The cell stress response

1. The cell stress response 9-1 Overview of the cell stress response - history, what causes it Regulation of the cell stress response - in prokaryotes, eukaryotes The Unfolded Protein Response (UPR) and ER Associated Degradation (ERAD)

2. Chronology of the cell stress response 9-2 1962 1974 1982 1985 1986 1988 • Italian scientist Ferrucio Ritossa discovers new puffing pattern in the giant chromosomes found in Drosophila salivary glands: many of the normal (pre-existing) ‘puffs’ were gone, and he saw new ones. He later found that a colleague had increased the temperature of the incubator. Also observed new RNA synthesis • Alfred Tissières ‘rediscovered’ these results and observed new pattern of (radiolabeled) protein synthesis following a heat-shock by SDS PAGE • Several heat-shock inducible genes are cloned • Alfred Goldberg shows that production of abnormal proteins in E. coli activates the heat-shock response • Richard Voellmy discovers the trigger for the heat-shock (stress) response • Sambrook and Gething discover the Unfolded Protein Response (UPR) of the endoplasmic reticulum • Molecular chaperone functions and structures are being elucidated

3. Agents/treatments that induce the stress response 9-3

4. Stress response: cellular changes viability temperature temperature viability time time 9-4 • transcriptional upregulation of the synthesis of some genes - major proteins produced have the following sizes: <30, 40, 60, 70, 90, ~100 kDa • downregulation of the production of most proteins • most dramatic example: - Pyrodictium occultum thermosome (chaperonin) accumulates to ~70% or more of total cellular protein; other proteins not produced • acquired thermotolerance

5. Trigger of cell stress response 9-5 • What triggers the cell stress response? - direct sensing of external agents, e.g., heat, by protein(s)? - indirect sensing of external agents? translational downregulation of the production of most proteins • Experiment: co-injection of purified proteins and hsp gene reporter transcription start Hsp70 promoter β-galactosidase gene Ananthan et al. (1986) Science232, 522-524.

6. Regulation of the stress response:prokaryotes 9-6a • stress-inducible genes have promoters that differ from genes that are not induced under stress conditions: TCTCNCCCTTGAA CCCCATNTA stress-inducible gene(s) - sometimes part of an operon, e.g., GroEL-GroES or DnaK-DnaJ-GrpE -35 HS promoter -10 • σ70 (sigma-70) binds RNAP and activates transcription of genes • σ32 (sigma-32) can also assemble with RNAP, but it is unstable and rapidly degraded under physiological conditons; under stress conditions (e.g., heat-shock), it is stabilized and it assembles with RNAP • RNAP-σ32 binds HS promoters, upregulating their transcription • DnaK (Hsp70), DnaJ (Hsp40) and GrpE E. coli mutants have a constitutively active stress response

7. feedback control 9-6b cellular condition physiological stress physiological chaperones chaperones inactive active inactive inactive Transcription Factor Transcription Factor Transcription Factor Transcription Factor folded proteins denatured proteins denatured proteins folded/degraded proteins

8. Regulation of the stress response:eukaryotes 100bp M S L... CATGAGCAT TGAATGTTCTAGAAT TCGAATGTTCTAGAG ATGTCACTT TATA TATA GTACTCGTA ACTTACAAGATCTTT AGCTTACAAGATCTC TACAGTGAA ... M L M 9-7 • Heat-shock transcription factor (HSF) binds heat-shock elements (HSE). The HSE has been conserved throughout evolution, from yeast to humans • HSEs consist of the sequences nGAAn and its complement nTTCn, and occur in tandem (multiple copies) - an example of a potent, bi-directional heat-shock promoter is from the C. elegans Hsp16-1 and Hsp16-48 operon: • at least 2-3 HSE sequences are required for HSF binding; HSF binding to HSEs is cooperative: the more HSEs present, the stronger the binding • many heat-shock protein promoters have been used to control gene expression - e.g., nematode biosensor

9. Regulation of the stress response:HSF Active trimer cellular stress HSF has a Winged Helix-Turn-Helix Motif trimerization via leucine zippers binding to HSEs reversal to monomers following stress HSEs stress-inducible gene HSF DNA binding domain (monomer) in complex with HSE sequence 9-8 Inactive heat shock transcription factor (HSF) monomer • activation of transcription by HSF requires phosphorylation • monomer-trimer transition, and activity while bound to DNA is regulated by molecular chaperones

10. The Unfolded Protein Response 9-9 Figure 1. The unfolded protein response (UPR): a model for this ER sensing and response pathway derived from the studies referenced in the text. When the burden of unfolded proteins is low, ER chaperone Kar2p binds to the lumenal domain of the Ire1p protein, thus limiting Ire1p self-association and activity of the protein. When the lumenal unfolded protein burden is increased as a result of pharmacological, genetic or developmental perturbation, the Kar2p molecules (and/or other chaperones) are 'distracted' from binding Ire1p, allowing self-association and activation of Ire1p. Active Ire1p participates in splicing of inactive HAC1 mRNA, called HAC1u, into a form, HAC1i, that is efficiently translated, allowing production of Hac1p transcription factor and increased synthesis of myriad ER-related genes. Adapted from Hampton (2000) Curr. Biol.10, 518.

11. ER Associated Degradation (ERAD) 9-10 Figure 2. Two fates for unfolded ER proteins controlled by the UPR. Unfolded proteins in the ER lumen or the ER membrane can either be folded (left branch) or degraded by ER-associated degradation (ERAD), by the appropriate proteins dedicated to these functions. An increased level of unfolded proteins increases the 'tone' of the UPR, causing concomitant increases in activity of each process. Importantly, the studies with null mutants indicate that these two fates both operate continuously in normal cells, such that loss of capacity to perform either branch results in measurable cell stress.