bch3atb notes

7/25/2019 Bch3atb Notes

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MEMBRANES AND CELL BIOLOGY

Introduction to Biomembranes

• Where are membranes found?o Bind the cell, many internal membranes (organelles)o Have an exoplasmic face and a cytosolic face – each have different properties

• F !"#$%! %F &'&B !'*o "ompartmentation+Barrier – enables compartmentali ation of en ymes, metabolites and particular path-ayso *pecific transport – allo-s specific compounds to cross the barrier and may be passive or active ( #. dependent

pumps)o &aintenance of electrical potential – many membranes can generate and store an electrical potential difference

across the t-o sides (such as mitochondria)o #rapping light energy – some membranes can trap light energy and convert it to electrical or chemical energy (such

as chloroplasts)o .rovision of specific environment – for such things as synthesis and signal transductiono *ignal ecognition+#ransduction – cell to cell interaction etco *hape &aintenance – membrane has a particular shape -hich is often dictated by the cytos/eletal proteins attached

to the membrane -hich constantly trac/ and move around• .hospholipids are the main lipid constituents of most biomembranes

o Have an amphipathic structure – hydrophilic head group and hydrophobic fatty acyl tailso %ne tail is straight and the other is bent this is all dependent on the saturation

0ouble bond cause unsaturation and increase fluidity*aturated allo-s denser pac/ing and therefore ma/ing it more solid

• "1 **'* %F 1$.$*o PHOSPHOGLYCERIDES – phsophatidylcholine one of the main constituents of the plasma membrane

.H%*.H #$021"H%1$!', .H%*.H #$021'#H !%1 &$!', .H%*.H #$021*' $!' !0

.H%*.H #$021$!%*$#%1o SPHINGOLIPIDS (sphingomyelin)o STEROLS (cholesterol)o Huge number of lipids – over 344 species identified differing in the length of their tails and the degree of saturation

in the human plasmao &ost are derivatives of glycerol565phosphateo Fatty acids can vary in length and contain no double bonds if saturated and in unsaturated contain 7,8 or 6 double

bondso .lasmalogen contains one fatty acyl chained lin/ed by ester and one by ether to glycerol

Highly abundant in heart and brain ho-ever don9t /no- physiological significance• *.H$!:%1$.$0*

o 0erivatives of sphingosine -ith an amino alcohol -ith a long hydrocarbon tailo %ther fatty acyl chains are lin/ed by an amide bondo %ther sphinolipids have sphingosine lin/ed to a sugar residue or branched oligosaccharide

• *#' %1*o 'xample cholesterolo nalogous sterols have been found in plants and fungio Basic structure is a ;5ring hydrocarbono mphipathic – single hydroxyl group -hich is e<uivalent to the polar head groups in other lipidso #end to intercalate bet-een the acyl chains of the phospholipidso llo- the membrane to be fluid but also maintain some solid structure

• .H%*H.H%1$.$0* 0$*.' *'0 $! ='% * *%1 #$%! – they form &$"'11'*, 1$.%*%&' !0 B$1 2' o 0ependent on the length of the fatty acyl chains and the degree of saturation on the termperature, on the ionic

composition of the a<ueous medium, and on the mode of dispersal of the phospholipid in the solutiono &icelles rarely form from natural phosphoglycerides as they are too long instead -ould form a liposome



o

• 0eveloping the model for biomembrane structureo Bilayer has been noted for a long time 7>34s by obertson Hypothesi ed that the appearance of the membrane

came from the binding of osmium tetroxide to proteins and polar groups of lipidso obertson model -as missing the proteinso Formed free e5etching to confirm the structure of the bilayer

Free e biological sample and cut -ith a cooled /nife in a vacuum*ample splits along the interior membrane plane revealing t-o fracture faces and vie- through '&

o 7>@4 got an idea of -hat the bilayer loo/s li/e.hospholipid bilayer studded -ith proteins:iven that membranes are fluid in principle the attached proteins should be mobile

• ' . %#'$! !0 1$.$0* &%B$1' $! #H' B$1 2' ?o "an thin/ of mobility in t-o different -ays

1 #' 1 0$FF *$%! – can move -ithin a membrane leaf # !*A' *' 0$FF *$%! – Flip from one bilayer to another

o &ost energetically favorable is lateral foro #ransverse diffusion is very slo- for lipids and is completely unfavorable for proteinso 0'&%!*# #$%! %F 1 #' 1 0$FF *$%! – F 2' !0 '0$0$!

se hetero/aryon cells -here they -ould fuse cells from t-o different species -ith different mar/ers onthe cell surface so able to differentiateFused mice and human cells -hich had proteins eachAisualise through a flruorescence -here it -as seen that the proteins -ere on separate sides but after a fe-hours they -ould start to mix and get a mosaic cell

"ool at the end to stop diffusion and then add fluorescent antibodies – proteins are free to move and is notinhibited by protein synthesis (no ne- proteins being made) and its not an active process (deplete #.).urely diffusion of the proteins 'ssentially no membrane proteins are freely moving in the plane of the membrane at 74 degrees or lo-er Higher the temperature higher the fluidity

• 7>@8 *$!:' !0 !$"H%1*%! . %.%*' F1 $0 &%* $" &%0'1o #he phospholipids form a continuous lipid bilayer o #he proteins float freely in the sea of lipido .roteins may interact -ith the surface (peripheral) or be embedded in the bilayer (integral)



o .roteins are asymmetrically organi edo .rediction is that because the lipids and proteins are able to flo- freely through the lipid membrane then the

distribution of integral proteins should be random. %B1'&* W$#H #H$* . '0$"#$%! – proteins diffuse faster in artificial membranes than in plasmamembranes by a factor of 84

• *i e of the complex should not matter if they move freely• F . – F1 % '*"'!"' '"%A' 2 F#' .H%#% B1' "H$!:

o ll fluorescent dyes emit light at one length and after they have absorbed light of another -ave lengtho Ho-ever if a high intensity light is delivered to the dye the dye -ill photobleach meaning that the high intensity

light has rendered the dye unable to fluoresce (destroy the dye)o #he idea behind F . is to measure the ability of a molecule to move around over time so it can be used for

loo/ing at the distribution of molecules in a membranedd a fluorophore -hich must be covalently attached to -anted molecule – can visuali e using a lo- lightintensity 5 add high intensity light to destroy the fluorophore then see if it recovers over time$f there9s fluorescence over time that means ne- molecules are moving into the space

o .rovides <uantitative dataHo- much light returns relative to the amount of light that -as there before photobleaching – .' "'!#'"%A' 2Ho- fast did the fluorescent molecules migrate bac/ to the photobleached area – measurement of diffusional mobility

• . %B1'&* W$#H F1 $0 &%* $" &%0'1o Why do membrane proteins diffuse faster in artificial membranes than in natural plasma membraneso Ho- do molecular complexes become immobili ed at the cell surface or diffuse at a much slo-er rate than in a

single moleculeo

• 1ateral mobility of integral membrane proteins is increased in spherocytic erythrocyteso *.H' %"2#'* – smaller rounded red blood cells -ith a smaller surface area and is less flexible and durable

0efect in cytos/eletal proteins (spectrin) – can use to loo/ at diffusion ratesB" get their shape by a net-or/ of integral and peripheral membrane proteins anchored to the

cytoplasmic face of the B" membraneo "ompared the lateral mobility of membrane proteins in normal vs spherocytic miceo &any membrane experiments are done on erythrocytes as the only contain a plasma membraneo 1abelled the glycoproteins -ith a fluorescent dye and use F . to loo/ at diffusion rates

0iffusion -as much <uic/er in the ghosts of sperocyteso H2.%#H'*$* – normal erythrocyte have spectrin spanning nodes containing integral protein

"ytos/eletal structure leads to a non random distribution• F1 $0 &%* $" &%0'1 "H !:'*

o &ore mosaic than fluido .rotein content is much higher o Have microdomain structures that aren9t diffusing as freely as previously thought



The Structure of the Bilayer

• *inger and !icholson fluid mosaic model different no- as there more proteins in the bilayer than originally thought• .ercentages of proteins compared to lipids in the lipid bilayer are essentially 34+34

o

o $n mitochondria protein percentage is higher than lipid ma/ing it more mosaic than fluid• no- that proteins can form micro5domain in the membranes have evidence -ith the experiments done on spherocytes that

they are limited in ho- much they can diffuse laterally Cust because of the cytos/eletal structure• Fluid mosaic model suggests that lipids should be randomly distributed due to the fluidity

o !%# #H' " *' – 'A$0'!"' F %& :.$ !"H% * F% . %#'$!*.roteins that contain a lipid group attached and anchors the protein into the membrane – integral proteins&any :.$ anchors ranging in their functionality – en ymatic, antigenic and adhesion also play a criticalrole in a variety of receptor mediated signal transduction path-ays

• *2!#H'*$* !0 # FF$" $!: %F :.$ 1$! '0 . %#'$!*o &embrane bound proteins so synthesi ed on the rough endoplasmic reticulum and transported through vesicles from

' through golgi and then ta/e their role in the plasma membraneo :.$5anchored proteins are sorted into sub domainso 'D.' $&'!# – B %W! !0 %*' 7>>8

1oo/ at ho- :.$ proteins are traffic/ed and the micro environment they are inFound that the :.$ lin/ed proteins are solubili ed poorly in non5ionic detergents such as #riton D5744Would expect that phosphatidylinositol (:.$) -ould partition really -ell in detergent micelles1oo/ed at a :.$ protein – soluble and insoluble

• Found that initially it -as soluble but then start seeing more and more insoluble – someho- thesolubility is changing over time

$nsolubility -as not due to protein5protein interactions as it -as still insoluble in high salt or al/alinecarbonate buffer as they disrupt the protein5protein interactions #his means that the microenvironment isaffecting its solubility#a/e the fraction and visuali e though high performance thin layered chromatography to loo/ at thecomposition – less lipid lane one compared to lane 8 but proportionally cholesterol is much higher in the

insoluble fraction all relate to *.H$!:%1$.$0* – all proportionally upregulated – very much different proportion that contain the :.$ protein& "H H$:H' *.H$!:%1$.$0* $! #H' $!*%1 B1' F "#$%!"holesterol and sphingolipids also sho-n to aggregate in artificial membranes

• 1$.$0 F# &%0'1o #here are microdomains of lipids laterally -ithin the membrane not Cust one big sea of lipidso *phingolipids are only in the outer leaflet of the plasma membrane bilayer o Fatty acids and sphingolipids are very different from those of glycolipids, consisting of very long chain largely

saturated acyl chainso *phingolipids tend to have more hydroxyl groups, both in the long chain bases and fatty acid components than

glycerolipids, and these enter into hydrogen bonding and contribute to the stability of rafts



• 1oo/ing at the composition of the outer and inner leaflet – vastly different• 0ata obtained on different leaflets byG

o 0'#' &$!$!: 1$.$0 *2&&'# 2.H%*.H%1$. *' ** 2"an use phospholipases to clip the lipid on the outer membrane"1' A'0 F% & W% 10 "%!# $!G F ##2 "$0 !0 12*% .H%*.H%1$.$0!"1' A'0 – !% & 1 .H%*.H%1$.$0$nner leaflet is protected from the en yme#a/e the membranes and dissolve in an organic solvent and separate through thin layer chromatography"an loo/ at symmetric and asymmetric distribution – by seeing -hat is intact (large cleavage product or not)$f cell is apoptotic start seeing a loss in the asymmetry – have mar/ers

o 1#' ! #$A' &'#H%0 F% 0'#' &$!$!: * F "' 'D.%*'0 .*

nnexin A human intracellular protein – binds .*ecombinant fluorescent annexin A – Binds to exposed .* can use as a probe to measure apoptosis by flo-cytometry

• &'"H !$*& F% & $!# $!$!: *2&&'# 2o #W% H2.%#H'*'*G

#hat the rate of flip flop (transverse movement) is very slo-#here is a specific mechanism the operates to maintain amino phospholipids at the inner leaflet

• &' * $!: F1$. F1%. #'*o "an measure ho- long it ta/es to flip from one leaflet to another o $ncubate erythrocytes -ith phospholipid vesicles containing fluorescent or radiolabelled phospholipids – get

incorporated into the outerleafleto $ncubate the B" at 6@degrees (in the presence of an energy source)o emove samples at different ssess .1 asymmetry using phospholipase treatment or bac/ extraction of

phospholipids$n the table liposomes are artificial membranes that don9t contain any proteins and it ta/es days to flip flop$n B" -ith the presence of proteins flip flop -as much faster indicating that transverse movement is

protein dependent



o

o .hosphotidylserine and phosphotidylethanolomine are translocated very rapidly to the inner leaflet -hilst phosphatidylcholine remains mainly at the outer leaflet

.* and .' must be facilitated by facilitated or active transport• F "$1$##'0 # !*.% # B2G

o F1$.. *'* for .* and .' and are #. 0'.'!0'!# (% #' #% $!!' )o F1%.. *' for ." and sphingolipids and are #. 0'.'!0'!# ($!!' #% % #' )o *" &B1 *' for cholesterol and are #. $!0'.'!0'!# ('$#H' W2)

ctivation of scramblase cause loss of phospholipid asymmetry eat me signal that is important for cellular senescence or apoptosis

• 0'#' &$!$!: # !*1%" #$%! #'*o .repare membrane (erythrocyte ghosts) – can -ash, -ash and reseal, disrupt by sonicating

and reseal0epending on ho- you treat them they can be lea/y or disrupted by sonication and reform -here you getinside out vesicles

o dd fluorescent analogue and incubate -here it -ill readily incorporate into the outer leafletsually have one longer acyl chain and one shorter acyl chain -hich contains the fluorescent molecule

o

Wash membranes -ith buffer containing B* (very efficient at binding free fatty acid) binds only to the outer leaflet – bac/ extraction of analogueo ecover membranes by centrifugation and solubili e in detergento 0etermine the fluorescence of the extract

• B* " ! B' *'0 #% B " 'D# "#o B* preferentially binds to the longer acyl chain of the analogue and then can extract

o

• 0oing these experiments to determine translocation of a flippase it -ould be recommended to use a molecule that doesn9thave any modification to the head group as the flipping proteins recogni e the different lipids through their groups $f there9sa modification you -ould al-ays -ant it to be bound to the acyl tail

• &odifications can ma/e the lipid more polar and therefore may slightly change results of an experiment• &%0$F$"#$%!* "H !:' . %#'$! !0 #H' 'F% ' " ! "H !:' $!#' . '##$%! %F #H'

'D.' $&'!#• $* # !*1%" #$%! . %#'$! 0'.'!0'!#

NOTE:Erythrocyte

Ghosts Are Just

the Membraneof the

ErythrocyteWhere

aemo!"ob#nas Been



o Have four populations of cells – t-o treated -ith n5ethylmaleimide (rective to-ards thiols, going to modify cysteineand therefore destroy en yme activity) and have plus or minus B* (after bac/ extraction) – other population of cells are biotenilated (mar/er) Cust to differentiate

o #hrough flo- cytometry get differentiation of cellso !'& /noc/ out all en ymatic activity including flippase – before B* there9s a high amount of fluorescence

because it has all moved to outer leaflet nothing translocated in – add B* no fluorescence as its bac/ extracted out

o

Membrane Proteins

• &embrane proteins constitute 64 of cellular proteins• ey components of all living organisms, constituting more than 64 of all cellular protein – signal transduction• &ore than half of F0 approved drugs bind to membrane proteins yet they remain the most challenging targets for structural

determination due to the hydrophobic nature of interacting -ith lipids• Biomembranes have an extremely high amount of proteins• #2.'* %F &'&B !' . %#'$!*

o ssociated -ith the membrane in many -ays – broadly defined as membranes that are integral – they themselvesare associated -ith the membrane in some -ay

Have a hydrophobic part that allo- them to anchor into the membrane%ther broad group are the peripheral proteins – only for a protein5protein interaction

• ssociated -ith something that is integral*ome membrane protein interactions are <uite transient

o

• ! #%&2 %F ! $!#': 1 &'&B !' . %#'$!o Has a transmembrane section that is hydrophobico

#here is al-ays an absolute asymmetry of proteins – can never be flipped around the other -ay, only has oneorientation in the membrane

o Have a protein that interacts -ith the extracellular side – can contain disulfide bridges for non5reducing conditionsalso have modifications that are added in the golgi

• *%1 B$1$*$!: &'&B !' . %#'$!*o #ypically using non5ionic detergents to disrupt the bilayer o .rotein enters the solution as a protein lipid detergent complexo #he lipid also gets solubili edo !eed to reconstitute bac/ into the membrane and get full activity bac/

0etergent to solubili e the integral proteins (to bind to the hydrophobic parts) using an amphipathic li/emolecule go through a process of dialysis to get rid of the detergent add phospholipid bac/ to try andmimic its native environment and then do activity assays



• $F F% .' $.H' 1 . %#'$!*o Bonds holding proteins – !oncovalent interactions such as ionic bonds, hydrophobic interaction, hydrogen bonds

and van der -aals forceso ust need to disrupt these bondingso 'D# "#$!: .' $.H' 1 . %#'$!*

#reatment -ith al/aline buffers (&%*# "%&&%!) or acidic buffersse of metal chelators#reatment -ith high ion strength or denaturing agents#reatment -ith organic solvents or sonication of membrane fractions

• .' $.H' 1 % $!#': 1

o

o .: – is al-ays soluble and is therefore peripheralG *'03. only predominantly in supernatant -ith triton thereforeis integralG *'"63. – peripheral due to being in the supernatant -ith high salt and high buffer (can be disrupted)

activity for integral inactiveneeds to reconstituted – peripheral is active $!#': 1 – High hydrophobic amino acids at externalsurface

o $ntegral proteins are very difficult to -or/ -ith ho-ever are extremely important to function as they are insoluble,can only be extracted -ith detergents – -hen extracted are often flexible and unstable (precipitate) – also have manymodifications so cannot Cust express in a bacterial cell

• "an perform hydropathy plots to predict transmembrane regionso #he polarity+hydropathy scale describes that change in free energy associated -ith transfer from a non5polar

environment to an a<ueous environmento negative change in free energy implies that a transfer -ill be favourableo #he more positive the value from the plot the more hydrophobic are the amino acidso Hydropathy plot predictions -or/ -ell for single transmembrane segment proteinso .' %! .1%# 5 H20 %.H%B$"o . %B1'&* – some transmembrane segments are not predicted due to amphipathic helices in ion channels

pea/ in a hydropathy plot does not prove the existence of a transmembrane helix"an9t be used for beta barrel

Techniques to Study Membranes



• 1$.%0%&$"*o *ub5branch of metabolomics -hich includes lipids, sugars, toxins etco 1ipidomics is further bro/en do-n intoG lipid isolation, lipid analysis, path-ay analysis and lipid5protein

interactionso "H 11'!:'* – a lot of chemical diversity, not coded for, no good predictive toolso 'merging field -ith slo- progress but advancing -ith advances in technologyo s opposed to genomic and proteomics there is no information that can predict the number of individual lipid

molecules present in an organismo lot of structural and chemical diversity in the lipidome for example glycerophospholipids are one class that can

have ten different headgroups and t-o tails in -hich can have 64 or more molecular structures giving incrediblediversity

o %ver 344 distinct lipid species identifiedo Have heterogeneity and different classes but membrane lipids and sphingolipids have a complex life they can

undergo extensive en ymatic as -ell as non5en ymatic modifications -hich can change their properties andfunctions – 00$!: !%#H' 1 2' %F "%&.1'D$#2

o #here9s also a large dynamic range in lipid concentrations -ithin a membrane and the concentration changes arehighly variable bet-een biological replicates

• $*%1 #$%! %F &'&B !'*o For example -ant to purify rat liver mitochondriao First need to homogeni e the tissue also Cust -ant one mitochondrial membrane and not the membranes of

everything else inside the cello &itochondria ma/e a -ide reticulated net-or/ o &itochondria and the ' are stuc/ together they have contact points so it is difficult to isolate Cust mitochondrial

membraneo eleasing the organelles – ta/e the tissue and $*%1#' the cell type by dissection or digestion then do differential

centrifugation after isolation need 0$* .#$%! by shearing (homogeni ation) or hypo5osmotic shoc/ to brea/ open and release the organelle

!eed to choose the correct disruptiono #o separate the organelles use differential centrifugation (process of repeating centrifugation of supernatants at

selectively higher speeds) – probably can9t ever purify Cust enrich the purification (going to have somecontaminations of other membranes) – larger particles settle faster than smaller and use density gradientcentrifugation ( #'5I%! 1 "'!# $F : #$%!)

• WH # #% 0% B% # ' !0 &$#%"H%!0 $ &$D$!:o sually say that mitochondria are in the post 74,444g pellet – -ill enrich for mitochondria but have an enriched

heavy membrane fraction as it contains significant portions of the ' o Brea/ ' 5mitochondria contacts -ith limited proteolysis follo-ed by a gradient centrifugation

' after homogeni ation J &icrosomeso %nce homogeni ed need to assess the degree of purity – can be done by Cust loo/ing at it

=uantitatively can use antigenic mar/ers (protein, lipid and carbohydrate), en yme mar/ers or microscopyo 1oo/ at different en yme mar/ers of different organelles to chec/ for contamination

• 12*%*%&' . $F$" #$%!o "an do antigenically and use -estern blot – issue for using -estern blot is the limit of detection is un/no-n

!eed a standard for comparison of diluted, enriched fraction to see -hen it disappears• WH2 1$.$0 "%&.%*$#$%! $! &'&B !'* $* $&.% # !#

o Ho- do lipids interact -ith membrane proteins to affect their function?:ross changes in the transmembrane regions of membrane proteins do not occur -hen membrane proteinscarry out their functions – must be subtle effects

o &embrane cross5over experiments#he influence of membrane lipid composition on the molecular activity of a maCor membrane protein (!aK

pump) -as examined as a test of the membrane pacema/er theory of metabolism$solate microsomal membranes from the /idneys of cattle and crocodiles -here similar !aK pumpconcentration -ere found but cattle membranes sho-ed a four to five fold higher en yme activity at

physiological conditions&olecular activity of the !aK pumps from both species -ere fully recoverable -he delipidated pumps -erereconstituted -ith (heat inactivated) membranes from the original source0id cross over -here the membranes -here reconstituted -ith the opposite species



• When cattle reconstituted -ith crocodile membranes activity -ent do-n but -hen crocodilereconstituted -ith cattle membranes activity -ent up

esults had supported the membrane pacema/er theory of metabolism and suggest the membrane lipidsand their polyunsaturates play a significant role in determining the molecular activity of the sodium pump

o &embrane proteins are not rigid entities around -hich the lipid bilayer distorts to provide the strongest interactionsather both the lipid and the protein molecules -ill distort to provide the best interaction, -ith the result that proteinfunction -ill be affected by the structure of the surrounding lipid bilayer

o #here are specific re<uirements for a small number of tightly bound lipids acting as co5factors for the proteinso #he conformation adopted by a membrane protein in a lipid bilayer -ill be such that the hydrophobic thic/ness of

the protein -ill be close to the hydrophobic thic/ness of the surrounding lipid bilayer o #here are specific interactions bet-een the lipid headgroups and the protein

• A$* 1$*$!: &'&B !'*o 1$:H# &$" %*"%.2 – easy to perform but limited resolutiono F1 % '*"'!"' &$" %*"%.2 – label a particular component in a cell -ith a fluorescent tag and vie- using a

microscope -ith a fluorescence attachmento $&& !%F1 % '*"!'"' &$" %*"%.2 – .repare a specimen for light microscopy by placing a specimen

-hich is usually fixed or permeabilised onto a microscope slide add an antibody that recogni es a particular component and allo- it to bind -ash a-ay the excess JL add secondary antibody that is fluorescently labeledantibody -ith F$#" allo-ing it to bind to primary antibody -ash a-ay excess vie- fluorescence microscopy

o $&& !%F1 % '*"'!"' W$#H !#$ " 0H' $! !#$B%02 – cadherins are members of a multigenefamily mediating calcium dependent cell5cell adhesion (plasma membrane mar/er)

o F1 % '*"'!# "H$&' $" . %#'$! – :F. as a probeo "H$&' $" F1 % '*"'!# . %#'$! – &a/e chimeric gene coding :F. lin/ed to protein D transfect cell

-ith gene construct :F.5D is produced and correctly locali ed by the cell fluorescent mar/er examine byfluorescence miscroscopy

o "%!F%" 1 &$" %*"%.2 – allo-s imaging of a particular focal plane -ithin the specimen"an generate time lapse and 60 reconstructions

o '1'"# %! &$" %*"%.2 !0 $&& !%'1'"# %! &$*" %*"%.2

Introduction to Cell Signalling

• #here are diverse signaling events in living organismso %rganisms are able to interact -ith their environment and are able to sense the extracellular environment viaG

&olecules dissolved in -ater or air that bind to cell surface receptors or enter through pores or transporters'xtracellular solid substrates&echanical interactions&onitoring light, temperatire, pressure and movement

o ble to sense neighbouring cells viaG0irect cell to cell Cunctions through -hich molecules are exchanged'xchange of diffusible molecules -hich bind to cell surface receptors or enter through pores or transporters&echanical interaction

• What happens -hen a signal arrives in terms of signal transduction?o !eeds to be a primary signal usually by a receptor (some on cell surface some inside the cell) need transmission

of that receptor into the cell caused by a conformational change to its protein follo-ing ligand binding signaltransduction (passing the message through different components) get an amplification (8 nd messenger molecules) canget a cascade reach final destination response



o

o "an affect existing mechanisms or expression of particular genes• H%W 0% "'11* '*.%!0 #% '!A$ %!&'!# 1 *$:! 1*

o • #2.'* %F *$:! 11$!:

o

o utocrine could be an example of a cancer cell• $!#' "'11 1 "%&& !$" #$%!

o What ma/es a good receptor? *pecific to the receptor, high affinity of ligand to receptor, rapid response, short half5life – very varied bet-een cells and receptors

o B' B1' #% # A'1 F %& *$#' %F & ! F "# ' #% # :'# *$#' '1 #$A'12 ' *$12o & 0', &%B$1$*'0 !0 # !'0 %FF '1 #$A'12 = $" 12o *.'"$F$"$#2 !0 H$:H FF$!$#2 (don9t need high concentration to initiate a reaction)

• 0 '! 1$!' !0 #H' F$:H# % F1$:H# '*.%!*'o fter a threat is detected a sympathetic response is relayed from the hypothalamus to the adrenal glandso ch is released from the pre ganglionic fibers increasing the sodium conductance of the modified post ganglionic

neuronso 0epolari ation of the cell membrane leads to an influx of calcium through voltage sensitive channelso Hormones stored in granules are released by the regulated secretory path-ay into the blood streamo #he increase in adrenaline and noradrenaline occur -ithin seconds and their effect is exerted -ithin secondso drenaline is also removed rapidly -ith a half5life of 74 seconds -ith various mechanisms to degrade adrenaline

• #he concentration of a molecule can be adCusted <uic/ly if the half5life is shortMo $f you -ant a rapid response the half life must be shorto 0ifference in half life changes the response time in molecules

• 'FF'"#* %F 0 '! 1$!'



o #here9s a plasticity in responseG adrenaline has many affects in different target organso $n the liver it increases the conversion of glycogen to glucose (increasing blood sugar levels)o $n the heart it increases the rate and force of the heartbeat and causes arterioles to dilate (can send more blood and

%8)o $n the lungs the breathing rate is increased and the arterioles in the lungs dilateo Blood vessels of the /idney, s/in and gut constrict (send less blood to unnecessary organs)o nd the pupils of the eye dilateo N* &' &%1'" 1' 'D' #$!: 0$FF' '!# 'FF'"#* $! 0$FF' '!# #$** '*

"omes do-n to the fact of different receptors in different tissues ( '"'.#% 0'.'!0'!#)• $s adrenaline a good signaling molecule? 2'*

o #ravels through the blood and is soluble so it can easily move aroundo *tored in the adrenal medulla and is released and degraded very <uic/lyo 'licits different responses through different receptors

• #H '' " "$ 1 " $#' $ #% :%%0 '"'.#% o #he receptor has to have *.'"$F$"$#2 – detecting only the molecule that the cell -ishes to perceiveo #he binding FFF$!$#2 must be such that is can detect the signaling molecule at the concentration at -hich it is

li/ely to be found in the vicinity of the cello #he receptor must be B1' #% # !*&$# #H' &'** :' that the signaling molecule conveys to the cell

• '"'.#% 1$: !0 *.'"$F$"$#2o #he structure of the beta adrenergic receptor -ith agonist bound has been found and all bind in the poc/et in the

same particular -ayo *eries of polar and non5polar interactions in binding of ligand to receptor

• '"'.#% 5*$:! 1 B$!0$!: FF$!$#2o Aie- a ligand binding to a receptor as a simple reversible reactiono Have a ligand receptor complex that is at e<uilibrium -ith free ligand and free receptor

o

o t e<uilibrium the rate of dissociation is the same as the rate of formation and can be formulated as• d J /off+/on

• #he lo-er the d the tighter the binding and this is seen by a lo-er off as compared to on – tighter binding implies a more stable receptor5ligand complex

• 'D.' $&'!# 112 0'#' &$!$!: 0$**%"$ #$%! "%!*# !#o .' F% &$!: 0$ '"# B$!0$!: ** 2*

1oo/ing at the '.% receptor ta/e out cells lines and incubate -ith increasing concentrations of radiolabelled '.% centrifuge to remove unbound '.% "ount reactivity stuc/ to cells (not all theradioactivity -ill be due to binding Cust to receptor) some -ill be non5specific binding (must eliminate)generate three curves Jtotal binding BJ non5specific binding "Jspecific binding

o

:et non5specific binding by adding unradiolabelled '.% and flood the cell -ith it and you -ill lose the non5specific binding sites

o Wor/s -ell -ith high affinity binding – many ligands don9t bind to such a high affinity and the approach -on9t -or/ o $f d is greater that 745@& -here off is relatively large -hen compared to on then need to perform a

"%&.'#$#$%! ** 2o "%&.'#$#$%! ** 2 – #o detect -ea/ binding of a ligand to its receptor -here you use another ligand -hich

binds -ith a high affinity$f using alprenol (antagonist) use adrenaline as the competitor se constant amount of radiolabelled alprenolol0isplace the alprenolol by adding increasing amounts of the competitor #he concentration of -hich the competitor inhibits the binding by about 34 approximates the d

• *ignaling systems have evolved so that a rise in the level of signaling molecule gives a proportional physiological response



o #herefore the binding affinity of a receptor for a ligand must be H$:H' than the normal circulating levels of theligand

• Fractional occupancy J O1P + d K O1Po .rovides information on the proportion of receptors that are bound to a ligand at any given time

ece!tors " Basic Princi!les and Ty!es

• "%!"'!# #$%! %F #H' '"'.#% o &aximal physiological response occurs -hen only a fraction of receptors are bound to the ligando %nly need a small amount of receptor bound to get a physiological responseo *ensitivity of a cell to signals

0etermined by the number of receptors and their affinity for the ligand#he fe-er receptors present, the less sensitive the cell is to the ligand#he fe-er receptors present, the higher the ligand concentration re<uired to induce a response

• What ma/es a good receptor?o !eeds to parallel -hat ma/es a good signalo H A' *.'"$F$"$#2 – 0etecting only the molecule that the cell -ishes to perceiveo Have an appropriate B$!0$!: FF$!$#2 to detect the signaling molecule at the concentrations in -hich it is li/ely

to be foundo &ust be able to transmit the message that the signaling molecule conveys to the cell – usually by modulation of the

further components in a signaling cascade• eceptors can be on the cell surface or intracellular depending on the location and -hat they -ill bind to• &ost receptors are activated by the binding of the ligand (some are activated by hear, light, pressure etc)• 0ifferent cell types have different sets of receptors -hich respond to the same ligand – to illicit different responses• #he same receptor in different cell types can illicit different responses by activating a different signal cascade (the

do-nstream components that differ)• 11 '"'.#% "#$A #$%! $!A%1A'* "%!F% & #$%! 1 "H !:' $! #H' '"'.#% • "%!F% & #$%! "H !:'* $! '"'.#% *

o eceptors transition from an inactive state to an active N stateo gonists drive the conformational e<uilibrium to-ards No .artial agonists do the same but less efficientlyo $nverse agonists drive the receptor to-ards the stateo $n the absence of any such molecules, the e<uilibrium is biased to the inactive state although a small fraction of

receptors remain activeo *olving a receptor structure in the and N state has been a holy grail for protein chemists

$f both structures are /no-n then it can be /no-n ho- to manipulate for drug developmentAery difficult because -or/ing -ith membrane bound proteins0%!' W$#H #H' B'# 8 0 '!% '"'.#% – used a nanobody to stabili e the active state

• drenoreceptor is a :5. %#'$! 1$! '0 '"'.#% *o :." s constitute a superfamily of diverse proteins -here all members have a seven transmembrane domainso :." s act as receptors for a multitude of different signals %ne maCor group, referred to as chemosensory :." s,

are receptors for sensory signals of external origin that are sensed as odors, pheromones or tasteo &ost other :." s respond to endogenous signals such as peptides, lipids, neurotransmitters or nucleotideso Human genome consists of >44 :." so "6 and " terminal domain are -hat interact -ith the : .roteins

o

• B *$" &'"H !$*& %F :." o eceptor is coupled to a heterotrimeric :#. binding protein (: protein)



o eceptor occupation promotes interaction bet-een the receptor and the : protein on the interior surface on themembrane

o #his induces an exchange of :0. for :#. on the : protein a subunit and dissociation of the alpha subunit from the beta gamma heterodimer

o 0epending on its isoform, the :#. alpha subunit complex mediates intracellular signaling either indirectly by actingon effector molecules such as adenylyl cyclase or phospholipase ", or directly by regulating ion channel or /inasefunction

• "%!F% & #$%! 1 "H !:' $! B'#8 0 '!' :$" '"'.#% o ctivated -hen bound by agonist molecules, but are inactive in the presence of inverse agonistso n antibody binds at the : protein binding site and acts as a substitute for beta8 adrenergic receptors : proteino #ransition from to the N state, induced by agonist binding results from a contraction of the agonist binding siteo "ontraction promotes changes in the pac/ing of amino acid residues bet-een transmebrane helices 3 and Qo #hese changes coincide -ith rotation of parts of these heliceso "onformational shits create a binding site for the " terminus of the : protein

o • #2.'* %F * F"' '"'.#% *

o : . %#'$! "% .1'0 '"'.#% * – lin/ed and have different combinations of subunitso $%! "H !!'1* '"'.#% *o #2 %*$!'5 $! *' 1$! '0 '"'.#% *o '"'.#% * W$#H $!# $!*$" '!I2& #$" "#$A$#2o 1$: !0 '"'.#% *

• *ignal transduction allo-s for amplification of the signal• %ften there is cross tal/ bet-een the path-ays• .hosphorylation of molecules is very important in transmitting the signal

Molecular S#itches and Introduction to $ nd Messengers

• $%! "H !!'1 1$! '0 '"'.#% *o 0etection of neurotransmitter molecules – transmitter gated ion channels

( cetylcholine eceptor)o Binding of the ligand to the receptor changes the ion permeability of the plasma

membrane$mportant in signal transduction in nerve cells

o llo-s the influx of specific ionso %pening of the pore is a transient event – the receptor returns to an inactive state

rapidlyo #he receptors ma/e up a family of related proteins – distinguishing feature is they

contain several polypeptides -hich can pass through the membrane#o study have fre<uently used electric fish such as #orpedo marmorata(sting ray)"an purify the receptors

o "'#21"H%1$!' '"'.#% &'"H !$*& – *tudied and characteri ed <uite -ellWhen a ligand binds get a conformational t-ist in the transmembrane regions leading to selective

permeabilisation of ions*electivity is due to the presence of various charged side chains at the bottom of the pore to openeceptors in this class -ith different ligand binding specificities all contain polypeptides -ith a high degreeof similarity – genes -ith high homology or produced by alternative splicing events

NOTE:ALL

receptors

requirea

conformation

alchange



• lternative splicing gives different specificities to different ligands• '!I2&' 1$! '0 '"'.#% *

o ecogni ed initially through their role in responses to extracellular signal proteins that promote the gro-th, proliferation, differentiation, or survival of cells in animal tissues

%ften responding to gro-th factor stimuluso #hese signal proteins are often collectively called gro-th factors and they usually act as local mediators at very lo-

concentrations of ligando esponse to gro-th factors typically slo- and usually re<uire many intracellular signaling steps that eventually lead

to changes in gene expressiono *$D "1 **'* %F '!I2&'51$! '0 '"'.#% * $0'!#$F$'0G

#-o most important areG• eceptor #yrosine inases – phosphorylate specific tyrosines on a small set of intracellular

signaling proteins• #yrosine5/inase5associated receptors – associate -ith intracellular proteins that have tyrosine

/inase activity#he others are also /inases

• '"'.#% #2 %*$!' $! *'*o epresent a class of receptors that has an intrinsic en yme activity

'xtracellular side is important for ligand bindingo #he ligand binds (typically gro-th factors) to the activation site it allo-s them to dimerise and get an

autophosphorylation event tyrosine residues get phosphorylated on the cytosolic domain creating aconformational change in the receptors creates ne- protein binding sites recruiting more proteins

o

• $!# "'11 1 '"'.#% *o $mportant for ligands that can cross the membrane – steroids, thyroid hormones, fatty acids, prostaglandins and

leu/otrines12.%.H$1$"

o #he receptors are found inside the cello ctivation often leads to effects in the nucleus – alteration of transcription rates of specific genes or sets of geneso *ignal enters the cell and binds to receptor in the cytoplasm -hich than moves into the nucleus ("1 ** 7)o #he signaling molecule directly binds the receptor -ithin the nucleus ("1 ** 8) – sually bound to a promoter site

on 0!o *# "# ' – highly conserved

0! binding domain – allo-ing to bind to specifically the right part of 0! to modulate transcriptionalactivity1igand binding domain – hoo/ed to the 0! binding domain through a H$!:' region allo-ingconformational changes to allo- for regulation to ta/e place

o sually in the inactive form -ith bound inhibitory proteins that don9t allo- it to recruit the next steps in thetranscriptional machinery

1igand binding causes a maCor conformational change allo-ing for the loss of inhibitory proteins and therecruitment of co activator proteins and initiate transcription of its target

o "1 ** $ ! "1' '"'.#% *Binding the ligand in the cytosol then moving into the nucleus – <uite often re<uires dimeri ation thatallo-s transport into the nucleus to bind to specific se<uence of 0! recruits all transcriptionalmachinery*1%W "#$!: * $# '= $ '* "H !:'* $! :'!' 'D. '**$%!



esponse isn9t Cust the turning on+off of the gene it also needs the recruitment as secondary transcriptionalevents

o "1 ** $$lready bound to the 0! -ithin the nucleus as a heterodimer

• W% $!: W$#H '"'.#% *o =uite often can identify receptors through conserved domainso $dentifying the ligand it responds to is difficulto Have a c0! pool coding for the receptors and transfect these into cells getting them expressed into particular cells

and then screen -ith potential ligands and identify if they are binding or not (ligands often fluorescently labeled)• % .H ! :." s – not all have been characteri ed so this techni<ue is used to characteri e

o "lone %rphan :." (don9t /no- function) into cells – apply tissue extracts to see if you get activation – measureif second messenger arises of the receptor fractionate the tissue to try and isolate the ligand

o "an also perform in a more sophisticated -ay – using a reporter assay*ame techni<ue but use a dual reporter assay system by have the :." and a fluorescent proteins as -ellthat acts as an internal control so you /no- you are getting expression at the same time putting inanother fluorescent protein -hich is under the control of a response element cascade from :." -illinitiate that fluorescent protein

• *$:! 1 " *" 0'* 5 after activation of the receptor o "an lead to signal amplificationo #he role of protein phosphorylation in signal transduction – inases and phosphataseso Ho- :#.ase s-itch proteins -or/ o $mportance of 8 nd messengers

• "%&.%!'!#* %F *$:! 1 # !*0 "#$%!o "ommonly employed intracellular signal5transducing moleculeso .re5existing proteins (change in activity)

: . %#'$!*$! *'* !0 .H%*.H # *'%#H' "2*#%1$" '!I2&'* such as calmodulin or adenylate cylate

o 8 !0 &'**'!:' (change in concentration)"a8K"yclic nucleotides$nositol lipids%*+ !*

• $!# "'11 1 # !*0 "#$%! . %0 "' 1 :' &.1$F$" #$%!o eceptors are lo- in abundance but often need millions of 8 nd messenger or activated en yme molecules to elicit the

re<uired response 5 mplification at each step

o

•

. %#'$! .H%*.H% 21#$%!o "hanges to phosphorylation status is common to most signaling cascadeso .roteins can be phosphorylated on *' , #H , #2 and H$*o .roteins can be phosphorylated on multiple sites and by more than one /inase – convergence of signaling pat-ayso apid – ta/es as little as a fe- secondso tilises -hat is already there and is easily reversible

• H & ! $!%&'o $n the human genome there are 37E protein /inases and about 734 protein phosphataseso 64 of human proteins covalently bind phosphate – 34,444 phosphorylation sites

• .rotein /inases themselves share common structural domains – insertion points are -hat allo- the protein to have specificityand provide domains for protein5protein interactions

• .H%*.H% 21 #$%! – alters the structure and therefore possible the en ymatic activity+binding affinity



o .hosphoryl group adds a negative charge thus disrupting the electrostatic interactions of formation of ne-electrostatic interactions

o .hosphoryl group can form hydrogen bonds -hich can favour ne- conformationso Free energy change associated -ith phosphorylation may push the e<uilibrium from one conformational state to

another o %ften /inases are activated by other /inases such as in the & . path-ay allo-ing for amplification and cross tal/

• *' $!'+#H '%!$!' $! *'* 5 1argest group of /inaseso $ncludesG c &. dependent protein /inase, c:&. dependent protein /inase, protein /inase ", "a8K+calmodulin

dependent protein /inases, :." /inases, protein /inase B, &.5activated protein /inase• #2 %*$!' $! *'* – second largest group

o nalysis of phosphoamino acid content in the cell – only 4 43 -as phosphotyrosineo #-o broad groups – those -hich are part of the receptor and those -hich are solubleo #hey are responding to : %W#H F "#% * – highly studied

• #here are also cytoplasmic tyrosine /inases an example is the +*# # signaling path-ay• .H%*.H # *'*

o lthough intracellular signal transduction is often portrayed as a protein /inase the counterbablancing function of phosphatases, and the control of phosphatase activity is e<ually relevant to proper regulation of cellular function

o *imilar to the /inases they are specific to *er, #hr and #yr o *# "# ' – "ontains a structural subunit, catalytic subunit and regulatory subunit

egulatory and catalytic subunit can both be post translationally modified• :#.ase *W$#"H . %#'$!*

o : proteins act as molecular s-itches they are either off or ono :uanyl nucleotide binding proteins in the inactive state binds :0. -hich is exchanged for :#. $! #H' "#$A'

*##'o "an be classified into t-o main classesG &%!%&' $" F &$12 (important for receptor tyrosine /inases and

especially the & . path-ay) or H'#' %# $&' $" F &$12 (important in :." )• H'#' %# $&' $" : . %#'$!*

o "lassified according to : alpha sub5unit homologyo :s l-ays use the activation of adenylate cyclase as the next step in the signal chaino :i – inhibition of adenylate cyclaseo :< – proteins usually have phospholipase " as an effector proteino :78 – activated by thromboxane receptors and thrombin receptorso By the combination of alpha, beta and gamma a great variety of : proteins can be produced

o

• &%!%&' $" : . %#'$!*o lmost all monomeric :#.ases contain a covalently attached lipid group that helps to anchor the proteins to the

cytoplasmic face of the membrane



o

• *'"%!0 &'**'!:' &%1'" 1'*o elay signals received at receptors on the cell surface – such as the arrival of protein hormones, gro-th fators etc –

to target molecules in the cytosol and+or nucleuso *mall and rapidly diffusibleo *ynthesi ed <uic/ly and metaboli ed <uic/lyo !ormally relatively lo- concentration but greatly amplify the strength of the signal

•

"2"1$" ! "1'%#$0'*o " &. and ":&.o " &. is produced at the plasma membrane by adenylate cyclase and effects are concentration dependento #he molecules are small and rapidly diffusible and rapidly metaboli ed

Signal Transduction and Second Messengers

• "a8K stays locali ed -ithin a cell• *'"%!0 &'**'!:' &%1'" 1'* – molecules that relay signals received a receptors on the cell surface to target

molecules in the cytosol or the nucleuso #end to be small and rapidly diffusibleo *ynthesi ed and metaboli ed <uic/ly (al-ays has to be a corresponding inactivation path-ay)o !ormally relatively lo- in concentration and greatly amplify the strength of the first signal

• "%&&%! $!# "'11 1 *'"%!0 &'**'!:' &%1'" 1'*o CALCI&M' CYCLIC AM( !0 :&., DIACYLGLYCEROL an% INOSI)OL *'+',-)RI( OS( A)Eo "alcium is the universal secondary messenger

• "2"1$" ! "1'%#$0'* – cyclic &.o .roduced in the plasma membrane by the en yme adenylate cyclaseo ll these molecules must have a degradation path-ay – " &. degraded by " &. phosphodiesterase ma/ing it

inactiveo 'ffects are concentration dependent – usually at lo- concentrations but can be synthesi ed relatively <uic/lyo *mall and rapidly diffusible and rapidly metaboli ed

• #he en yme that ma/es " &. ( 0'!2121 "2"1 *') is activated by :." o Have a stimulatory ligand li/e adrenaline that -ill bind to the :." get activation of the heterotrimer

exchange of :0. for :#. ctivates adenylyl cyclaseo #here are also inhibitory : proteins



o

o %nce denylyl cyclase is activated synthesis of " &. can begin• " &. *$:! 1* #H % :H " &. 0'.'!0'!# . %#'$! $! *'

o . exists as a tetramer of t-o regulatory subunits and t-o catalytic subunitso Binding of " &. to the regulatory subunits induces a conformational change, causing these subunits to dissociate

from the catalytic subunits activating their /inase activityo elease of the catalytic subunits re<uires the binding of more than t-o cyclic &. molecules to the regulatory

subints in the tetramer o #here are t-o types of . G #2.' $ is in the cytosol

#2.' $$ bound via its regulatory subunit and special anchoring proteins to the plasma membrane, nuclear

membrane, mitochondrial outer membrane and microtubuleso %nce the catalytic subunits are freed and active, they can migrate into the nucleus or activate metabolic en ymeso Have a plethora of different targets – different range of rates <uic/ or slo-

o

o 'ach receptor subunit as t-o distinct sites for binding "amp called and B each are located in a different domain inthe subunit

o Binding of " &. to site B induces a conformational change that unmas/s site o Binding of " &. to site leads to the release of catalytic subunitso Binding of the first site lo-ers the d of the second site therefore small changes in the level of " &. can cause

proportionaltely large changes in the amount of dissociated " subunits and hence in the activity of a .o Has various effectso ctivation of adenylyl cyclase and increase in " &. has tissue specific effects

.lasticity – has a number of different roles in different tissues• 1$.$0* * *'"%!0 &'**'!:' *

o .hosphatidylinositol and its various phosphorylated derivatives are minor lipids of the cytoplasmic leaflet of the plasma membrane

o ll the hydroxyl groups on inositol have the potential to be phosphorylatedo !umber of different lipids that can act as secondary messengerso $n order to produce lipid second messengers you need to increase concentrations of particular species so are

en ymatically modifiedo #H '' "1 **'* %F '!I2&'* #H # &%0$F2 1$.$0*

.H%*.H%1$. *'* – cleave into fragments1$.$0 $! *'* – phosphorylate the inositol group1$.$0 .H%*.H # *'* – remove the phosphates

• Nall conceivable products produced by these en ymes from the parent lipids participate insignaling reactions



• #he second messengers produced by these reactions participate bet-een the a<ueous phase of thecytoplasm and hydrophobic phase of the membrane bilayer also important in anchoring proteins tothe bilayer

#-o maCor products from phosphatidylinositol that participate in signaling are diacylglycerol and inositoltriphosphate ($.6) 5 made through a series of phosphorylation reactions

o F #' %F 0 : !0 $.60iacylglycerol and inositol triphosphate are both inactivated en ymatically0 : is inactivated by phosphorylation ma/ing it phosphatidic acid$.6 is dephosphrylated to inositol – this process are inhibited by lithium

• " 1"$ &o niversal signal capable of activating many different cellular processes operating over a very -ide time domaino #he concentration of "a8K in cells at rest is approximately 744!m but this increases to 344!m or more follo-ing a

stimulus that activates the "a8K %! reactions When the stimulus is removed, the "a8K %FF reactions return theconcentration of "a8K to its resting levels

o $n the blood and extracellular fluid the concentrations of "a8K are <uite high -hich ma/es it very easy to increasethe intracellular "a8K concentrations by allo-ing it to come in from outside

o • $!# "'11 1 " 1"$ & * *$:! 1

o "ells use a large amount of energy expelling "a8K from the cell to maintain normal concentration levelso $n the extracellular space the "a8K concentration is about 8m& inside it is 744n&o #here are also "a8K stores -ithin the cell such as the ' as can mitochondria

o

o "ytosolic concentration of "a8K is very lo- in normal resting cells -hich are maintained by "a8K membrane pumps

o ' contains *' " pumps to se<uester "a8K there are also !aK+"a8K changes• "a8K H A' & !2 "%!# %1 .%$!#* F% "%!"'!# #$%! 1'A'1*

o "a8K entry channels, -hich control the entry of "a8K from the outsideo "a8K release channels, -hich control the release of "a8K from internal stores (' and mitochondria)o "a8K buffers ensure that the concentration of "a8K remains -ithin its operation range and does not rise to levels that

can induce cells deatho %nce "a8K has carried out its signaling function, there are "a8K pumps and exchangers that remove it from the

cytoplasm by either extruding it from the cell or returning it to the internal storeso "a8K signaling functions are carried out by various "a8K sensors and effectors that are responsible for translating

"a8K signals into a change in cellular activity• . %. : #$%! %F #H' " 8K *$:! 1



o $t is a small ion -ith a large diffusion coefficient in -ater but diffusion through the cytoplasm is very slo-o $t gets se<uestered and there abundant "a8K binding proteinso t a concentration of 4 7micro molar the half time for a free "a8K is 64 micro seconds and the range of diffusion is

4 7 micro meter o When bound to a protein messenger li/e calmodulin it has a -ider range

"almodulin binds four "a8Ko &any things bind "a8K and many do so through an 'F5 hand motif used by the proteins to sense and respond to

fluctuation in cellular "a8K 0ifferent 'F5hand se<uences found in proteins display "a8K sensitivities and /ineticsthat can vary by over 8 orders of magnitude

• " 1&%0 1$! ("a8K &%0 1 #'0 . %#'$!)o p to four "a8K ions are bound to calmodulin via its ; 'F hand motifso 'F hands supply an electronegative environment for ion coordination fter "a8K binding, hydrophobic &ethyl

groups from methionine residues become exposed on the target proteino #hese helices contain complementary hydrophobic regions,o #he flexibility of calmodulin9s hinged region allo-s the molecule to -rap around its target, allo-ing it to tightly

bind to a -ide range of different target proteins• . %#'$! $! *' " – "#$A #'0 B2 "a8K

o . " comprises a family of *er+#hr /inases that are involved in the transduction of signals for cell proliferation,differentiation, apoptosis and angiogenesis

#here is a -ide range of substrates

o

The M%P& Path#ay

• "H "#' $*$!: #H' & . $! *' . #HW 2o 0rosohpila have compound eyes that are made by E44 ommatidia in each compound eye

'ach ommatium has 88 cells -ith E photoreceptor cellsll develop from an epithelial sheet

o :et mutant flies that don9t have a @ photoreceptor -hich are usually made last@ has rhodopsin that is sensitive to A"an assay for missing photoreceptors by phototaxis

o "H "#' $*$!: #H' *'A'!1'** & # !#

#he photoreceptor cells are the first cells to differentiate follo-ed by the non5neuronal cone cells and pigment cells

E is the first photoreceptor to differentiate follo-ed by 8+ 3 and 6+ ; and 7+ Q@ is the last recruited*'A .rotein is re<uired for expression of all cells except @ -hich re<uires it for differentiation$n the absence of function *'A (protein) @ remains a non5neuronal cone cell*evenless -as cloned and expressed in a drosophila cell line*'A protein -as found to be a receptor tyrosine /inase

o t the same time another mutant -as found to also lead to the loss of the @ photoreceptor but unli/e sevenless thisgene also re<uired function in the E photoreceptor 5 @ and E interact

• $0'!#$F2$!: *$:! 1 # !*0 "$!: . %#'$!* $! #H' *'A # .#HW2



o $nvestigators produced mutant flies expressing a temperature sensitive *ev proteinso When maintained at a permissive temperature, all their ommatidia contained @ cellso When they -ere maintained at a non permissive temperature, no @ cells developedo t an intermediate temperature ho-ever, Cust enough of the *ev receptor tyrosine /inase -as functional to mediate

normal @ development But at this intermediate temperature, the signaling path-ay -ould become defective, andthus no @ cells -ould develop, if the level of another protein involved in the path-ay -as reduced

o recessive mutation affecting such a protein -ould have this effect because, in diploid organisms li/e drosophila, ahetero ygote containing one -ild type and one mutant allele of a gene -ill produce half the normal amount of thegene product Hence even if such a recessive mutation is in an essential gene, the organism -ill be viable

o #herefore, a fly carrying temperature5sensitive mutation and the sev gene and a second mutation affecting another protein in the signaling path-ay -ould be expected to lac/ @ cells at the intermediate temperature

o By use of this screen researchers identified gene encoding three important proteins in the *ev path-ayNA' 2 #$&' "%!* &$!: – andom screening – 7444s of drosophila lines

• #H' #H '' . %#'$!*o *on of sevenless (*%*) – a guanine neucleotde exchange factor identified and named in a previous screeno as7 – a monomeric : proteins (s-itch :0. to :#.)o 0r/ – an adaptor proteino #hese have orthologues in mammalsMMM "%!*' A'0 *2*#'& " %** 11 ' 2%#'*

o

o *ho-n for the first time the & . /inase path-ay and ho- its turned on – "%!*' A'0 in all eu/aryotes$!$#$ 1 . # %F . #HW 2 J Basically -or/ by dimeri ation of tyrosine receptors after binding itsligand tyrosines phosphorylated phosphotyrosines act as a doc/ing system recogni ed by adaptor

proteins and identified protein 0r/ (in mammalian system its called : B8) this protein has an –*H8domain -hich is important for doc/ing onto the phosphorylated tyrosine also has an *H6 domain -hich isimportant in doc/ing onto other proteins allo-s recruitment of *%* (guanine exchange factor)

activates as inase cascade• # !* #%.H%*.H% 21#$%! %F '"'.#% #2 %*$!' $! *'*

o .hosphorylation of tyrosines -ithin the /inase domain increase /inase activity of the en ymeo .hosphorylation of tyrosine outside of the /inase domain creates high affinity doc/ing sites for other signaling

components – recogni ed by *H8 domain – proteins that have a specific plug -hich allo-s them to bind to phosphotyrosines and other nearby proteins



o

o !ormal receptros dimeri e and the /inase domains cross phosphorylate each other, increasing the activity of the/inase domains, -hich can no- further activate the receptor dimer by phosphorylating other sites on the receptors

o mutant receptor -ith an inactivated /inase domain can still dimerise but cannot cross phosphorylateo $f mutant is over expressed this can cause a dominant negative inhibition effect and bloc/ signaling

"an see function by doing this pretty much li/e /noc/5out• 0$FF' '!# . %#'$!* " ! B$!0 #% #H' .H%*.H%#2 %*$!' *$#'*

o %nce you get phosphorylation of the cytoplasmic domain of tyrosine /inase can get a number of proteins that -ill bind

o "an recruit a large of molecules to bind most have the *H8 domaino .roteins also have *H6 domains to bind to other proteins acting as a scaffold

"an /noc/ out particular tyrosine residues to see -hat molecules bind and don9t• . %#'$!* B$!0 A$ *H8 0%& $!

o "lassic example is :ro-th Factor eceptor Bound .roteins 8 (: B8) is an adaptor protein containing an *H8domain and t-o *H6 domains

o #he : B8 lin/s the *%* :5activating protein to the receptor tyrosine /inaseo #he *rc Homology 8 *H8 domain is a protein domain of about 744 amino acidso *H8 domains function as regulatory modules of intracellular signaling cascades by interacting -ith high affinity to

phosphotyrosine5containing target peptides in a se<uence5specific manner o *H8 domains recogni e bet-een 65Q residues " terminal to the phosphorylated tyrosine in a fashion from one *H8

domain to another, nd strictly phosphorylation5dependent manner o *H6 domains direct complex formation -ith . %1$!' $"H regions of other proteinso *# "# ' %F *H8

Have a phosphotyrosine binding poc/et and a poc/et specific for other amino acids giving it specificity"an bind -ithout disturbing the protein they are binding toBecause each domain has distinct sites for recogni ing phosphotyrosine and for recogni ing a particular amino acid side chain, different *H8 domains recogni e phosphotyrosine in the context of differentflan/ing amino acid se<uences

• "#$A #$%! %F *o #he : B8 adaptor protein binds to a specific phosphotyrosine on the receptor and to the as guanine nucleotide

exchange factor (:'F) such as *%*, -hich stimulates as to exchange its bound :0. for :#. #he activated asthen activates do-nstream signaling path-ays



o

o * – &onomeric : s-itch proteins all -or/ by exchange of :0. to :#.:#. in the cytosol is 74 times greater than the :0. and as rapidly binds :#. once :0. has been eCected

o * $* "#$A #'0 B2 :'F (: !$!' 'D"H !:' F "#% *) – losing :'F is e<uivalent to loss of *• ctivation of * is transient – can be measured by having a fluorescent version of as and a fluorescent version of :#.

o When they bind in close proximity get F '#o *ustained signaling through do-nstream components

• #H' & . &%0 1' – .H%*.H% 21 #$%! " *" 0'o #hree /inases constitute the core module of the cascade #he last of the three is called a mitogen protein /inase

(& .5/inase)o feature of a & .5/inase is that its full activation re<uires the phosphorylation of both a threonine and a tyrosineo #he protein /inase that cataly es both of these phosphorylations is called a & .5/inase5/inase, -hich in

mammalian as signaling is called &' o #he re<uirement for both a tyrosine and a threonine phosphorylation ensures that the & .5/inase is /ept inactive

unless specifically activated but a & .5/inase5/inase, -hose only /no-n substrate is a & .5/inaseo & .5/inase5/inase is itself activated by phosphorylation by & .5/inase5/inase5/inase, -hich in the mammalian

as signaling path-ays is called af o af is activated by as

o

o ctivated as binds to the !5terminal domain of af, a serine+threonine /inase#he af family of serine+threonine /inases is central to this path-ays and is expressed in three forms, af57, 5 af and B5 af, -hich all share highly conserved !5terminal regulatory regions and a "5terminal



catalytic /inase domain ll three af proteins are ubi<uitously expressed although expression levels have been reported to differ greatly depending upon the tissue type

• 7;5656 . %#'$!*o $mportant in regulating these path-ays – bound holding things inactive and stuffo "an effect intracellular signaling in one of three -ays

By direct regulation of the catalytic activity of the bound proteinBy regulating interactions bet-een the bound proteins and other moleculesBy controlling the subcellular locali ation of the bound ligand

o :enerally operates as a dimer • "#$A #$%! %F F – still not clear but comprises different events, including lipid association, interaction -ith as,

phosphorylation+dephosphorylation events and the binding of 7;5656 adaptor proteins

'i(ersifying M%P& Path#ay Signal Out!ut

• 2' *# & #$!:o &ating in yeast is regulated in part by the & . path-ayo &ating signaling in budding yeast – .heromone binding to its :." leads to :alpha activation and dissociation

from the :beta gamma heterodimer, and activation of a conserved & . cascade that leads to the transcription of mating5specific genes, cell polari ation in the direction of partner cells and subse<uent fusion of mating pairs #hesignal is transmitted by the : beta gamma dimer, through : veta interactions -ith several effectors

o !otably : beta regulates the activity of t-o distinct scaffold proteins to activate the conserved & . (through*te3) and "dc;8 (through Far7) modules

o

o Fe- significant differences to -hat -as previously learntG !% #2 %*$!' '"'.#% $! *' $!A%1A'0 – instead is mediated through :." 'ffects are through both existing protein components and transcription& . path-ay in this sample is tethered to *te3 – important for isolation of different path-ays,eliminating cross tal/ – don9t get as much amplification as if it -as floating freely*te3 is a scaffolding protein that hold it all together

• "#$A#$%! #H % :H :." $!*#' 0 %F '"'.#% #2 %*$!' $! *'o "an get various signals -hich feed in through the & . path-ay from different types of receptors not Cust through

receptor tyrosine /inaseso "an get stimulation of as and+or af directly by other mechanisms such as "yto/ines, integrins, :." etc

o



•

• 8050$:'o Because you have fluorescently labeled the proteins you can perform at 80 differential gel electrophoresis -here

there are three dyes available• "an use mass spectrometry to identify the potential targets – many potential targets

o &any may or may not be involved to determine if they are is a lengthy process• 1$!' $#2 !0 " %** # 1 (!%#' – 'F' #% H$:H1$:H#'0 .$"# ')

o *everal cross5tal/s to other signaling path-ays including :F stimulated small : proteins and /inases, richlyregulate the & . signaling cascades

o *ame components involved in multiple path-ayso . %B1'&S $f -e stimulate one path-ay, -hy don9t -e stimulate all the path-ays

• #'&.% 1 !0 *. #$ 1 % : !$* #$%! %F #H' & . . #HW 2o #here are cross tal/ but there must be control of signal duration and also must have feed5bac/ loop inhibitiono Aery importantly there must be some sort of compartmentali ation to control the & . path-ay – 0% !%# H A'

&$D'0 "'11

o

• ': 1 #$%! %F *$:! 1 0 #$%!o #he amount of time a component is in the active state can be critical for formulating the final responseo *tudies in primary cells sho- that sustained activation of the & . path-ay precedes cellular differentiationo #ransient & . activation results from recruitment of the : B85*%* complex to ':F., but prolonged & .

activation is associated -ith the recruitment of the : B85*%* complex to the !:F5activated #r/ receptor o .rolonged & . activation results in & . translocation to the nucleus, -hich broadens the repertoire of

substrates compared to the transiently activated cytoplasmic & . o !ormally -hen -e get activation -e get dimeri ation of the receptor and activation 5 then it gets dephosphorylated

very <uic/ly % often the receptor -ill get internali ed by endosomes and degraded getting a loss of the signalo $f there is sustained activation of & . it can enter the nucleus and can get regulation of transcription factorso ! &B' %F W2* #% :'# * *# $!'0 "#$A#$%!

eceptor get se<uestered into recycling endosomes -here the receptor is /ept s-itched on by extension of gro-th factors signalutocrine loops -hich increase the receptor numbers and receptor activationecruit to a & . scaffold protein, &.7 to late endosomes*ustained activation of as by co locali ing to the ' or golgi

• !': #$A' ': 1 #$%!o #he capacity of the cell to differentiate bet-een true signals, -hich are meant to drive cellular outcome, and

inconse<uential noise, has been demonstrated to depend on mechanisms of feed5for-ard and feedbac/ regulation



o !egative feedbac/ can limit the duration of a signalG once a predefined threshold is reached #he signal induces itso-n negative regulators

o When incorporated in a path-ay the negative feedbac/ circuit confers output stabili ation, even -hen the system ischallenged -ith a high degree of environmental noise

• !': #$A' F''0B " 1%%.* $! #H' & . .#HW2o ctivated & . can,

$nhibit af57 by direct phosphorylation$nterfere -ith the coupling of the as exchange factor *%* to receptors by inducing inhibitory

phosphorylationcculumate in the nucleus inducing the transcription of & . phosphatases, -hich dephosphorylate ' activation sites$nduce *prouty expression -hich interferes -ith as and af activation

M%P& and Transcri!tion " )%S NOT ECO 'E'

• "%!# %1 %F "%&. #&'!# 1$I #$%! – #H' 0$*"%A' 2 %F * o 1arge5scale genetic screens performed in 0rosphila and " 'legans simultaneously led to the isolation of a ne-

compartment termed inase *upressor of aso * -as sho-n to be a positive effector of as signaling that appeared to act either upstream of af or in a parallel

path-ay, but the precise molecular mechanism by -hich * function to transmit signals to as -as un/no-no * had a putative /inase domain but biochemical and genetic studies -ere inconclusive

o #here is no * homologue in yeast• * *# "# '

o Was found to interact -ith af57, &' and & . Both &' 7 and 8 B$!0 0$ '"#12 #% #H' ca3 0%& $!*o ll /no-n * members contain five conserved domains

7 – ;4aa region uni<ue to * 8 – proline rich region6 – cysteine rich inc finger domain; – ser+thr rich region3 – putative /inase domain

• * *" FF%10$!: . %#'$!o protein -hose main function is to bring other proteins together for them to interact #hese proteins usually have

many protein binding domainso

Ho- to tell if a protein acts as a scaffold?• "H "#' $*$!: *" FF%10 . %#'$!o 0efined interaction bet-een interacting proteinso $. scaffold proteino $s the scaffold needed? se :*# fused to different scaffold domains to determine modular organi ation of scaffold

complexo 0oes loss of the scaffold protein lead to mislocalisation of the target proteins .erform an immunoblot

• * ': 1 #$%! "2"1'o *uggested model – * locali es &' to the plasma membrane in a as5dependent manner and promotes the

assembly of a multiprotein complex that brings &' into close proximity -ith its upstream activator af anddo-nstream substrate & .

o * provides a doc/ing platform at the plasma membrane onto -hich af, &' and & . can form a complex



o $n inactivated cells, * is maintained in the cytosol through an interaction -ith 7;5656o Follo-ing stimulation by gro-th factors, * translocates to the plasma membrane -here it facilitates activation of

&' and & . o * is able to regulate the spatial activation of the & . path-ayo ecently sho-n that & . induces dimeri ation * is re<uired for & . dimeri ation – monomers translocate

to the nucleus

•

• * is in the mammalian & . path-ay -hereas as seen before *te3 is in the yeast mating response of the & . path-ay

•

• #he *#'3 scafold also ta/es place in higher order regulatory systems• *caffold proteins can be a target of feedbac/ phosphorylation

o Feedbac/ phosphorylation of the * scaffold by activated & . bloc/s F binding and attenuated &' activation therefore decreasing the path-ay output

o

• B'!'F$#* !0 "%*#* #% #H' *" FF%10 &'"H !$*&o 1o-ers the entropic cost of signaling interactionsG the loss of independent translational and rotational degrees of

freedom is paid through binding interactions -ith the scaffoldo By restricting the conformational freedom of interacting proteins, scaffolds can orient these molecules to enhance

the rate of signal transfer o #ethering has potential dra-bac/S t high concentration, scaffolds may titrate en yme and substrate a-ay from one

another



o $ncreasing affinities can restrict substrate release and diffusion throughout the cell, potentially limiting signalamplification

• 11%*#' $" ': 1 #$%! B2 *" FF%10 . %#'$!*o *caffolds can allosterically modulate the conformation of en ymes and substrates to gate information flo-o $n & . signaling, * can directly bind to the af and influence its activity to-ard the &' #he /inase

homology domain of * dimeri es -ith af, altering the conformation of the "5helix on af so that its /inasedomain becomes catalytically active allo-ing af to phosphorylate &'

• Ho- can one path-ay be the effector of such different responses?o &'"H !$*&* "%!# %11$!: *$:! 11$!: *.'"$F$"$#2 %F #H' & . .#HW2

0uration and strength of the signal*caffold and locali ation"ross tal/ -ith other cascades&ultiple components at each step

• . %B1'&* F "'0 B2 # !*" $.#$%! F"#% o 'xpression, getting into the nucleus, finding the 0! binding site and getting access to it and recruit transcription

factors• #he phosphorylation and dephosphorylation of transcriptional regulators mediated by specific protein /inases and protein

phosphatases is the most common mechanism of controlling gene expressiono #here is a plethora of target & . can bind to

• & . *$:! 11$!: ': 1 #'* & 1#$.1' *.'"#* %F # !*" $.#$%!o & . can phosphorylate transcription factors and regulate their import and export from the nucleuso .hosphorylate, transcription factors often change conformation leading to the regulation of their functions including

0! binding and interactions -ith co5acitvators or co5repressor o #hese co5activators and co5repressors as -ell as chromatin remodeling components, can also be subCect to regulation

by & . cascadeso $n some cases the phosphorylation of transcription factors modulates their stability

• & . !0 '"%:!$*$!: $#* * B*# #'*o $nteract via a conserved doc/ing siteo 0oc/ing sites on transcription factors can act as sensors that detect the strength and duration of & . activityo .lay important adaptor roles nd directly recruit proteins into transcription factor complexes on gene promoters

• 0%" $!: $!#' "#$%!o "lassical doc/ing domain Follo-ing doc/ing phosphorylation of the *+#. motifs can occur

o

• "%&.1'D 0%" $!:+.H%*.H% 21#$%! $!#' "#$%!o *uggests that transcription factors utili e different combinations of & . doc/ing sites to direct phosphorylation

events that regulate their activities in response to direct stimuli• $!# "'11 1 1%" 1$* #$%!

o &any transcription factors permanently reside in the nucleus but other shuttle bet-een the cytoplasm and thenucleus

o !ucleocytoplasmic shuttling is an active process controlled by nuclear locali ation signals and nuclear exportsignals -ithin transcription factors

o #he phosphorylation status can directly regulate the accessibility of these !1* and !'* to nuclear import andexport proteins by mas/ing or unmas/ing signal se<uences on transcription factors

• "%!# %1 %F # !*" $.#$%! F "#% . %#'$! 1'A'1*o second important mechanism for regulating transcription factor it to control the amount of transcription factor byG

&odulating transcription factor expression levels*tability of the protein



Membranes and Cell Signalling

• & . path-ay important in cell proliferation and gro-th therefore dysregulation of the path-ay can cause substantialeffects

• WH # & '* "'11 B'"%&' " !"' % *?o $ndependence of proliferation signalso 'vasion of apoptosiso $nsensitivity to anti5gro-th signalso nlimited replicative potentialo bility to invade and metastasi eo bility to attract and sustain angiogenesis for nutrient supply

• !eed dysregulation of these six processes• "ancer progression – involves a series of mutation

o single stem cell sustains a particular mutation could be in the cell cycle gives slight gro-th advantage getsanother mutation damages increases more and more mutations full blo-n cancer

• %ften these mutations include B!% & 1$#$'* $! & . *$:! 11$!:o $mpinges on most, if not all of the processes critical for the development of cancer o bout a third probably ;4534 have dysregulation in the & . path-ay in all human cancers – <uite often the

early stages of the signal cascade so '"'.#% *, * and F• %ften see receptor overexpression – mutations in as (almost all pancreatic cancers) and af (many isoforms but most

commonly B F mutations)• & # #$%!* 1W 2* ' 12 *#'.* %F #H' . #HW 2• "%!*#$# #$A' "#$A#$%! %F & . *$:! 11$!:

o &ost cancer associated lesion that lead to constitutive activation of & . signaling occur at early steps of the path-ay

%verexpression of receptor tyrosine /inasesctivating mutations in receptor tyrosine /inases*ustained autocrine or paracrine production of activating ligands – cell is secreting its o-n ligand and thiscan also affect neighbouring cellsas mutations and B5 af mutations

• 02* ': 1 #$%! %F ': Fo %ften arises due to gene amplification or duplication eventso eceptor overexpression means an excess of receptors on the cells membrane or overexpression of ligand that bind

to the receptor #umour cells can overexpress both receptors and its ligands:enetic mutation can lead to dysfunctional receptors 5 permanently s-itched %!

• * :#.ases "# * &%1'" 1 *W$#"H'* #H# "%!# %1 #H' "#$A$#2 %F & !2 *$:! 11$!:.#HW2*

o no- that as involves the exchange of :0. for :#. after activation by :'Fso 0on9t Cust have dysregulation of as but also of its stimulating factors

• * & # #$%! – most common oncogenic mutations occur to as itself -here it is in a constitutively active stateo "an render as resistant to : . activity leading to decreased :#. hydrolysis and constitutive activation (!%

&% ' ': 1 #$%!) leading to & . stimulation• F – not all af isoforms are the same – one of the big problems is B5 F mutations

o B5 F has a negatively charged regulatory region Cust upstream of the " 6 domain and is constitutively phosphorylated

o $n B F it carries a constant negative charge in the ! region – has strong /inase activity any-ayo &ost common mutation is AQ44' changing the activation loop ma/ing it constitutively active

Q4 of malignant melanomas have this mutation in B Fo "an measure by loo/ing at the phosphorylation of & . substrate

nd see that -ith these mutations you get increased & . activity$f you have mutations in the germline you can get other disorders

• B5 F mutation on its o-n can confer a transforming ability• 0rug treatment of B5 af &utations



o sing Ielobraf a threonine /inase inhibitor – *pecifically targets the AQ44' B raf mutation%nly -or/s -ith this particular mutation*tops sustained activity of & .

o *ide effect of the drug – at certain concentration you see a bit of an increase of phosphor &e/ in the W#• "onstitutively activated & . obviously the effects are caused by transcription

o lot of the transcriptional events are dependent on the sustained activity of the & . path-ayo !eed phosphorylation of particular transcription factors to get the gene effectso *ome of the gene products are targets themselves for phosphorylation by & .

o

• .7 is an oncogenic #ranscription Factor made up of dimers of c5 ! and c5F%*• "an correlate the expression of transcription factors -ith cancers• Fra 7 overexpression is sustained by prolonged & . and is a part of c5F%*

Membrane .rote#n structure an% funct#on / my 0heroes1

What are membrane .rote#ns an% 2hat %o they %o3

• .roteins that sit in the membranes seen in pro/aryotic and eu/aryotic cells• #hey can be found in the outer membrane or the inner membrane as -ell• olesS

5 %btain nutrients and expel -aste5 *ense and respond to external stimuli5 &aintain and dissipate electrochemical gradients5 "ommunication bet-een cells

• $n Bacteria many membrane proteins haveS5 Airulence factors in pathogenic bacteria5 dhesion to host cells5 ntibiotic resistance5 #race metal ac<uisition (gaining of nutrients from the environment)

• &any membrane proteins are maCor drug targets• %ne of maCor membrane proteins acting as drug targets is the :5protein coupled receptors (:." s)

Membrane structure

• $ntegral membrane proteins must exist ThappilyU in a diverse membrane environment• #herefore this restricts the structure of membrane proteins – the proteins have to be able to cope -ith these different

environments (i e hydrophobic environment, hydrophilic environment, etc )



)hree 0fam#"#es1 of membrane . rote#n structures

• lpha helical protein J Found in the plasma membrane (inner membrane of mitochondria)• Beta barrel protein – Found in the outer membrane of mitochondria• &onotopic membrane protein J $ntegral membrane protein that has to be extracted via detergent – it doesn9t have any

structures that go right through the membrane (doesn9t traverse the membrane)• $n soluble proteins, you see a mixture of many different proteins• Ho-ever in membrane proteins you usually only see either alpha integral or beta integral membrane proteins – therefore lac/

structural diversity

Why such a "ac4 of structura" %#$ers#ty3

• &ainly because of the interior of the membrane• #he interior of the membrane is hydrophobic• We can9t expose any groups to the hydrophobic environment that are polar, because the peptides need to be TconcealedU• $n the case of alpha helices and beta sheets the protons that are on the peptide lin/age can be shielded by the secondary

structure that exists• 'n ymatic or other functions are therefore limited by the secondary structure

MONO)O(IC MEMBRANE (RO)EINS

Common features of monoto.#c membrane .rote#ns

• Have high structural homology -ith a soluble proteins• sually form homodimers – -hich are hydrophobic on one face (the green part is the hydrophobic face -hich is the part that

gets embedded into the membrane)



• sually present the membrane, revealing the active site• !ot easy to identify

M#tochon%r#a" e"ectron trans.ort cha#ns #n mamma"s an% yeast

• &ain focus is on yeast• 2east complex doesn9t contain complex 7 instead it has !0$57• $t can9t pump protons though – but in yeast this is not problem•

0A"ternat#$e1 Com."e5 *6 NAD -%ehy%ro!enase

• Has a hydrophobic part that is embedded into the membrane• Ho- might the en yme -or/?

5 !eeds to -or/ -ith t-o substrates5 !eeds to interact -ith ! 0H -hich is hydrophilic5 nd a <uinode -hich is hydrophobic5 #he ! 0H comes along and gets suc/ed into the active site and goes a-ay5 Whereas the hydrophobic <uinode can come up through the membrane, interact at the active site and then go bac/

into the membrane

AL( A ELICAL MEMBRANE (RO)EINS

•

Has a really diverse structure• Aaried T<uantitiesU of structure protrude from one membrane• lot of these oligomerise• #hey can either THomoU or THeteroU oligomers• #he interaction bet-een the subunits is mainly in the membrane, -hich is -hat holds them together

BE)A-BARREL MEMBRANE (RO)EINS

• 2ou can have all sorts of si es



7-ray crysta""o!ra.hy of so"ub"e .rote#ns

7>E49s57>>49s technological advances lead to maCor brea/throughs in soluble protein structural biologyS

5 ecombinant protein techni<ues5 "ryo5free ing techni<ues for protein crystals5 vailability of synchrotron radiation sources and advances in phasing techni<ues, e g & 0

5 dvances in computing

Membrane (rote#n crysta""o!ra.hy6 cha""en!es at each sta!e

.rotein expressionS

– &embrane proteins are expressed in lo- yield – 0ifficult to visualise by *0*5. :' etc

.rotein purification

– &ust be isolated in the presence of detergents "rystallisation

– 1o- yields limit screening – &ust be crystallised -ith detergents

0ata collection

– "rystals often fragile – :reat variability in crystal <uality – 1o- resolution dataG high solvent contents – Aery radiation sensitive

.hasing

– Heavy atom soa/ing difficult – 0ata <uality limits phasing – utomatic programs may not be applicable

(rof: Gunnar $on e#;ne

• #opology of membrane proteins• #opologyS

5 !umber of transmembrane helices5 %rientation -ithin the bilayer 5 1ocation of !5 and "termini and loops (i e are they cytosolic or 5 extracytosolic?)

• T.ositive $nside uleU5 "ytosolic (TinsideU) face of a membrane protein has the greatest concentration of positive charge (residues rg and

1ys)



• &aCor experimentS "an topology be determined for the membrane protein proteome of an entire organism?• #oo/ every single ' coli membrane protein and cloned each t-iceS

5 #hey added a "5terminal .ho tag (al/aline phosphatase)5 Which is only active in the periplasm

• lso added "5terminal :F.5 %nly fluorescent in the cytosol

• &easured .ho activity and :F. fluorescence for each clone• esults

Why 2as the 2or4 from $on e#;ne so #m.ortant3

• $t allo-ed the rare structural information gained on membrane proteins to be translated to structural predictions• $t allo-ed functional experiments to be designed for membrane proteins in the absence of structure• $t facilitated a maCor step for-ard in techni<ues for the recombinant overproduction of membrane proteins

Dr: Da$#% Dre2

• #echni<ues for overexpression and stabilisation of membrane proteins using :F. fusion proteins

<uant#t#es of .rote#n nee%e% for crysta""#=at#on an% structure so"ut#on

• :enerally -e need milligram <uantities of pure protein for crystallisation experiments• Homogeneous proteinS



5 Free of Xother9 protein contaminants5 Free of Xother9 protein forms

ctive+inactive protein mixturesWith+-ithout cofactor.roteolytic cleavage etc

(rote#n (ur#f#cat#on

•

se detergent to extract the protein from the membrane• 0etergent mimics the membrane structure• $t has a hydrophobic tail and hydrophobic head, Cust li/e the lipids• *o to disrupt these membranes, -e mix them -ith large <uantities of detergent, so that -e can extract the proteins into a

detergent micelle• $f -e use the right amount of detergent, -e can encapsulate the target structure and pull it out of the membrane

(rote#n crysta""#=at#on

• 0iffraction from a single protein molecule is too -ea/ to detect, so -e gro- protein crystals to enhance the diffraction po-er• Hanging5drop method

)y.es of membrane .rote#n crysta"s

• 2ou can get three different formsS5 80 crystal5 #ype $ 60 crystal5 #ype $$ 60 crystal

• #he ability to gro- protein crystals depends on ho- much of the structure is protruding from the membrane

Da$#%>s E5.er#ment

• "an the production of integral membrane proteins be streamlined in a high throughput manner?5 He set up an expression vector, -here you put in your membrane protein of interest5 $t9s under the control of the #@ promoter, so you can induce it -ith $.#:5 1in/ed to the same polypeptide -ith :F. and His5#ag for purification5 $n bet-een these there is a #'A cleavage site – -hich is a very highly active protease5 Ho- to purify a protein -ith a His5#ag?



.erform metal affinity chromatography (can be nic/el or cobalt)• #his system is applicable to bacteria, probably yeast and insect cells and mammalian cells• *o until you cleave the membrane protein you can tag -ith :F.• 2ou use :F. fluorescence toS

5 *creen expression levels in -hole cells5 *creen detergents for extraction of target protein from membrane5 "hec/ integrity of target protein5 "hec/ that target protein is correctly processed and targeted to the membrane5 1ocate target proteins by *0*5.:'

• :F.5tagging can also be used to ma/e sure that the protein goes to the correct membrane

• #he other thing -e can do is to screen the protein for detergent5 llo-ing us to extract the protein from the membrane5 lso allo-s us to chec/ that our protein isn9t aggregating (main problem -e have membrane proteins is that if -e

use the -rong detergent all the hydrophobic parts of the membrane come together and form aggregates)5 .rovides good extraction from the membrane

• .erform a gel filtration colum, -hich separates based on si e5 1arge protein comes out early

D#ffus#on

:ases (oxygen, carbon dioxide) can diffuse across the cell membrane -ith no energy

cost to the cell

(ass#$e $:s: Act#$e )rans.ort

• "an either be passive or active transport• .assive transport J :oes through a membrane protein, -ithout much energy cost• ctive transport J "ell has to produce energy to allo- the movement of a molecule against the concentration gradient

?ac#"#tate% %#ffus#on 8a4a (ass#$e trans.ort9

• &embrane proteins facilitate substrate translocation across the membrane at a faster rate than is possible -ith simplediffusion

• X.assive9 transport 5 minimal energy re<uirements from the cell

Act#$e trans.ort

• %ccurs against a concentration gradient• e<uires energy from the cell

E5am."e of Act#$e )rans.orters

• B" transporters5 se #. as energy for transport5 T lternating ccessU model of membrane transport5 "ataly ed by #. hydrolysis to 0. po-ering the transport of the substrate

(rofessor So I2ata

• .erformed crystalli ation of membrane proteins• *olved structures of respiratory complexes• ntibody technology for membrane protein crystalli ation



• "rystallisation reservoir containsS5 .recipitant (salt, .': etc )5 Buffer 5 dditives (salts, organics etc )5 0etergent concentration and type is an additional variable (only in the drop not added to reservoir)

• Hanging 0rop &ethodS5 We have a reservoir containing specific precipitant (salt, .': etc ), buffer, additives etc5 #a/e one drop of reservoir and mix it -ith an e<ual volume of protein

5 *uspend the drop above the reservoir and this is sealed in a chamber 5 *o overtime becauseG for example the .': concentration in this drop is half that in the reservoir, -ater diffuses fromthe drop to the reservoir to try and e<uali e those concentrations

5 #he drop shrin/s and crystals gro-• #here9s t-o -ays to set up your crystalli ation experimentS

7 ational screenS Where you determine that .': -ill be used for crystal formation8 *parse5matrix screeningS .urchase a screen based on the six most common conditions under -hich proteins gro-

Dr: Da$#% Dre2

• 0etergent stability screening to improve crystalli ation• 0etergent used to mimic membrane detergent (as mentioned in the previous lecture)• What he did -asS

5 .redicted the topology of a specific membrane protein using a server 5 .redicted that in this protein the !5terminus starts off inside the cytoplasm and then it crosses the membrane a

number of times5 nd the "5terminus is also in the cytoplasm5 %nce he predicted this topology he loo/ed at -here the cysteine residues might be in that protein5 He predicted that some of these cysteine residues -ill be in the transmembrane alpha helices5 He mixed the proteins in detergents -ith buffer and fluorescent molecules, heated them and -atched the

fluorescence increase over time5 He basically loo/ed at protein stability against different detergents5 From this he -as able to get a half life for the membrane protein unfolding and then plotted it5 What -ould the half5life tell you about the stability of the protein?

1onger the half5life more stable the protein

5 %nce he found the most stable proteins, he crystalli ed and measured the diffraction properties of the crystals

(rofessor So I2ata 8 e a"so %#sco$ere%9

0eveloped methods to ma/ing antibodies that might assist in ma/ing protein crystalli ation

(atho!ens' ant#!ens' e.#to.es 8Assume% 4no2"e%!e9

• n antigen is a foreign molecule that can interact -ith cells of the immune system and illicit an immune response• ntibodies recognise antigens and the specific part of the antigen that they recognise is called the epitope

Ant#bo%#es 8Assume% 4no2"e%!e9

• lso called immunoglobulins• Bind substances that are foreign to the body (antigens) and target them for destruction• "ontain ; chains

5 #-o heavy chains5 #-o light chains

• #he chains are lin/ed by non5covalent and disulphide bonds• Form a 25shaped molecule #otal complex is 734 /0a



• #he basal fragment of the 2 is called the Fc (Xcrystallisable9)• #he t-o branches are called Fab (Xantigen5binding9)• 'ach branch has a single antigen binding site• .arts of an antibody are X"onstant9 regions and some are XAariable9• "onstant regions have a conserved motif ($g fold)

5 6 in each heavy chain5 7 in each light chain

• Aariable domains have significant variation in their amino acid se<uence5 7 in each heavy chain- 7 in each light chain

• *o $-ata9s -or/ -as to use the Fab part of the antibody for crystalli ation

Ant#bo%#es #n the "aboratory

• 0ot .lot• '1$*• Western Blot• $mmuno5precipitation• Flo- cytometry• &embrane "rystalli ation• $f you9re going to generate an antibody, it doesn9t matter if your protein is properly folded or not• Why is this the case?

5 Because -hen you run it on *0*5. :' then Western blot, you9re denaturing the protein any-ay5 #he antibody only recogni es the se<uence not the tertiary structure

• But if you -ere going to use antibodies in membrane protein crystalli ation, it does matter if the protein is folded or not5 #his is because the proteins are folded in the crystals5 #he antibody needs to recognise the folded protein in this case

(ro%uct#on of ant#bo%#es

• %ne -ay to produce antibodies is to hiCac/ the natural antibody5production path-ay• se an animal to raise antibodies against a specific antigen• "hoose antigenS

5 0ecide -hat part of the antigen you re<uire the antibody to be against5 ' g domain, binding poc/et, -hole protein, peptide5 #he antigen chosen depends on the end use (e g for Western Blot, the protein+peptide antigen does not need to be

folded) – for membrane proteins you need to immuni e -ith an animal -ith a folded protein• *o the procedure is to immuni e -ith an antigen, ta/e the serum and you can either isolate the antibodies from that, using a

server

?or membrane .rote#ns' ho2 %o you ma4e sure that the membrane .rote#n #nocu"ate% #nto the an#ma" #s fo"%e%3

• $-ata, thought to encapsulate the membrane proteins into proteo5liposomes• #a/e lipids and extrude it into the proteo5liposomes• #his does t-o thingsS

7 &a/es sure that the membrane protein is folded because the hydrophobic parts of the membrane protein,needs to interact -ith the hydrophobic part of the liposome

5 1i/e-ise for the hydrophilic parts5 #his cannot happen if the protein is unfolded

8 $t9s sitting in the proteo5liposome5 &ost of the structure is shielded but there9s little bits of hydrophilic structure that are revealed5 We only -ant antibodies that stic/ to the ends to ma/e crystal contacts5 #his is all that -e are revealing -hen -e put it into proteo5liposomes

• %nce they thin/ they found some antibodies that bind to our membrane protein in liposome, the chec/ that that is true byadding *0* and doing the -hole '1$* again

• What the *0* does, is brea/s up the proteo5liposome and unfolds the membrane protein



• *o he loo/ed at the antibodies that stuc/ to the folded situation and not the unfolded situation



INBORN ERRORS O? ME)ABOLISM

Introduction

• 0espite genetic variation the chemical composition of our tissues and our metabolic processes are remar/ably similar • .athology tests utili ed to chec/ for deviations seen in blood or urine -here most deviations are due to ac<uired dysfunction

such as infection• &aCor deviations in our homeostatic state are due to "= $ '0 02*F !"#$%! such as heritable disorder

o !%W! * $!B% ! ' % * %F &'# B%1$*&epresent a subset of genetic disordersS some disorders may arise due to developmental issues

• B1%%0 #'*#* – measure aemias such as high blood ammonia H2.' &&%!'&$• $!' #'*#* – measure urias• *ome metabolic tests can be determined through biopsies as some metabolic activities only occur in special tissues

o Biopsy allo-s for direct measure of the defective en yme• B$%.*$'* . %B1'&*

o *ome metabolic activities are found in all tissues such as glycolysis, #" cycle, %D.H%* but some metabolicactivities are #$** ' *.'"$F$"

%nly liver and /idney cortex ma/e glucose%nly liver can convert ammonia into urea etc"!* can only ma/e neurotransmitters

o #a/ing a biopsy sample from many of these tissues is ris/yo &ore common no- to ta/e a * $! B$%.*2 and *'= '!"' #H' 0!

• 'D. '**$%! %F :'!'#$" 0$*% 0' *o !ot all mutations express themselves as metabolic defects can beG

*2&.#%& #$" – *ilent mutations that do not change the aa se<uence of a proteins, mutations thatma/e conservative changes to the aa se<uence % genetic variants that do not affect health*2&.#%& #$" 'D"'.# F% ""$0'!# 1 "$ " &*# !"'* – blood clotting defects such asHaemophilia -here trauma is re<uired for the expression, some diseases such as haemolytic anaemia9srevealed only on exposure to drugs, lactose intolerance only presented if consume dairy&$10 #% &%0' #' – problematic but not incompatibleG

• H '&%"H %& #%*$* – body absorbs too much irono utosomal recessiveo Body absorbs a lot more iron from their food than necessary and starts building up iron

stores in various areas of the body including the liver, bone marro-, heart and Cointo .rolonged can cause disease

• :% # – characteri ed by recurrent attac/s of acute inflammatory arthiritis -ith red, tender, hots-ollen Coints

o "aused by elevated levels of uric acid in the blood -here the uric acid crystalli es anddeposits in Coints, tendons and surrounding tissues

o "an be caused by diet or genetic predispositiono >4 caused by under5excretion and 74 from overproduction

*'A' ' #% 1'#H 1 – mutations of genes critical for development• 0o not affect development but become serious to lethal after birth

• D51$! '0 & # #$%!* – $f a gene encoded by the D5chromosome is mutated the mother -ill have one unaffected geneand one mutated gene 'ven if the mutated gene is 744 affected, the female still has 34 of maximum activity &any casesthis is enough to ma/e it asymptomatic

• '!I2&'* !0 $!#' &'0$ 2 &'# B%1$*&o We have about 64,444 genes, each one coding for a protein Ho-ever due to differential splicing and other factors

-e probably have about 744,444 proteins'*#$& #'0 844,444 . %#'$!*&utations in any one has the possibility of causing defects in metabolism

• .%#'!#$ 1 "%!*'= '!"'* %F "H !:'* $! '!I2&' F !"#$%!

•

• $f the gene coding for 'n yme " is defective in the reaction path-ay potential conse<uences could beGo 1 " %F F% & #$%! %F 0 % '!0 . %0 "# F for exampleG



A%! :$' '9* 0$*' *', en yme " -ould be glucose Q phosphatase leading to the inability to formglucose 5 explanation the en yme glucose Q phosphatase is needed for brea/do-n of glycogen to glucose

• A%! :$' '9* 0$*' *' – 0'F'"# J :1 "%*' Q .H%*.H%# *'o "auses – hypoglycemia (lo- blood glucose), hypomeagly (enlarged liver), hyperlipidemia, gro-th failure, lactic

acidosiso ffects 7+34444

• $f the gene encoding for 'n yme " is defective in the reaction path-ay can get accumulation of ", the immediate precursor of the bloc/ed reaction

o "ommon exampleG :12"%:'! *#% :' 0$*' *' – defective en yme are phosphorylases -hich areresponsible for the brea/do-n of glycogen

'D &.1' – H' 9* 0$*' *' – 0'F'"# – 1$A' :12"%:'! .H%*.H% 21 *'• "auses – hypoglycemia, hypomeagly, hyperlipidemia• bility to synthesi e glycogen but don9t have the ability to brea/ it do-n

o "an be treated by cornflour • ffects 7+Q3444

'D &.1' 8 – 0'F'"# 5 :1 " %!21 # !*F' *' ( :#)• ccumulation of many toxic compounds leading to accumulation of bilirubin therefore causing

liver damageo %!' %F 1$A' 9* %1'* J 0'#%D$F$" #$%! %F * B*#!"'*

• eaction cataly ed by :# en ymes involves the addition of glucuronic acid moiety toxenobiotics and is the most important path-ay for the elimination of the top 844 drugs 1*% themaCor removal path-ay for foreign substances

• $f the gene encoding for 'n yme " is defective in the reaction path-ay can get accumulation of and Bo $n Aon :ier/e9s disease -here glucose Q phosphatase is bloc/ed there is a gross build up of heptatic glycogen -hich

can cause liver damage*ame en yme defect can cause build up of blood lactic acid

• $f the gene encoding for en yme " is defective in the reaction path-ay can get production of products -ith normally minor importance

o &ay cause the use of a normally minor path-ay or an entirely inappropriate path-ay.H'!21 '#%! $ – due to a defect in the en yme phenylalanine hydroxylase 'n yme is re<uired for the conversion of phenylalanine to tyrosine – .henylalanine accumulates and is converted to

phenylpyruvate•

!eed to eat a diet lo- in phenylalanine but containing tyrosine• utosomal recessive• $ssue not -ith tyrosine as it is ac<uired in the diet• $ssue is the build up of its substrate .H'!21 '#%!' -hich can cause permanent brain damage

& .1' *2 . $!' 0$*' *' – 0ue to defect in an en yme complex -hich oxidises branch chainamino acids

• #he branched amino acids are the most abundant amino acids in proteins, so dietary inta/e tendsto be <uite high of these amino acids

• *2&.#%&* !0 0$ :!%*$*o Because of the enormous number of these diseases and -ide range of systems affected, nearly every presenting

complaint to a doctor may have a congential metabolic disease as a possible diseaseo 0o ens of congenital metabolic diseases are no- detectable by ne-born screening tests

•

# ' #&'!#*o *ome of the disorders in amino acid metabolism have been treated -ith restriction of protein inta/eo :ene therapy is being loo/ed into too 0ialysis to remove toxic compounds from blood -or/s, only useful for a short timeo Bone marro- or organ transplantation

'iabetes Mellitus

• Fasting blood glucose is elevated above the normal 744mg+d1• 'ither the . !" ' * 0%'* !%# . %0 "' * FF$"$'!# $!* 1$! or "'11* 0%!9# '*.%!0 #% $!* 1$!



o $nsulin promotes absorption of glucose• 1oss of insulin production is usually caused by a destruction of pancreatic beta cells• 1oss of insulin sensitivity is often caused by loss of insulin receptor sensitivity• 0iabetes is a condition -here glucose is excreted into urine leading to fre<uent urination and increased hunger • #2.' $ 0$ B'#'* – " !9# . %0 "' $!* 1$!

o esults from the body9s failure to produce insulin and currently re<uires the person to inCect insulin or -ear aninsulin pump

o "aused by a loss of the insulin producing beta cells of the islets of langerhan in the pancreas1oss often caused by auto5immunity

o esults in a catabolic state -ith fat and muscle -asting• #2.' $$ 0$ B'#'* – $!* 1$! '*$*# !"'

o "ondition in -hich cells fail to use insulin properlyo sually late onset and associated -ith obesity – represents >4 of all diabetes

• $* * **%"$ #'0 W$#H 0$ B'#'* &'11$# *o 0amage to blood vessels resulting in cardiovascular diseaseo *tro/e, blindness and end5stage renal diseaseo $mpacts on the nervous system – in severe cases it affects the circulation and diabetic foot ulcers often leading to

lo-er extremity amputations• &'# B%1$" *2!0 %&' – "luster of conditions – increased blood pressure, high blood sugar levels, excess body fat

around the -aist and abnormal cholesterol levels – increasing the ris/ of heart disease, stro/e and diabeteso Having three or more at the same time can lead to metabolic syndromeG

%B'*$#2 – 1 :' W $*# *$I', H2.' #'!*$%!, H$:H B1%%0 * : , H$:H "H%1'*#' %1o $* F "#% * – ge (increases -ith age), obesity, history of diabetes, other diseases

• H% &%! 1 "%!# %1 %F B1%%0 :1 "%*'o Aerterbrates use glucose, fatty acids and amino acids as fuel to produce #.o 1$A' !0 . !" ' * are the human fuel supplieso .ancreas is made of t-o maCor tissue typesS "$!$ (prod digestive en ymes) and $*1'#* %F 1 !:' H !*

(B'# . %0 $!* 1$!, 1.H . %0 :1 " :%!)o :1 " :%! – raises blood glucose by stimulating the release of glucose by liver by gluconeogenesiso $!* 1$! – lo-er blood glucose through increased upta/e by tissues and deposition of glycogeno :lucagon is not the only hormone to release glucose into the blood but insulin is the only hormone that can lo-er

blood glucose levelso $n a non5diabetic blood glucose is maintained at 744mg+d1o $nsulin increases after a meal and stores glucose in the liver and to lesser extent muscle

0ifferent tissues use different fuels – muscle and adipose tissue use fatty acid:1 "%*' & *# B' *'0 B2 B $!

o :lucose not needed for energy is stored as glycogen or converted to triglycerides• n increased insulin resistance raises blood triglyceride level and other blood fat levels lso interferes -ith /idney function

leading to an increased blood pressure• $!* 1$! "#$%!

o "ontrols blood glucose in t-o -ays"#$A #$%! %F # !*" $.#$%!

• ctivation of transcription is through the & . path-ay through tyrosine /inase receptors



•

': 1 #$%! %F '!I2&'* #H # " *' :1 "%*' #% B' *#% '0 * :12"%:'!• *timulates upta/e of glucose through :1 #; transporter • ctivates glycogen synthase

• 1o-ering and increasing blood glucose

•

o %nly the liver is capable of creating glucose through gluconeogenesis – muscle does not contain the right en ymes(glucose Q phosphatase) to allo- glucose to enter the bloodstream

• #2.' $ 0$ B'#'* – nable to eat fato #here is insufficient insulin to allo- him to ta/e up any glucose from dietary sources so if he had any fat he -ould

use this as an energy source and -ould become /etotico $nsulin is used to allo- glucose upta/e by tissues but because there9s no insulin glucose can9t be ta/en up and so in

turn begins to brea/ up fatty acids into /etone bodies as an alternative fuelo "onstantly having to oxidise fat ma/es him thino &aintain blood glucose through gluconeogenesis by eating protein

• '#%5 "$0%*$* *2&.#%&*o bdominal pain, nausea and vomiting, deep and rapid breath, s-eet smelling breath, dro-siness

• $n type $ diabetes and /eto5acidosis gluconeogenesis due to untreated diabetes slo-s do-n the #" cycle due to the removalof oxaloacetate and enhancing the conversion of acetyl5"o into /etone bodies



•

• #H' W B : 'FF'"#o "ancer cells change their metabolism to enable them to leave their normal place of residence and invade other

tissues re<uiring them to become less dependent on oxidative metabolism as a source of energyo #-o types of metabolism – ! ' %B$" (:12"%12*$*) and ' %B$" (%D.H%*)o When cancer cells become transformed they become dependent on anaerobic respiration

'nd product of this is lactic acid -hich convert to pyruvate and then to aceyl5"oo #ransformation of cells is a set of changes that cause a cell to become freed of the normal constraints that control a

diploid cell1oss of regulation of mitosis and cell cycle chec/points

bch3atb notes

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