lys

1. lys Two strategies for protein degradation 1) Send the protein to a degradative compartment 2) selective degradation of individual molecules

2. Cellular protein degradation highly processive 5-8 a.a. protein extremely specific same cellular compartment, half life can vary from 2 min to many days

3. “an old hammer is made from two heads and three handles…”

4. Protein steady state kinetics the steady state can be due to high or low k’s

5. synthesis degradation protein Protein steady state in vivo to change the concentration of a protein the cell can alter either process

6. +H3N +H3N COO- COO- COO- COO- +H3N COO- Proteases are well known enzymes…

7. Proteases for degradation classic protease: highly site specific not processive chambered protease: processive and protective

8. Many chambered proteases Thermoplasma Saccharomyces E. coli Rhodococcus versions found in all organisms all function in protein degradation

9. Eukaryotic 20 proteasome 0.5 to 1% of the cellular protein alternative subunits for specialized P’somes can not degrade folded proteins

10. x 90o Eukaryotic 20 proteasome active sites inside; chamber can hold 100kD 28 subunits, 14 separate proteins how is specificity attained??

11. Eukaryotic 26S proteasome substrate recognition ATP-dependent unfolding proteolysis

12. Eukaryotic 26S proteasome

13. ubiquitin Ub 76 a.a. 8,000 mw only in eukaryotes very highly conserved Specificity in protein degradation covalent addition of a small tag to mark the target protein for destruction

14. ubiquitin Ub 76 a.a. 8,000 mw only in eukaryotes very highly conserved Specificity in protein degradation

15. Ub Ub Ub Ub - Ub C O 2 Ub Protein Protein PROTEASOME Ubiquitination Ub side chain lysine NH3+

16. Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub 20S core 19S caps proteolysis binding, unfolding ATP hydrolysis Ubiquitin-proteasome degradation

17. 2004 Chemistry Nobel Prize Aron Ciechanover Avram Hershko Irwin Rose www.nobelprize.org

18. Ubiquitin enzymology

19. Ubiquitin ligase (E3) RING domains

20. GST-Ub Ub multi-Ub addition In vitro E3 activity of a RING domain

21. The Cell Cycle and Ubiquitin L. Hartwell, 2001 Nobel (with Nurse and Hunt)

22. The Cell Cycle and Ubiquitin Ub-mediated degradation M-cyclin cyclin- dependent kinase Ub-mediated degradation S-cyclin plus at least another layer

23. The Cell Cycle and Ubiquitin Cloning and analysis of CDC genes reveals two main classes of E3 involved in cell cycle control: 1) SCF complex E3 subunits 2) APC - anaphase promoting complex over 10 subunits, substrates include B cyclins, Pds1p

24. F box adaptors for E3 specificity

25. Ub E2 RING domain RBX1 CUL1 substrate SKP1 F box adaptor F box adaptors for E3 specificity In many cases, the substrate-F box interaction is mediated by phosphorylation BUT

26. Ub E2 RBX1 CUL1 HIF1a B/C VHL Cullin1/Rbx1 Elongin B/C VHL tum. sup Oxygen sensing and ubiquitination High O2 HIF1a Low O2 HIF1a

27. proline hydroxylase OH O2 Low O2 (HIF stable) proline hydroxylase HIF1a HIF1a HIF1a Oxygen sensing and ubiquitination High O2 (HIF degraded) binding to CBCVHL E3 oxygen-dependent modification to an E3-binding sub.

28. Ub E2 RBX1 CUL1 p27 SKP1 SKP2 An SCF complex in detail

29. Coordinates plus docking…

30. SCF theme is broadly used in biology Ub E2 RBX1 CUL substrate SKP1 Adaptor Numerous cullins, numerous SKPs, over 50 E2s and a varitey of adaptors: 80 F box 200 BTB domain proteins 40 SOCS/BT (AYU Box)

31. Ubiquitination and HIV Vpu Vpu CD4 HIV-encoded Vpu programs proteasomal degradation of CD4

32. Ubiquitination and HIV F WD40 Vpu Vpu CD4 Cul Skp1 Rbx E2 HIV-encoded Vpu programs proteasomal degradation of CD4 by recruiting the cytoplasmic F-box protein bTRCP

33. E6 p53 E6AP HECT domains: a distinct E3 family HPV 16, 18: cancer correlated, p53 degrading HECT: Homology to E6ap C-Terminus

34. MDM2 p53 Homology-driven discovery: p53 again MDM2: known regulator of p53 and transcriptional target MDM2 has a functional RING domain

35. MDM2-p53 regulation

36. MDM2-p53 inhibitors In Vivo Activation of the p53 Pathway by Small-Molecule Antagonists of MDM2 (2004) Vassilev et al. Science 303: 844

37. MDM2-p53 inhibitors In Vivo Activation of the p53 Pathway by Small-Molecule Antagonists of MDM2 (2004) Vassilev et al. Science 303: 844

38. Degradation and immunology plasma membrane ER membrane

39. DRiPs: the source of immune antigens?

40. DRiPs: the source of immune antigens?

41. production production Parkin defective Parkin pathological protein(s) pathological protein(s) Homology-driven discovery Familial Parkinsonism: single gene defect Responsible gene: Parkin with RING-H2 Model based on E3 function symptoms

42. distributed information “degron” Destruction signals direct recognition rec. of modification (F box)

43. Degradation in quality control and protein regulation altered structure regulatory events

44. Quality control in disease Alzheimer’s Huntington’s ALS (Lou Gehrig’s) Parkinsonism cystic fibrosis long Q-T syndrome retinitis pigmentosa etc...

45. Ubiquitin in Alzheimer’s plaques ubiquitinated proteins amyloid

46. Quality control degradation -operates continuously in all cells -particularly important in non-dividing cells such as neurons -important part of stress-response pathways -significant therapeutic potential

47. Quality control ligases of the ER

48. Ub Ub Ub Ub Ub Ub Alternative linkages for polyubiquitin lysines: 6, 11, 27, 29, 33, 48, 63

49. Ub Multiple linkage sites in ubiquitin multi-ubiquitin chains 48 linked C-48-C-48-C-48 29 linked C-29-C-29-C-29 63 linked C-63-C-63-C-63 K63-linked chains are not targeted to proteasome

50. Ub 63 linkage in TNF signaling