Uncovering an unfolded protein response in

Trypanosoma brucei by a computational approach

Shai CarmiBar-Ilan University

Deptartment of physics and the faculty of life sciences

June 2009

The endoplasmic reticulum (ER) The ER is a membrane enclosed Eukaryotic organelle that provides an environment in which secreted or other proteins with hydrophobic domains can be properly folded and targeted without aggregating.

Responsible for protein•Folding•Modification•Transport•Degradation

•Lipid and steroid synthesis•Calcium storage

The unfolded protein response (UPR) ER stress in the form of

high concentration of misfolded proteins triggers the unfolded protein response:A signaling pathway which relieves stress by selective gene upregulation and other mechanisms.

Prolonged ER stress leads to programmed cell death (Apoptosis).

UPR in protozoans (such as yeast) has only the IRE1-HAC1 system.

From Allen Volchuk’s lab website

Metazoan UPR

nonconventional splicing

UPR sensors and signal transduction The three ER stress sensors respond differently to high concentration of unfolded proteins:

IRE1 oligomerizes, autophosphrylates and activates its RNase domain to remove an intron from HAC1 mRNA to facilitate its translation. hac1p then binds to specific promoters to activate transcription of UPR genes.

PERK phosphorylates eIF2α to block the formation of ribosomal preinitiation complex and to attenuate translation, while at the same time preferentially translate the ATF4 transcription factor.

ATF6 migrates to the Golgi upon UPR induction, where it is cleaved and activates transcription from other promoters.

Metazoan Xbp1 nonconventional splicing results in frame shift and activation.

Bernales et al., 2006

UPR components The ultimate goal of the response to decrease the

concentration of misfolded proteins in the ER.

Upregulate chaperones that increase the chances of proper folding by preventing aggregation and covalent modification.

Express lipid biosynthesis genes to expand the ER. Remove proteins from the ER by accelerated degradation and

transportation out of the ER. Relieve ER load by global translation attenuation and specific

mRNA degradation of ER client proteins. Genes which participate in other processes (e.g., protection from

oxidative stress, signaling, and metabolism) are also upregulated.

More facts about the UPR

The three arms of UPR induction are not just independent on/off switches but are modulated by interconnections and external factors.

ER stress can be artificial (‘pharmacological’) or biological. The entire UPR or selected branches are induced under normal

physiological conditions (development of B cells into ‘antibody factories’ plasma cells, nutrient deprivation) as well as in disease: diabetes, viral infection, retinal degeneration, cancer (hypoxia).

All three branches also upregulate genes that elicit programmed cell death after prolonged stress via standard pathways (e.g. calcium release from the ER, cytochrome c release, caspase activation, dephosphorylation of eIF2α, reactive oxygen species production).

Pro-survival and pro-apoptotic pathways compete. For example: PERK also phosphorylates eIF2α but at the same time upregulates CHOP and consequently GADD34 which dephosphorylates eIF2α.

Trypanosoma brucei

Trypanosomes are parasitic eukaryotes that diverged 200-500 million years ago.

Pathogens of the African Sleeping sickness. Live in the gut of the Tsetse fly

(the “procyclic form”), then transfer to the bloodstream of humans and cattle.

Have unique biology: - Kinetoplast - RNA editing with gRNA- trans-splicing- Variable surface glycoproteins

From Mark Field’s lab website

S.C.

mRNA processing

mRNA is processed by trans-splicing and polyadenylation.

T. brucei genes have no promoters.

Gene expression is regulated by controlling splicing,mRNA stability, and translation.

Gene1 Gene2 Gene3 Gene4

PolycistronicTranscript

AAAA

AAAA

AAAAAAAA

SL

Itai Dov Tkacz

Trans-Splicing=And

Polyadenylation=

Unfolded protein response?

No homolouge of IRE1, the ER stress sensor, was identified to date in T. brucei.

T. brucei cannot regulate gene expression by transcription activators.

Does T. brucei have unfolded protein response and how is it executed?

Applying ER stress

Silencing the translocon components. Treating with the reducing agent Dithiothreitol (DTT).

DTT reduces disulfide bonds (S-S) which are essential for the proper folding of many proteins.

Leads to UPR in various organisms.

After translation initiation, proteins processed by the ER are attached to the ER membrane by the signal recognition particle (SRP), after which they are transported into the ER lumen through a channel called the translocon.

RibosomemRNA

ER membrane

© Saggit Goldshmidt

Sec63

SL-RNA Silencing (SLS)

How does SLS compare to classic UPR in other organisms? How is the ER stress signal transmitted into the nucleus?

SL-RNA is the only gene whose expression is controlled by interaction between a promoter and an activator- the tSNAP complex.

0 60 120 240

SL

SR1

min4mM DTT

.Anat Kabi, 2007

ER stress

DNASL SL SL

4226

50SL

RNANucleoplasm

No protein

No mRNA

SLS induction

4242 42 42

4242

?

No trans-splicing

Cell death

Itai Dov Tkacz. Itai Dov Tkacz.

Increase in levels of the chaperone BiP is also obsreved.

Lustig et al., EMBO reports, 2007

Microarrays Microarrays are chips on which thousands of DNA

oligos are printed in an array. Each oligo represents a fragment of one gene.

Total RNA is isolated and then reverse transcribed to cDNA, during which it is also labeled with fluorescent dye.

The cDNA is hybridized with the array after which the array is read by a scanner. The higher the concentration of a given gene, the brighter will be the corresponding spot in the array.

Expression profiles of entire genomes can be obtained within a single experiment.

Other uses include ChIP or construction of specialized arrays to detect alternative splicing.

Wikipedia

The transcriptome of ER stressed cells

Use microarrays to track the change in mRNA levels of stressed cells in comparison to untreated cells.

Every gene is a dotEvery gene is a line

Arrays for 1 hour and 3 hours are consistent.

DTT

r=0.85

Regulation exists: many genes are up and downregulated.

Functional analysis of regulated genes Genes upregulated at least 1.5-fold after both 1 hour and 3 hours

DTT treatment were classified to functional categories. No filtering can be done as in other organisms.

The class with the largest number of upregulated genes is the secretory pathway.

Many signaling genes were upregulated, suggesting the process is regulated.

Genes related to transport, metabolism, gene expression, mitochondria, and cell cycle were also upregulated.

We also observed upregulation in DNA repair, oxidative stress, and calcium homeostasis genes, as expected in cells committing apoptosis; and in motility genes, raising the hypothesis that trypanosomes have evolved a ‘chemotaxis’-like mechanism.

Functional analysis of regulated genes Upregulated genes in T. brucei belong to similar functional

categories as UPR genes from other organisms.

Genes downregulated after 1 hour treatment with DTT are enriched with ER client proteins (P<10-4).

Hanoch Goldshmidt, S.C., 2009

ER stress leads to programmed cell death

Hanoch Goldshmidt, Dvorah Mattas, 2009

DNA laddering-genomic DNA digested at specific intervals

Loss of mitochondrial membrane potential

Production of reactive oxygen species

Propidium iodide (PI)- correlates with membrane permeability.Annexin V binds to Phosphatidylserine (PS). Exposed PS indicates apoptosis.

Also observed endoG release

Concentration of cytoplasmic calcium ions increases after treatment also indicating apoptosis

How to identify the genes which mediate SLS?

The transcriptomes of DTT treatment and Sec63 silencing are quite dissimilar even though both lead to SLS.

The few commonly upregulated genes may participate in the SLS pathway.

Choose two candidate genes for further experimental analysis.

Candidates Tb10.6k15.1520-

small GTPase. Homolog of human RagC. Homolog participates in

amino-acid starvation signaling to mTOR and localizesin the nucleus and the cytoplasm.

Essential inT. brucei.

Tb927.1.2100-Caplain-like cysteine peptidase.

Calcium binding protein. Lost its proteolytic activity. Membranal, not essential. Role in apoptosis signaling?

Ephrat ben-Mayor

S.C.

Experimentally test candidate genes

Prepare double silenced cell line where both the candidate gene and Sec63 are silenced by RNAi.

Test for SLS by northern blot.

Silencing is induced by Tetracyclin.

Conclusions

T. brucei respond to ER stress by upregulating specific genes in a pattern that resembles the response of other organisms.

In addition, mRNA metabolism is arrested in synergy with programmed cell death that takes place at later times.

We conclude that T. brucei has UPR with trypanposome specific elements.

Computational approach suggests proteins that participate in the signaling pathway. More experimental work is required to establish a concrete statement.

Special thanks to all Michaeli’s lab members:

Ephrat ben-Mayor

Hanoch Goldshmidt

Nasreen Hag-Yahia

Ronen Hope

Sachin Kumar Gupta

Asher Pivko

Mali Romano

Itay Dov Tkacz

Pawel Tuliniski

Damian Visnovezky

Vadim Volcov

Liat Wulffhart

Dr. Devorah Mattas

Dr. Chaim Wachtel

Prof. Shulamit Michaeli