Uncovering an unfolded protein response in Trypanosoma brucei by a microarray approach

1. Uncovering an unfolded protein response in Trypanosoma brucei by a microarray approach Shai CarmiBar-Ilan UniversityDepartment of physics- Prof. Shlomo Havlin The faculty of life sciences- Prof. Shulamit Michaeli Ohalo,June 2009

2. 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. • Unique biology: - Kinetoplast - Antigenic variation - trans-splicing S.C. IAEA From Mark Field’s lab website

3. The endoplasmic reticulum (ER) • The ER is a membrane enclosed organelle in which nascent proteins are processed (‘protein processing factory’). • Stages in protein maturation: • Folding • Modification • Transportation • Degradation

4. The unfolded protein response (UPR) • 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.

5. 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. • Do Trypanosoma brucei have unfolded protein response?

6. A problem • Stress response is usually mediated by a genome-wide 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.

7. DNA chips- Microarrays Treated cells • 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.

8. 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.

9. 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

10. Biochemical evidence DNA laddering • Persistent ER stress leads to programmed cell death. • Apoptosis seem to be accelerated by shutoff of mRNA production by inhibiting transcription of SL-RNA which is necessary for splicing. • Upregulation is mediated by mRNA stability. Hanoch Goldshmidt, Dvorah Mattas, 2009 Phosphatidylserine exposure Anat Kabi, 2007 Lustig et al., EMBO reports, 2007

11. Conclusions • We conclude that Trypanosoma brucei have unfolded protein response with some unique elements. • How does the ER stress signal reach the nucleus?

12. 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