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Uncovering an unfolded protein
response in Trypanosoma brucei by
a microarray approach
Shai Carmi
Bar-Ilan University
Department of physics- Prof. Shlomo Havlin
The faculty of life sciences- Prof. Shulamit Michaeli
Ohalo,June 2009
Trypanosoma brucei

Parasitic eukaryotes that diverged
200-500 million years ago.

Pathogens of the African Sleeping Sickness.
Transfer from the gut of the Tsetse fly to the
bloodstream of humans and cattle.
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Unique biology:
- Kinetoplast
- Antigenic variation
- trans-splicing
.
S.C
IAEA
From Mark Field’s
lab website
The endoplasmic reticulum (ER)

The ER is a membrane enclosed organelle in which nascent proteins
are processed (‘protein processing factory’).
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Stages in protein maturation:
Folding
Modification
Transportation
Degradation
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The unfolded protein response (UPR)
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High concentration
of misfolded proteins
triggers the
unfolded protein response:
A pathway that relieves
stress by selective gene
upregulation and other
mechanisms.

Prolonged ER stress
leads to programmed cell
death (Apoptosis).
nonconventional splicing
From Allen Volchuk’s lab website
Microorganisms has only
the IRE1 pathway.
Unfolded protein response?

No homolouge of IRE1, the ER stress sensor, was
identified to date in Trypanosoma brucei.

Trypanosoma brucei cannot regulate gene expression by
transcription activators.
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Do Trypanosoma brucei have unfolded protein
response?
A problem

Stress response is usually mediated by a genomewide remodeling of the gene expression program.

To understand the response, we need to know all
the genes which are affected.

Standard biochemical methods probe only one
gene at a time.
DNA chips- Microarrays
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Expression profiles of entire genomes can
be obtained within a single experiment!
Microarrays are chips on which thousands
of short DNA sequences are printed. Each
fragment represents one gene.
RNA is isolated, reverse transcribed, and
labeled with a fluorescent dye.
The cDNA is hybridized with the array and
the array is scanned. The higher the mRNA
level of a given gene, the brighter will be
the corresponding spot in the array.
Microarray unit exists in Bar-Ilan.
Drawbacks: costly, noisy, not at protein
level.
Treated cells
Microarray study of ER stressed cells

We use microarrays to track the changes in mRNA
levels of stressed cells in comparison to untreated cells.
DTT
Each bar is one gene.
Arrays for 1 hour and 3 hours
are consistent (r = 0.85).
Regulation exists: many genes are up and downregulated.
Functional analysis of regulated genes

Upregulated genes in T. brucei are involved in processes
similar to UPR genes from other organisms.
Hanoch Goldshmidt, S.C., 2009
Biochemical evidence
DNA laddering

Persistent ER stress leads to
programmed cell death.
Hanoch Goldshmidt,
Dvorah Mattas, 2009

Phosphatidylserine exposure
Apoptosis seem to be accelerated by shutoff
of mRNA production by inhibiting transcription
of SL-RNA which is necessary for splicing.
4mM DTT
min
0
60
120
240
SL
SR1
.
Anat Kabi, 2007

Upregulation is mediated by mRNA stability.
Lustig et al., EMBO reports, 2007
Conclusions

We conclude that Trypanosoma brucei have unfolded
protein response with some unique elements.

How does the ER stress signal reach the nucleus?
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 Volkov
Liat Wulffhart
Dr. Devorah Mattas
Dr. Chaim Wachtel
Prof. Shulamit Michaeli