1. Molecular Chaperones The Hsp70 Super-family

2. Pathways For Protein Folding

3. Protein Folding in Cells Occurs in a Crowded Environment where Misfolding & Aggregation are Favored

4. Hsp 70 Family Proteins Assist Protein Folding in an Energy Dependent Process A.Hsp70 domain structure & Co- chaperone sites B.Hsp70 chaperoning Cycle

5. The Hsp 70 Super-Family Contains Diverse Members With Related Domain Structures

6. Protein Folding in Eukaryotes Involves Multi- Chaperone Systems Hsp90 assisted Hsp60 assisted

7. The Hsp70 Super Family Contains Distinct Sub-Families

8. The C. elegans Hsp70 Chaperones Cytoplasmic & Nuclear –Hsp70 –Hsp-1 –Hsp110 homologue Endoplasmic Reticulum –Hsp-3 –Hsp-4 –Grp170 homologue Mitochondria –Hsp-6 Hsp-5 is a pseudogene

9. Hsp 70 Family in Drosophila Heat-shock-protein- 70Ba Heat-shock-protein- 70Bb Hsp70Bbb Heat-shock-protein- 70Bc 87B12-87B12 87B14-87B14 87B14-87B1587A3-87A3 70 kDa Hsps Locus Hsc70-3 Heat shock protein cognate (Grp78) 87A3 Large MW Hsp70s Hsc70 Cb (hsp110) 70C-b

10. Hsp70 Family Members are Found in All Major Cellular Compartments Cytoplasm & Nucleus –Hsp70 (multiple paralogs) –Hsp110 (Multiple Paralogs) Mitochondria & Chloroplasts –Hsp70 Endoplasmic Reticulum –Grp78 (hsp 70 multiple paralogs) –Grp170

11. Cells Respond to Stress by Increased Synthesis of Molecular Chaperones Misfolded proteins in the cytoplasm & nucleus induce the heat shock response, synthesis of additional heat shock proteins - Hsp’s Misfolded proteins in the endoplasmic reticulum induce the unfolded protein response, synthesis of additional ER chaperones - Grp’s

12. IRE-1 XBP-1 The Unfolded Protein Response (UPR) The UPR occurs when proteins are misfolded in the endoplasmic reticulum (ER). Reducing agents, such as DTT, interfere with disulfide bond formation while drugs can interfere with glycosylation; both agents cause proteins to misfold in the ER thus triggering the UPR. The product of the ire-1 gene is the sensor of misfolded proteins and when activated removes an intron from the pre mRNA from the xbp-1 gene. Active xbp-1 protein (from spliced mRNA) activates the genes that code for ER chaperones, such as hsp-4. Hsp4 (grp78) grp170

13. Cellular Stresses Promote Misfolding and Aggregation of Proteins High temperatures Heavy metals Alcohol Anoxia Osmotic stress Energy starvation Reducing agents

14. The Structure of Yeast Hsp110

15. Hsp110 is a Nucleotide Exchange Factor for Hsp70

16. Summary Efficient folding of many newly synthesized proteins depends on assistance from molecular chaperones, which serve to prevent protein misfolding and aggregation in the crowded environment of the cell. Nascent chains binding chaperones, including trigger factor, Hsp70, and prefoldin, stabilize elongating chains on ribosomes in a Non-aggregated state. Folding in the cytosol is achieved either on controlled chain release from these factors or after transfer of newly synthesized proteins to downstream chaperones, such as the chaperonins. These are large, cylindrical complexes that provide a central compartment for a single protein chain to fold unimpaired by aggregation. Understanding how the thousands of different proteins synthesized in a cell use this chaperone machinery has profound implications for biotechnology and medicine. -