Transcript talk_UPR - Columbia University

Uncovering an unfolded protein
response in Trypanosoma brucei by
a computational approach
Shai Carmi
Bar-Ilan University
Deptartment of physics and the faculty of life sciences
June 2009
The endoplasmic reticulum (ER)
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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)
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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).
Metazoan UPR
nonconventional splicing
From Allen Volchuk’s lab website
UPR in protozoans (such as yeast) has only
the IRE1-HAC1 system.
UPR sensors and signal transduction
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The three ER stress sensors respond differently to
high concentration of unfolded proteins:
Bernales et al., 2006
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.
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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.
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ATF6 migrates to the Golgi upon UPR induction,
where it is cleaved and activates transcription from
Metazoan Xbp1 nonconventional splicing
other promoters.
results in frame shift and activation.
UPR components
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The ultimate goal of the response to decrease the
concentration of misfolded proteins in the ER.
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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.
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More facts about the UPR
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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
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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
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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
Polycistronic
Transcript
SL
Trans-Splicing=
And
Polyadenylation=
AAAA
AAAA
AAAA
AAAA
Itai Dov Tkacz
Unfolded protein response?
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No homolouge of IRE1, the ER stress sensor, was
identified to date in T. brucei.
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T. brucei cannot regulate gene expression by
transcription activators.
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Does T. brucei have unfolded protein response and how
is it executed?
Applying ER stress
Silencing the translocon components.
Ribosome
Treating with the reducing
agent Dithiothreitol (DTT).
mRNA
ER membrane
© Saggit Goldshmidt
Sec63
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.
DTT reduces disulfide
bonds (S-S) which are
essential for the proper
folding of many proteins.
Leads to UPR in various
organisms.
SL-RNA Silencing (SLS)
Lustig et al.,
EMBO reports,
2007
SLS induction
Nucleoplasm 42
42 50
26
?
SL
42
42
SL
RNA
42
SL
DNA
SL
42
Itai Dov Tkacz.
No trans-splicing
42
No mRNA
No protein
Increase in levels of the chaperone BiP is also obsreved.
Cell death
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SL-RNA is the
only gene whose
expression is
controlled by
interaction
between a
promoter and an
activator- the
tSNAP complex.
4mM DTT
min
0
60
120
SL
SR1
.
Anat Kabi, 2007
How does SLS compare to classic UPR in other organisms?
How is the ER stress signal transmitted into the nucleus?
240
Microarrays
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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
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Use microarrays to track the change in mRNA levels of
stressed cells in comparison to untreated cells.
DTT
r=0.85
Every gene is a dot
Every gene is a line
Arrays for 1 hour and 3 hours are consistent.
Regulation exists: many genes are up and downregulated.
Functional analysis of regulated genes
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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.
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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.
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Functional analysis of regulated genes
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Upregulated genes in T. brucei belong to similar functional
categories as UPR genes from other organisms.
Hanoch Goldshmidt, S.C., 2009
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Genes downregulated after 1 hour treatment with DTT are
enriched with ER client proteins (P<10-4).
ER stress leads to programmed cell death
Loss of mitochondrial membrane potential
Production of reactive oxygen species
DNA ladderinggenomic DNA digested
at specific intervals
Concentration
of cytoplasmic
calcium ions
increases after
treatment also
indicating
apoptosis
Also observed
endoG release
Propidium iodide (PI)- correlates with membrane permeability.
Annexin V binds to Phosphatidylserine (PS). Exposed PS indicates apoptosis.
Hanoch Goldshmidt,
Dvorah Mattas, 2009
How to identify the genes which
mediate SLS?
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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
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Tb927.1.2100Caplain-like cysteine
peptidase.
Calcium binding protein.
Lost its proteolytic activity.
Membranal, not essential.
Role in apoptosis signaling?
.
S.C
Ephrat ben-Mayor
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Tb10.6k15.1520small GTPase.
Homolog of human RagC.
Homolog participates in
amino-acid starvation
signaling to mTOR and
localizes
in the nucleus
and the
cytoplasm.
Essential in
T. brucei.
Experimentally test candidate genes
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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
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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