BMB 174
BMB 174 Regulated Proteolysis 3 April 7, 2015 The behavior of a cell is determined by its repertoire of proteins Nerve cell Muscle cell A cell’s protein repertoire is determined by a balance between production of new proteins and elimination of existing ones New proteins made Old proteins degraded s 10’ 10’ Processes & Diseases Linked to Ubiquitin System Cellular functions: cell cycle, localization, transcription, apoptosis, signaling, glucose sensing, circadiam rhythms cancers: sarcomas, leukemias: Mdm2 (ubi-ligase) renal carcinomas: VHL tumor suppressor (ubi-ligase) breast cancer: tumor suppressor BRCA1 (ubi-ligase) neurodegenerative diseases: Parkinson’s: UCH-L1 (de-ubi enzyme), Parkin (ubi-ligase) Angelmann’s syndrome: E6-AP (ubi-ligase) autoimmune disease: polyendocrinopathy, Aire (ubi-ligase) muscle atrophy: atrogin-1/MAFbx and MuRF1 (ubi-ligase) The principal means of degrading proteins in cells is via the ubiquitinproteasome system (UPS) The ubiquitin-proteasome system is vast. Of the ~22,000 genes encoded in each of your cells, about 1000 of them are involved in ubiquitin metabolism The Ubiquitin–Proteasome System altered function Ub Ub E1 E2 Ub E1 E2 E3 UbR protein protein E3 ATP E2 Ub Ub Ub Ub DUB Ub protein Ub + Ub ~dozen UBLs ~50 E2s ~600 E3s ~90 DUBs ~25 UbRs 3 26S Different ubiquitin modifications do different things Ub substrate endocytosis, transport K63 K63 K63 substrate DNA repair ribosome function K48 K48 K48 K48 substrate proteolysis RING domains comprise the lion’s share of E3s RING (300) HECT (28) HECT and RING E3s have different mechanisms cb HECT E3 RING E3 Ub E2 E3 substrate substrate ca Ub E2 substrate E3 Ub E2 substrate E3 Ub Ub E2 substrate E3 E2 E3 E3s and E1 bind competitively to E2s ca E2 Ub substrate substrate Ub E3 E3 E2 Ub Ub Ub e E3 E1 d E3 Ub f E2 E2 substrate substrate Ub c b Ub E2 Ub E2 E2 Ub E2 The reaction cycle of ubiquitination E2 E3 e c Ub d E3 substrate E2 substrate substrate Ub Ub Ub E3 b f E3 Ub a E2 substrate E3 substrate Ub Ub E2 E3 Regulation of RING-dependent ubiquitination by phosphorylation P E2 P substrate substrate E2 Ub E2 APC/C Cdh1 adaptor Cdc20 adaptor substrate E2 Ub Ub Adaptor inhibition Ub APC/C SCF E3s P cc Cdh1 adaptor E2 E3 activation substrate substrate Ub cb substrate Substrate activation Cdc20 adaptor ca Ub E2 Other modes of regulation of RING E3s E3 binding inhibitor (cullins + CAND1) substrate ca substrate Ub E2 E2 + SCF cb K K substrate Pseudosubstrate inhibitor (APC + Acm1) Ub E2 APC/C cc Ub E2 no Lys + no Lys substrate Allosteric activator (Ubr1 + dipeptides) Ub substrate substrate Ub E2 + Ubr1 E3 Ub E2 E3 Other modes of regulation of RING E3s cd Ub substrate substrate Small molecule co-substrate (SCFTIR1 + auxin) E2 + SCF ce Ub E2 E3 Substrate ordering (APC/C) Time of proteolysis Substrate 1 Substrate 2 Substrate 3 late Substrate 3 low Substrate 2 + APC Substrate 1 E2 early Ub processivity Substrate 1 high Ub E2 An example of how regulated destabilization controls a biological process: TheG1/S G1/Stransition transition in The in budding buddingyeast yeast S phase U U U U U An example of how regulated stabilization controls a biological process: hypoxic signaling O2 O2 O2 O2 high HIF1a O2 O2 OH Oxygen level O2 low HIF1a RNAP In low oxygen, HIF-1 accumulates and drives expression of proteins that induce red blood cell production (Epo) and proteins that promote blood vessel formation (VEGF) Gabi Alexandru RING domains constitute the lion’s share of ubiquitin ligases, and about half of them comprise cullin–RING ligases (CRLs) CRL (~270) RING (300) HECT (28) Diversification of the SCF repertoire G1-Cdk Srb10 P ~20 FBPs in yeast ~70 FBPs in human P GCN4 SIC1 P CDC4 GRR1 CDC4 F SKP1 CUL1 F H Ub CDC34 Different kinases for targeting SKP1 CUL1 CLN1,2 H Ub CDC34 Different F-box proteins Diversification of the SCF-like E3s ~50 SOCS box proteins in humans ~200 BTB domain proteins in humans HP VHL HIF1 S CDC4 MEL26 SOCS S El-C H CUL2 BTB Ub CUL3 MEI-1 H Ub CDC34 CDC34 Cul2 Cul3 What SCF looks like Ub E2 Skp1 b-cat peptide RING Cul1 Cul1 Ning Zheng, Brenda Schulman & Nikola Pavletich How does Ub transfer happen across a gap? How are ubiquitin chains assembled? How is chain specificity achieved? Ub-sepharose depletes more than E1 non-E1 factor eluted by salt only when applied in absence of ATP E1, E2, E3 promote Ub conjugation E2, but not E3, sticks to Ub-sepharose covalently in presence of E1 & ATP E1 + ATP protect E2 from thiol inactivation Ubiquitin flows from E1->E2->substrate Figure 13 +CSN Rbx1 cc low activity substrate cb high activity substrate ca +Cand1 Ub E2 Cand1 substrate Cul1 N8 adaptor no activity + N8 N8 E2 cd What SCF looks like Ub E2 Skp1 b-cat peptide RING Cul1 Cul1 How does Ub transfer happen across a gap? How are ubiquitin chains assembled? Ning Zheng, Brenda Schulman & Nikola Pavletich
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