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Protein Degradation

Protein Degradation. BL4010 10.7.05. Proteins have variable life-spans. Life-span factors. Natural stability ("genetically encoded") an inherent biophysical characteristic Change in environment temperature pH Active degradation specific mechanism location partners. Terminology.

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Protein Degradation

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  1. Protein Degradation BL4010 10.7.05

  2. Proteins have variable life-spans

  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

  7. Ubiquitination DEGRADATION

  8. 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

  9. Ubiquitin • 76 amino acids • Highly conserved • 3 amino acid changes yeast to human • Thermostable

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

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

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

  13. 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.)

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

  15. SUMOylation • SUMO = small ubiquitin related modifier (1996)

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

  17. 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

  18. Yeast proteasome

  19. 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

  20. Thermoplasma proteasome

  21. 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)

  22. 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)

  23. Htr Protease serine protease (chymotrypsin family)

  24. Unlike proteasome, most proteases are specific

  25. Proteolysis as a regulatory mechanism

  26. Proteolysis regulates cell death

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

  28. 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.

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