Intracellular Protein Degradation

1. Intracellular Protein Degradation Chris Weihl MD/PhD weihlc@neuro.wustl.edu Department of Neurology

3. How is trash handled?

4. Protein Degradation in the Cell Ub Nucleus Autophagy Ub Aggresome Ub UPS Ub Endocytosis

5. Consequence of impaired protein degradation • Protein aggregates • Ubiquitinated inclusions • Vacuolation • Damaged organelles • Secondary impairment in other cellular processes • Cell Death • Underlying pathogenesis of degenerative disorders (neurodegeneration, muscle degeneration, liver degeneration, lung disease, aging)

6. Protein Degradation • Turnover of protein is NOT constant • Half lives of proteins vary from minutes to infinity • “Normal” proteins – 100-200 hrs • Short-lived proteins • regulatory proteins • enzymes that catalyze committed steps • transcription factors • Long-lived proteins • Special cases (structural proteins, crystallins)

7. May depend on tissue distribution • Example: Lactic Acid Dehydrogenase • Tissue Half-life • Heart 1.6 days • Muscle 31 days • Liver 16 days • Protein degradation is a regulated process • Example: Acetyl CoA carboxylase • Nutritional state Half-life • Fed 48 hours • Fasted 18 hours Protein Degradation

8. Protein Degradation • Ubiquitin/Proteasome Pathway • 80-90% • Most intracellular proteins • Lysosomal processes • 10-20% • Extracellular proteins • Cell organelles • Some intracellular proteins

9. How are proteins selected for degradation?

10. UBIQUITIN • Small peptide that is a “TAG” • 76 amino acids • C-terminal glycine - isopeptide bond with the e-amino group of lysine residues on the substrate • Attached as monoubiquitin or polyubiquitin chains G K

11. Ubiquitination of proteins is a FOUR-step process • First, Ubiquitin is activated by forming a link to “enzyme 1”(E1). AMP • Then, ubiquitin is transferred to one of several types of “enzyme 2”(E2). • Then, “enzyme 3”(E3) catalizes the transfer of ubiquitin from E2 to a Lys e-amino group of the “condemned” protein. • Lastly, molecules of Ubiquitin are commonly conjugated to the protein to be degraded by E3s & E4s

13. The UPS is enormous! The UPS is enormous! The genes of the UPS constitutes ~5% of the genome • E1’s- 1-2 activating enzymes • E2’s- 10-20 conjugating enzymes • E3’s- 500-800 ubiquitin ligase- drives specificity • DUBs- 100 ubiquitin specific proteases- regulators of pathway The genes of the UPS constitutes ~5% of the genome • E1’s- 1-2 activating enzymes • E2’s- 10-20 conjugating enzymes • E3’s- 500-800 ubiquitin ligase- drives specificity • DUBs- 100 ubiquitin specific proteases- regulators of pathway

14. PROTEASOME COMPONENTS 20S Proteasome 19S Particle ATP 26S Proteasome

15. Hydrolysis peptide bonds after: hydrophobic a.a. =CHYMOTRYPSIN-LIKE - 5 acidic a.a. = (-) CASPASE-LIKE -1 basic a.a. = (+) TRYPSIN-LIKE -2

16. DEUBIQUITINATION De-ubiquitinating

17. Pathways controlled by regulated proteolysis

18. Mechanism of muscle atrophy

19. MURF/Atrogin

20. Knockout of Atrogin Rescues atrophy

21. Proteasome inhibitors proteasome ub-ub-ub-ub ub-ub-ub-ub ub-ub-ub-ub ub-ub-ub-ub proteasome ub-ub-ub-ub

22. Proteasome inhibition increases Usp14 ubiquitin-hydrolase activity Usp14 Uch37 Borodovsky, A et al EMBO J. 20:5187-96 2001

23. The proteasomal DUB Usp14 impairs protein degradation Lee, BH et al Nature 467:179-84 2010

24. Decrease steady-state levels of aggregate prone proteins in the absence of Usp14 Lee, BH et al Nature 467:179-84 2010

25. Lyosomal degradation • Autophagy

26. Autophagy • Lysosomal degradation of proteins and organelles • Occurs via three routes • Macroautophagy • Microautophagy (direct uptake of cellular debris via the lysosome) • Chaperone mediated autophagy (selective import of substrates via Hsc70 and Lamp2a)

28. Yeast Genetics meets Human Genetics • Identification of >50 autophagy essential proteins with mammalian homologs

29. Macroautophagy FOXO3 Beclin ATG7 Lysosome mTOR ATG5-ATG12-ATG16L1 Autophagosome Induction Nucleation Trafficking & Cargo loading Autolysosome Phagophore Sequestration Degradation Fusion “Autophagic Flux”

30. Genetic knockout of autophagy initiating proteins Complete loss of ATG5 leads to lethality

31. Tissue specific knockout of autophagy • Degeneration of CNS tissue; Hara et al 2006 • Hepatomegaly in Liver; Komatsu et al 2005 • Atrophy and weakness of skeletal muscle; Masiero et al 2009 • Pathologic similarities • Ubiquitinated inclusions • Aberrant mitochondria • Oxidatively damaged protein

33. Basal Autophagy • Autophagy has a “housekeeping” role in the maintenance of cellular homeostasis • Autophagy is responsible for the clearance of ubiquitinated proteins

34. Selective Autophagy • Aggregaphagy– p62/SQSTM1, Nbr1 • Mitophagy – Parkin, Nix • Reticulophagy – endoplasmic reticulum • Ribophagy – translating ribosomes • Xenophagy – e.g. Salmonella via optineurin • Lipophagy – autophagy mediated lipolysis • Performed by an expanding group of ubiquitin adaptors

38. p62 as an autophagic tool • p62 associates with ubiquitinated proteins and LC3 • p62 is an autophagic substrate

39. LC3 as an autophagic tool LC3-I (18kD) LC3-II (16kD) GFP-LC3 starved

40. IBMPFD myopathy

41. Autophagosome proteins are elevated in IBMPFD 2 p62 protein levels (A.U) 1 0 Con WT RH9 RH12 2 LC3II protein levels (A.U) 1 0 Con WT RH9 RH12 Ju et al, JCB 2009

42. Autophagosome accumulate in IBMPFD cells Ju et al, JCB 2009

43. Why do autophagosomes accumulate? • Upregulation of functional autophagosomes • Decrease in autophagosome degradation or “autophagic flux” • Phagophore closure • Autophagosome-lysosome fusion • Absence of functional lysosomes

44. Functional VCP is required for “autophagic flux” VCP

45. IBMPFD mutant VCP impairs “autophagic flux” Ju et al, JCB 2009

46. Autophagosomes and lysosomes coalesce in IBMPFD

47. IBMPFD has “blocked” autophagy Ub Nucleus

48. Therapeutic interventions to treat autophagic disorders

49. Rapamycin as an inducer of autophagy • Immunosuppressant used to treat transplant rejection • Inhibits the mTOR pathway • mTOR integrates extrinsic growth signals and cellular nutrient status and energy state • Active mTOR • Protein synthesis and cell growth • Inactive mTOR (or rapamycin treatment) • Inhibition of protein synthesis and increased autophagic degradation of protein

50. Rapamycin enhances autophagy in skeletal muscle