1. Protein Degradation BL4010 10.7.05

2. Proteins have variable life-spans EnzymeHalf-lifeHours Ornithine decarboxylase0.2 RNA polymerase I1.3 Tyrosine aminotransferase2.0 Serine dehydratase4.0 PEPcarboxylase5.0 Aldolase118 GAPDH130 cytochrome c150

3. Life-span factors Natural stability ("genetically encoded") –an inherent biophysical characteristic Change in environment –temperature –pH Active degradation –specific mechanism –location –partners

4. Terminology Half-life - Average time for half of the protein pool to become denatured or degraded (depends on what you measure) Turnover - Lifespan of a protein from synthesis to degradation Stability - Subjective property of a proteins natural tendency to denature under certain conditions Denaturation - Unfolding, partial or total of a polypeptide Degradation - Proteolysis of a peptide Ubiquitination = Ubiquitylation Protease = peptidase

5. Two routes to digest proteins Lysosomes – Receptor mediated endocytosis & phagocytosis Proteasomes: for endogenous proteins –transcription factors –cell cycle cyclins –virus coded proteins –improperly folded proteins –damaged proteins Cystic fibrosis is due to the accelerated degradation of chloride transporter

6. Ubiquitin mediates degradation for many but not all proteins

10. Ubiquitination DEGRADATION

11. Ubiquitination Ubiquitinating enzymes E1,E2, E3 - thiol ester bond Final target - isopeptide bond between a lysine residue of the substrate (or the N terminus of the substrate) and ubiquitin

12. Ubiquitin 76 amino acids Highly conserved –3 amino acid changes yeast to human Thermostable

13. Ubiquitin is first activated Ubiquitin is adenylated Forms bond at Cys of E1 activating enzyme E1 transfers Ubq to E2 conjugating enzyme

14. Polyubiquitination E2 conjugating enzyme is bound by E3 ligase which transfers Ubq to the target protein

17. Why have a 3-step ubiquitination process? Ubiquitin E1 (1) E2 (12-30) E3 (>200?) –HECT-type –RING-type –PHD-type –U-box containing

18. N-termini Acidic N-termini –Arg-tRNA protein transferase –conversion of acidic N-terminus to basic! VAST MaGiC (Val, Ala, Ser, Thr, Met, Gly, Cys) resistant to Ubiquinitation WHEN sQuiDs FLY tend to have short half-lives ( <30 min.)

19. Signals for degradation (degrons) PEST sequences (Pro, Glu, Ser, Thr) FREQK nonessential under starvation conditions DUBS (de-ubiquinating enzymes) provide additional regulation

21. SUMOylation SUMO = small ubiquitin related modifier (1996)

22. The proteasome Alfred Goldberg & Martin Rechsteiner in 1980's Similar in structure to GroEL chaperone Unfolding and proteolysis Much more specific –Why?

23. Eukaryotic Proteasome 26S (200 kD) complex – 2OS (673 kD) proteasome or multicatalytic protease complex (MCP) as the key proteolytic component –19S complex containing several ATPases and a binding site for ubiquitin chains. 19S particle "caps" each extremity of the 20S proteasome –Unfolds the protein substrates –Controls entry into the 20S proteasome –Stimulates proteolytic activity In yeast, only 3 out of 7 subunits are proteolytically active

26. Yeast proteasome

27. Bacterial proteasomes do not require ubiquitin T. acidophilum 20S proteasome 14  -subunits and 14  -subunits in a four stacked ring –2 outer rings of seven  subunits/2 inner of seven  subunits Central channel with three chambers –2 antechambers located on opposite sides of a central chamber. –14 catalytic sites within the central chamber –N-terminal threonine is catalytic residue –Covalent modification of Thr by lactacystin, a natural inhibitor of the proteasome. Unspecific proteolysis but products always 6 to 9 residues. This corresponds to the length between adjacent catalytic sites in the central chamber

28. Thermoplasma proteasome

32. Other Proteases Cell cycle control/stress response proteases –Proteasome –HtrA Calcium activated proteases (Calpains) Apoptotic proteases –ICE family (caspases) Autocatalytic proteases Nutrient regulated proteolysis (lysosome) Intramembrane cleave protease (ICLiPs)

33. What is proteolysis? Proteolysis = peptide hydrolysis (facilitated nucleophilic attack of water on peptide bond) Four mechanistic categories of protease –serine proteinases chymotrypsin family subtilisin family –cysteine proteinases (e.g. papain, caspsases) –aspartic proteinases (e.g. pepsin) –metallo proteinases (e.g. thermolysin)

34. Htr Protease serine protease (chymotrypsin family)

35. Unlike proteasome, most proteases are specific

36. Proteolysis as a regulatory mechanism

37. Proteolysis regulates cell death

38. Proteolysis as a regulatory mechanism (sequestration of sterol response element transcription factor)

39. Why make proteins that have short half-lives? It seems wasteful to try to maintain the concentration of a protein while it is simultaneously being degraded.