1. PROTEIN FOLDING AND DEGRADATION Kanokporn Boonsirichai

2. Proteins must fold up into their unique 3-D conformation  To be able to perform their specific function  To assemble correctly with other proteins  To bind with small-molecule cofactors that are required for their activity  To be appropriately modified by protein kinases or other protein- modifying enzymes

3. When a protein folds:  Most of its hydrophobic residues are buried into an interior core.  A large number of noncovalent interactions are formed.  The final conformation is usually of the lowest free energy.

4. Proteins begin to fold as they exit from the ribosome  Secondary structures are formed and aligned roughly within a few seconds   “Molten globule”  Side-chain adjustment (slow) to form the appropriate tertiary structure Cytochrome b 562

5. By the time it is released from the ribosome, much of the folding has already been completed.

6. Molecular Chaperones A class of proteins which mediate protein folding

7. Heat-Shock Proteins (Hsp)  Firstly identified in E. coli  Increased synthesis at elevated temperature (42 o C)  What happens at elevated temperature? Why does the cell need more chaperones?

8. Heat-Shock Proteins (Hsp)in Eucaryotes  Two major families: Hsp60 and Hsp70  Members are organelle-specific: cytosolic, ER- associated, mitochondrial  Work with their own set of assiciated proteins  Show an affinity for exposed hybrophobic patches  Hydrolyze ATPs

9. Hsp70 Works with Hsp40 Performs its function as the target protein is being translated Binds to a string of seven hydrophobic amino acids Hydrolyzes an ATP as it binds; releases ADP and rebinds ATP as it dissociates

10. Hsp60 (Chaperonin) Acts after its target protein has been fully synthesized. Provides a favorable environment for the target protein to refold. Binding of ATP and GroES may transiently stretch the misfolded protein. ATP hydrolysis triggers the release of the target protein.

11. Choices of Protein Quality Control

12. Proteasome  A machinery for protein destruction  An abundant ATP-dependent protease (= 1% of cellular proteins)  Found in the cytosol and the nucleus

13. Only marked proteins are targeted to the proteasome Ubiquitin A 76-amino-acid tag Activated through a high-energy thioester linkage to a cysteine residue Is transferred to the lysine residue of the target protein

14. biology.caltech.edu/Members/Deshaies ~ 30 distinct kinds > 100 kinds E3 recognizes specific degradation signals in the target proteins. Multiubiquitin chain is recognized by the proteasome. Targets Denatured/ misfolded proteins Proteins with oxidized/ abnormal amino acids

15. Regulated Destruction of Proteins  Some proteins turn over rapidly at all time.  Some proteins are stable most of the time but become unstable under a certain condition. Mechanisms Activation of specific E3 Activation/ exposure of the degradation signal in within the target protein

16. Activation of Ubiquitin Ligase

17. Activation of Degradation Signal

18. Abnormally folded proteins may form protease-resistant aggregates Some protein aggregates form a fibril structure Cross-beta filaments: layered of polypeptide chains with continuous stacks of beta sheets Prion disease

19. Protein-aggregates-induced Diseases  Huntinton’s disease  Alzheimer’s disease  Creutzfeldt-Jacob disease (CJD) in humans  Bovine spongiform encephalopathy (BSE) in cattle (i.e. mad cow disease) Prion diseases

20. Regulation of cellular protein levels can occur at many different points Transcription RNA processing RNA transport Translation Protein degradation