Ubiquitin Presentation- Jacob Patterson

1. The Role of Ubiquitin in Protein Degredation and Signal Transduction

2. Consists of 76 amino acids, 8.5 kDa Found in all eukaryotic cells (ubiquitously) Highly Conserved Used in post-translational modification Ubiquitin

3. 26S Proteasome Abundant in nucleus and cytoplasm destroys proteins marked by Ubiquitin through Lysine 48-linked polyubiquitination

4. 26S Proteasome Consists of central hollow cylinder (20S) 4 stacked “rings” of 7 proteins each Capped by regulatory particles (19S) that recognize ubiquitin through ubiquitin binding domains (UBDs)

5. Core structure

6. Three types of Ubiquitination

7. Monoubiquitination Adds one ubiquitin molecule to one substrate protein residue Required before a poly chain can begin to form Membrane Trafficking, Transcription, Endocytosis

8. Polyubiquitination Requires one Ub linked to substrate before chain begins to form. Chains made by linking Glysine residue of Ub to a Lysine of a Ub bound to a substrate. Linking to different position on Ub leads to different results.

9. Lysine 48-linked polyubiquitination Linked by 48th amino acid (Lysine) Marks proteins for destruction Requires at least 4 Ub to be recognized by proteasome

10. Lysine 63-linked polyubiquitination Binds to allow coordination of endocytic trafficking Bound to ESCRT-0 to prevent binding to proteasome

11. Ubiquitination

12. Ub activating enzyme (E1) E1 binds ATP and Ub. Transfers Ub to an active site cysteine residue, releasing AMP Thioester linkage between C-terminus of Ub and E1 cysteine sulfhydryl group One E1 can transfer Ub to several different E2 enzymes

13. Ub conjugating enzyme (E2) Ub is transferred from E1 to E2 through a trans(thio)esterification reaction. Binding to both the activated Ub and the E1 enzyme before releasing E1. Each E2 can transfer Ub to a hundred different E3 enzymes

14. Ub ligase enzyme (E3) Attaches Ub via isopeptide bond to a lysine on target protein

15. E3 ligases The most varied of the three enzymes. Each E3 can attach to many different substrate proteins. Different E2, E3 pairings will recognize different proteins by distinct degradation signals.

16. Deubiquitinating enzyme (DUB) Use catalytic diads or triads of cysteine, histidine, and asparagine to catalyze hydrolysis of the isopeptide bond

17. Deubiquitinating enzyme (DUB) Around 100 in the human genome Some cleave the whole chain, some only cleave a set amount of Ubs DUB USP5 selectively binds a 4-ubiquitin chain and severs it.

18. Ubiquitin in Protein Degradation

19. Ubiquitin in Protein Degradation After a protein is Ubiquitinated, it must be recognized by the 19S regulatory particle Ubiquitin Binding Domains exist to interpret signals from Ubiquitinated substrates. ~20 different UBDs exist to bind to different specific shapes of Ubiquitin chains and different monoubiquitinated locations on a protein.

20. Ubiquitin in Protein Degradation Narrow gate formed by the N-terminus tails of the alpha ring subunits Protein must be partially unfolded, at least their tertiary structure Must be deubiquitinated first Order not clearly known, depends of specific substrate

21. Proteolysis Threonine-dependant nucleophilic attack Central chamber releases typically 7-9 residue polypeptides. Sometimes produce functioning molecules

22. Regulation of Protein Degradation One means of controlling Ubiquitination is regulating the activation of E3 ligases.

23. Regulation of Protein Degradation Another way for a protein to avoid degradation by the proteasome is to mask the residues that release the degradation signal. Phosphorylating the area or creating an unstable N-terminus will let nearby E3’s know

24. NF- k B A protein complex that controls transcription of DNA. Synthesized as p105 and p100, C-termini inhibit entry into nucleus. Ubiquitinated and processed by the Proteasome into their active forms, p50 and p52.

25. Circadian Rhythm and Aging Ubiquitin is responsible for the degradation of the “master circadian protein.” Also damaged proteins that arise due to aging, stress, and oxidative damage.

26. Ubiquitin in Signal Transduction

27. RIP1 Complexes with a polyubiquitin chain As long as the signaling protein is ubiquitinated, it acts to prevent cell death. Once deubuquitinated by the A20 enzyme, RIP1 is free to drive forward the cell death process.

28. PCNA Monoubiquitination activates PCNA to restart DNA synthesis, but very error prone. In yeast, lysine 63-linked polyubiquitination leads to an error free pathway.

29. Epidermal Growth Factor Receptor Cell-surface receptor that auto-phosphorylates to activate downstream activation cascade. Leads to DNA synthesis, cell proliferation, and cell adhesion. Important for innate immune response Ubiquitination by Lysine 63-linkages required for endocytosis and post endocytic sorting Mutations to EGFR lead quickly to cancer, proper ubiquitination prevents out of control mutations.

30. Defects in Ubiquitination Pathway Tumor suppressor proteins like p53 and p27 are stabilized by Ubiquitin Defects in Ubiquitin system accelerate degradation of suppressors, increasing risk of cancer causing mutations

31. Defects in Ubiquitination Pathway Alzheimer's Huntington’s Parkinson’s Kennedy’s Syndrome (Spinobulbar Muscular Dystrophy)

32. Lewy Bodies Parkinson’s Displace other cell components

33. Ubiquitin-like Proteins (UBLs) Little is known about most of them Enzyme cascade is almost the same SUMO- Small Ubiquitin-like Modifier Attaches in a manner similar to Ubiquitin, only used in signal transduction. ISG15- Interferon Stimulated Gene 15 Expressed in response to interferons or viral dsRNA Used in JAK-STAT signalingpathway

34. Prokaryotes Prokaryotic Ubiquitin-like Proteins (PUP) Attach to substrates in the same manner Only requires 2 enzymes

35. Prokaryotes