sample preparation, chromatographic

fractionation if necessary, mass spectrometry analysis as well as data interpretation. obviously we provide identifications for 1d- or 2d-gel isolated proteins. We have been involved in the characterization of disulfide-bridges, preoteolytic cleavage sites, phosphorylation as well as ubiquitination. We have been developing novel analytical methods for the characterization of secreted as well as intracellular o-glycosylation.

We also provide services for a fee: protein identifi-cation as well as mass measurements.

our resources: analytical HPLc system for sample preparations; a nanoHPLc system, equipped with an autosampler (sunchrom/eldex) coupled with an LcQ fleet 3d ion trap (Thermo fischer scientific); a reflex III maLdI-tof ms (Bruker); and a nanoacQuItY uPLc system coupled with a Q-tof Premier mass spectrometer (Waters) (the latter two are shared with the researchers of the szeged university); in-house mascot and ProteinProspector servers.


185

LABO

RATO

RIES

OF

CORE

FA

CILI

TIES

bIoInfoRmaTICs GRoUp

Sándor PONGOR / Principal Investigator, Group Leader

Zoltán HEGEDŰS / Principal Investigatorádám KERÉNYI / staff scientistBeáta REIZ / Phd student

Molecular networks

structural descriptions in molecular sciences consist of entities such as atoms, residues, domains as well as (binary) relationships such as chemical bonds, sequential and spatial vicinity etc. The repre-sentation of genomic data follows the same rule, but the resulting molecular network descriptions (mnd) greatly vary in size and contain a large number of elements, many of which are uncertain (figure 1a). similarly to molecular structures, mnds can be studied in terms of patterns and similarities, but their most intriguing property is the analogy with networks found in other areas such as electrical and social networks, the Internet, etc. mnds allow one to ask questions about stability, robustness, error and attack tolerance, which are relevant to biological or biochemical systems. our group is interested in ap-plying this general approach to protein folding, pro-tein similarity analysis as well as the transcriptional regulatory networks of living cells.

Proteins, described either as sequences or as 3d structures, form a similarity space in which domain types appear as tightly connected clusters reminis-cent of small world networks. The distribution of the random similarities that are found between unre-lated groups, on the other hand, follows a scale free distribution, found in many other biological and

non-biological networks (figure 1B). our group is involved in collecting domain sequence data. results of a similarity search can be considered as adding a subnetwork to the existing network of similarities, and protein domain prediction schemes can be based upon the comparative analysis of the underlying similarity networks.

Figure 1A

Figure 1B

The current paradigm of molecular biology is shifting towards the interpretation of data produced by high-throughput methods. The new data sources allow one to study system-wide properties in molecular terms. We are developing novel, generalized knowledge representation schemes for the study of the topological proper-ties of molecular networks such as the regulatory networks in living cells and the similarity space of protein structures.

186

LABO

RATO

RIES

OF

CORE

FA

CILI

TIES

Systems biology: generic models of biological systems

Generic models of biological systems strive to cap-ture the logical structure of biological processes. In the field of visual pattern recognition we develop sim-plified models of neural activities that can capture the differences between continuous/discontinous, open and closed contours that are well distinguished by hu-man vision (collaboration with the cognitive science Group of Has-Bme and with the cognitive science department of Bme, Budapest). In the field of bacte-rial communication we develop models that include chemical communication between bacterial cells – the chemical language – as well as competition between cells, which will allow us to describe the development of local structures in bacterial communities (collabo-ration with IcGeB, trieste). our long-term goal is to apply the principles of generic models in the interpre-tation of bioinformatics, i.e. in biological datamining.


Protein family annotation

The identification of the tribbles family mem-bers’ role as regulators of signal processing systems and physiological processes, including development, together with their potential involvement in diabe-tes and cancer has generated considerable interest in these proteins. tribbles have been reported to regu-late the activation of numerous intracellular signal-ing pathways, with roles extending from mitosis and cell activation to apoptosis and modulation of gene expression. Within the framework of an international collaboration with the cardiovascular research unit of the university of sheffield (uK) we are involved in the bioinformatic investigation of the tribbles family using phylogenetics and functional protein pattern identification methods. our current research activity is focused on the bioinformatic analysis of the syn-theny, and the chromosomal regions where the genes are located.

Genome Bioinformatics

The Bioinformatics Group of Brc has special ex-pertise in large-scale bioinformatics data management systems that have become an integrating force in sys-tems biology, by providing common platforms and databases for different high-throughput experimen-tal technologies. one of the major focus points of the ongoing research activity is the bioinformatic evalu-ation of experimental data from new transcriptome profiling approaches using next generation sequenc-ing technology. We participate in an international col-laborative project with the aim of identifying marker genes involved in the immune response of Zebrafish in different pathological conditions.

Bioinformatics infrastructure resources

most biologists today need some kind of assistance in accessing and analyzing the data available through the Internet and in keeping up with the new analysis methods. Bioinformatics is relatively new in the Brc, and our group has been involved in establishing and maintaining the biocomputing infrastructure. We have developed Brc Bionet, an HttP-based intranet resource, which is accessible from any of the over four hundred Pcs and workstations in the laboratories. Brc Bionet's tools include:

• emBoss bioinformatic softwaresuit (~100 pro-grams);

• PIse graphical interface for emBoss package; • Library information and online access to scien-

tific journals;• medline alerting service;

Sándor PONGOR BIOINFORMATICS GROUP

187

LABO

RATO

RIES

OF

CORE

FA

CILI

TIES

• citation database; • a repository of local teaching materials • scientific toolbox (periodic table, fact sheets

about isotopes, vectors, restriction sites, etc.) • a collection of more than a thousand WWW

links of biological interest.We also provide institute-wide access to various

molecular modeling tools including the sYBYL pro-gram package of tripos Inc. covering the scientific discipline of molecular mechanics, molecular dynam-ic simulations, homology modeling, ligand docking and threading. Contact: [email protected]

Sándor PONGOR BIOINFORMATICS GROUP

188

LABO

RATO

RIES

OF

CORE

FA

CILI

TIES

CellUlaR ImaGInG laboRaToRY

Ferhan AYAYDIN / Principal Investigator, Group Leader

Edit KOTOGáNY / Phd studentZsuzsanna KóSZó / technician

our modern imaging center is equipped with a state-of-the-art confocal Laser scanning microscope, a fluorescence stereo microscope, a real-time Imag-ing Workstation and powerful image analysis com-puters with imaging software. With these modern microscopes we can perform protein localization and mobility analyses, three dimensional, time course dy-namic analyses of live cells, tissues and organisms. Thanks to the new imaging techniques and the devel-opment of new fluorescent dyes and proteins, today’s biological and medical research has increasingly be-come dependent on microscopy and image analysis. It is now possible to specifically label virtually any mol-ecule and directly probe its function in live cells by light microscopy.

This ability to visualize the dynamics of proteins in vesicles, organelles, cells and tissue has begun to pro-vide new insights into how cells function in health and

disease. such work yields unique mechanistic insight by directly illustrating the complex spatial-temporal dynamics of fundamental cellular processes such as mitosis, morphogenesis, polarization, embryonic de-velopment, membrane trafficking and cytoskeleton dynamics. many of these processes are highly dynam-ic and are challenging to image by traditional means. In this aim, we are strongly committed to the develop-ment and application of optical imaging methods that will enable us and others to understand the complex organization within and between cells.

Microscopy and cytometry application of a new cell proliferation assay

Labeling, detection and quantification of cells in the s-phase (dna synthesis) of cell cycle progression are crucial in characterizing the cellular responses to various treatments and genetic modifications. Bromo-deoxyuridine (Brdu) labeling of cells followed by an-tibody staining is the standard method for detecting cells in the s-phase. antibody detection of Brdu in-volves harsh treatments or nuclease digestion to facili-tate epitope access. moreover in plants cells, cell wall digestion is also necessary. These steps could interfere with cellular morphology and are time-consuming. We have optimized the recently developed ethynyl-

At the Cellular Imaging Laboratory, we are strongly committed to the development and application of modern optical imaging methods that will enable us and others to understand the complex organization within and be-tween cells. Our lab’s special interest is the functional analysis of cell division related proteins using both human cancer cells and tumor-like callus tissues of plants. In human cancer cells we analyze the role of SUMO (small ubiquitin related modifier) proteins. We are especially interested in their role during cancer development and me-tastasis. Our lab also takes part in various scientific collaboration projects with different research labs.

189

LABO

RATO

RIES

OF

CORE

FA

CILI

TIES

deoxyuridine (edu) method on plant cell cultures and seed-derived roots as well as on isolated plant nuclei using confocal laser scanning microscopy and flow cytometry.

SUMO and Human Cancer Therapy

small ubiquitin-related modifiers (sumo) are post-translational protein modifiers that are ligated to target proteins in a manner similar to ubiquitin. Post-trans-lational modifications play a cardinal role in carcino-genesis affecting important regulatory proteins, such as transcriptional factors, growth factors or oncopro-teins. sumo conjugation (sumoylation) also impacts many cellular pathways through modulation of tran-scriptional activity, protein stability and protein subcel-lular localization. sumo conjugation is necessary for cell division and several substrates of sumoylation are major players in oncogenesis, tumor suppression and cancer metastasis. Thus, it is conceivable to suggest that

alterations of the sumoylation network ultimately affect cancer-related molecular pathways.

Therefore the discovery of novel sumoylation-mod-ulatory molecules could yield potential medical appli-cations such as cancer treatment. In collaboration with the Laboratory of functional Genomics, Brc, szeged, we aim to exploit this possibility to discover new syn-thetic molecules that change the course of carcinogen-esis by modifying the sumo pathway. to achieve this goal, we are screening diverse small molecule libraries to discover novel compounds that interfere with the lo-calization and conjugation patterns of human sumo proteins using high-throughput live-cell confocal laser scanning microscopy. We have already identified many novel chemicals that affect sumo-1 localization, cell viability, morphology and cell division. We have also discovered new non-toxic autofluorescent chemicals that can be used as live cell organelle probes.

E-mail: [email protected]

Ferhan AYAYDIN CELLULAR IMAGING LABORATORY

190

LABO

RATO

RIES

OF

CORE

FA

CILI

TIES

Sándor Bottkaducza, e., Gáspár, r., márki, a., Gyula, P., Bottka, s., falkay, G. (2001) use of antisense oligonucleotides to verify the role of the α(1a)-adrenergic receptor in the contractility of the rat uterus post partum. Mol Pharmacol. 59: 1235-42.

Zádor, e., Bottka, s., Wuytack, f. (2002) antisense inhibition of myod expression in regenerating rat soleus muscle is fol-lowed by an increase in the mrna levels of myod, myf-5 and myogenin and by a retarded regeneration. Biochim Biophys Acta. 1590: 52-63.

Laczkó, I., váró, G., Bottka, s., Bálint, Z., Illyés, e., vass, e., Bertrand, J.r., malvy, c. and Hollósi, m. (2006). n-terminal acylation of the sv40 nuclear localization signal peptide en-hances its oligonucleotide binding and membrane transloca-tion efficiencies. Arch. Biochem. Biophys. 454: 146-154.

oroszi, L., Galajda, P., Kirei, H., Bottka, s., ormos, P. (2006) direct measurement of torque in an optical trap and its appli-cation to double-strand dna. Phys Rev Lett. 97(5): 058301.

Zhiponova, m.K., Pettkó-szandtner, a., stelkovics, e., neer, Z., Bottka, s., Krenács, t., dudits, d., fehér, a., szilák, L. (2006) mi-tosis-specific promoter of the alfalfa cyclin-dependent kinase gene (medsa;cdKB2;1) is activated by wounding and ethylene in a non-cell division-dependent manner. Plant Physiol.140: 693-703.

Bálint, Z., nagy, K., Laczkó, I., Bottka, s., végh, G.a., sze-gletes, Z. and váró, G. (2007). adsorption and self-assembly of oligodeoxynucleotides ontoa mica surface. J. Phys. Chem. c. 111: 17032-17037.

Wilhelm, I., nagyőszi, P., farkas, a.e., couraud, P.o., romero, I.a., Weksler, B., fazakas, c., dung, n.t., Bottka, s., Bauer, H., Bauer, H.c. and Krizbai, I.a. (2008). Hyperosmotic stress in-duces axl activation and cleavage in cerebral endothelial cells. J. Neurochem.107: 116-126.

Bertrand, J.r., malvy, c., auguste, t., tóth, G.K., Kiss-Ivánko-vits, o., Illyés, e., Hollósi, m., Bottka, s. and Laczkó, I. (2009). synthesis and studies on cell-penetrating peptides. Bioconjug. Chem. 20: 1307-14.

László PuskásKitajka, K., Puskás, L.G., Zvara, Á., Hackler, L. Jr,, Barceló-coblijn, G., Yeo, Y.K. and farkas, t. (2002). The role of n-3 polyunsaturated fatty acids in brain: modulation of rat brain gene expression by dietary n-3 fatty acids. Proc. Natl. Acad. Sci. U.S.A. 99(5): 2619-2624.

Gu, r., fonseca, s., Puskás, L.G., Hackler, L. Jr., Zvara, Á., du-dits, d. and Pais, m.s. (2004). transcript identification and profiling during salt stress and recovery of Populus euphratica. Tree Physiol. 24(3): 265-276.

Kitajka, K., sinclair, a.J., Weisinger, r.s., Weisinger, H.s., mathai, m., Jayasooriya, a.P., Halver, J.e. and Puskás, L.G. (2004). effects of dietary omega-3 polyunsaturated fatty acids on brain gene expression. Proc. Natl. Acad. Sci. U.S.A. 101(30): 10931-10936.

Jayasooriya, a.P., ackland, m.L., mathai, m.L., sinclair, a.J., Weisinger, H.s., Weisinger, r.s., Halver, J.e., Kitajka, K. and Puskás L.G. (2005). Perinatal omega-3 polyunsaturated fatty acid supply modifies brain zinc homeostasis during adulthood. Proc. Natl. Acad. Sci. U.S.A. 102(20): 7133-7138.

Kelemen, J.Z., Kertész-farkas, a., Kocsor, a. and Puskás, L.G. (2006). Kalman filtering for disease-state estimation from microarray data. Bioinformatics 22(24): 3047-3053.

tímár, J., mészáros, L., Ladányi, a., Puskás, L.G. and rásó, e. (2006). melanoma genomics reveals signatures of sensitivity to bio- and targeted therapies. Cell. Immunol.244(2): 154-157.

faragó, n., Kocsis, G.f., fehér, L.Z., csont, t., Hackler, L. Jr., varga-orvos, Z., csonka, c., Kelemen, J.Z., ferdinándy, P. and Puskás L.G. (2008). Gene and protein expression changes in response to normoxic perfusion in mouse hearts. J. Pharmacol. Toxicol. Methods. 57(2): 145-154.

Katalin F. MedzihradszkyHunyadi-Gulyás, É. and medzihradszky, K.f. (2004). factors that contribute to the complexity of protein digests. Drug Dis-covery Today 3: s3-s10.

Békési, a., Zagyva, I., Hunyadi Gulyás, É., Pongrácz, v., Kovári, J., nagy, a.o., erdei, a., medzihradszky, K.f. and vértessy, B.G. (2004). developmental regulation of dutPase in Drosophila melanogaster. J. Biol. Chem. 279: 22362-22370.

medzihradszky, K.f., darula, Z., Perlson, e., fainzilber, m., chalkley, r.J., Ball, H., Greenbaum, d., Bogyo, m., tyson, d.r., Bradshaw, r.a. and Burlingame, a.L. (2004). o-sulfonation of ser-ine and threonine - mass spectrometric detection and characteri-zation of a new posttranslational modification in diverse proteins throughout the eukaryotes. Mol. Cell. Proteomics 3: 429-440.

csizmók, v., Bokor, n., Banki, P., Klement, É., medzihradszky, K.f., friedrich, P., tompa, K.a. and tompa, P. (2005). Primary contact sites in intrinsically unstructured proteins: The case of calpastatin and microtubule-associated protein 2. Biochemis-try-US 44: 3955-3964.

ott, P.G., varga, G.J., szatmári, a., Bozsó, Z., Klement, É., medzihradszky, K.f., Besenyei, e., czelleng, a. and Klement, Z. (2006). novel extracellular chitinases rapidly and specifi-cally induced by general bacterial elicitors and suppressed by virulent bacteria as a marker of early basal resistance in to-bacco. Mol. Plant Microbe Interact. 19: 161-172.

Kurucz, É., márkus, r., Zsámboki, J., folkl medzihradszky, K., darula, Z., vilmos, P., udvardy, a., Krausz, I., Lukacsovich, t., Gateff, e., Zettervall, c.J., Hultmark, d. and andó, I. (2007). nimrod, a putative phagocytosis receptor with eGf repeats in Drosophila plasmatocytes. Curr. Biol. 17: 649-654.

Kiss, a., Balikó, G., csorba, a., chuluunbaatar, t., medzihrad-szky, K.f. and alföldi, L. (2008). cloning and characterization of the dna region responsible for megacin a- 216 production in Bacillus megaterium 216. J. Bacteriol. 190: 6448-6457.

szájli, e., fehér, t. and medzihradszky, K.f. (2008). Investigat-ing the quantitative nature of maLdI-tof ms. Mol. Cell. Pro-teomics 7: 2410-2418.

szöllősi, e., Bokor, m., Bodor, a., Perczel, a., Klement, É., medzi-hradszky, K.f., tompa, K. and tompa, P. (2008). Intrinsic structur-al disorder of df31, a Drosophila protein of chromatin deconden-sation and remodeling activities. J. Proteome Res. 7: 2291-2299.

Zsigmond, L., rigó, G., szarka, a., székely, G., Ötvös, K., darula, Z., medzihradszky, K,f., Koncz, c., Koncz, Z. and szabados, L. (2008). Arabidopsis PPr40 connects abiotic stress responses to mitochondrial electron transport. Plant Physiol. 146: 1721-1737.

CenTRal laboRaToRIesseleCTeD pUblICaTIons:

191

LABO

RATO

RIES

OF

CORE

FA

CILI

TIES

Sándor PongorKurucz, É., Zettervall, c.J., sinka, r., vilmos, P., Pivarcsi, a., ekengren, s., Hegedűs, Z., andó, I. and Hultmark, d. (2003). Hemese, a hemocyte-specific transmembrane protein, affects the cellular immune response in Drosophila. Proc. Natl. Acad. Sci. 100(5): 2622-2627.

vlahovicek, K., Kajan, L., murvai, J., Hegedűs, Z. and Pongor, s. (2003). The sBase domain sequence library, release 10: do-main architecture prediction. Nucleic Acids Research 31(1): 403-405.

Hegedűs, Z., czibula, Á. and Kiss-tóth, e. (2006). tribbles: novel regulators of cell function; evolutionary aspects Cell. Mol.Life Sci. 63(14): 1632-1641.

Hegedűs, Z., czibula, Á. and Kiss-tóth, e. (2007). tribbles: a family of kinase-like proteins with potent signalling regula-tory function. Cellular Signalling 19(2): 238-250.

Kuzniar, a., van Ham, r.c.H.J., Pongor, s. and Leunissen, J.a.m. (2008). The Quest for orthologs: finding Gene corre-spondences across Genomes. Trends Genet. 24: 539-551.

stockhammer, o., Zakrewska, a., Hegedűs, Z., spaink, H. and meijer, a. (2009). time resolved transcriptome profiling and functional analyses of the zebrafish embryonic host response to salmonella infection. J. Immunol. 182(9): 5641-5653.

Kuzniar, a., Lin, K., He, Y., nijveen, H., Pongor, s. and Leunissen, J.a.m. (2009). ProGmap: an integrated annota-tion resource for protein orthology. Nucl. Acids Res. 1-7 (in press).

netotea, s., Bertani, I., steindler, L., Kerényi, Á., venturi, v. and Pongor, s. (2009). a simple model for the early events of quorum sensing in Pseudomonas aeruginosa: modeling bacte-rial swarming as the movement of an "activation zone". Biology Direct 4: 6-6.

Ferhan AyaydinKovács, I., ayaydin, f., oberschall, a., Ipacs, I., Bottka, s., Pon-gor, s., dudits, d. and tóth, e.c. (1998). Immunolocalization of a novel annexin-like protein encoded by a stress and abscisic acid responsive gene in alfalfa. Plant J. 15(2):185-197.

seregélyes, c., mustárdy, L., ayaydin, f., sass, L., Kovács, L., endre, G., Lukács, n., Kovács, I., vass, I., Kiss, G.B., Horváth, G.v. and dudits, d. (2000). nuclear localization of a hypoxia-inducible novel non-symbiotic hemoglobin in cultured alfalfa cells. FEBS Lett. 482(1):125-130.

ayaydin, f., vissi, e., mészáros, t., miskolczi, P., Kovács, I., fehér, a., dombrádi, v., erdődi, f., Gergely, P. and dudits, d. (2000). Inhibition of serine/threonine-specific protein phos-phatases causes premature activation of cdc2msf kinase at G2/m transition and early mitotic microtubule organization. Plant J. 23(1):85-96.

ayaydin, f. and dasso, m. (2004). distinct in vivo dynamics of vertebrate sumo paralogues. Mol. Biol. Cell 15(12):5208-5218.

mukhopadhyay, d., ayaydin, f., Kolli, n., tan, s.H., anan, t., Kametaka, a., azuma, Y., Wilkinson, K.d. and dasso, m. (2006). susP1 antagonizes formation of highly sumo2/3-conjugated species. J. Cell Biol. 174(7):939-949.

székely, G., Ábrahám, e., cséplő, Á., rigó, G., Zsigmond, L., csiszár, J., ayaydin, f., strizhov, n., Jásik, J., schmelzer, e., Koncz, c. and szabados, L. (2008). duplicated P5cs genes of Arabidopsis play distinct roles in stress regulation and devel-opmental control of proline biosynthesis. Plant J. 53(1):11-28.

snyrychová, I., ayaydin, f. and Hideg, É. (2009). detecting hy-drogen peroxide in leaves in vivo, a comparison of methods. Physiologia Plantarum 135(1): 1-18.

Kotogány, e., dudits, d., Horváth, v.G. and ayaydin f. (2009). a rapid and robust assay for detection of s-phase cell cycle pro-gression in plant cells and tissues by using ethynyl deoxyurid-ine. Plant Methods (submitted)

CenTRal laboRaToRIesseleCTeD pUblICaTIons:

192

IMPR

INT

BRCBaja

55

Belgrád 5

Arad43

Budapest5

Debrecen47

Újszeged

Szeged

Tisza

Tisza

Tisza

Liget

Belvárosi híd

Kossuth Lajos sugárútKossuth Lajos sugárút

Bertalan híd

Derkovits fasor

Temesvárikörút

The location of bRC:

2009Published byanikó PáyPhone: +36-62-599-774email: [email protected]

PicturesBrc

Artwork and layout

Printed byJuhász nyomda, szeged

http://[email protected] a kiadvány a tÁmoP–4.2.3–08/1–2009-0009 projekt

keretében, az európai unió támogatásával, az európai szociális alap és az európai regionális fejlesztési alap társfinanszírozásával valósult meg.

table of contents table of contents6 i nno v ation lászló vÍgh deputy director general for...

Documents