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1、Protein Targeting and Function:Ubiquitin Modifications,Jim CollawnOctober 17th, 2019,Protein Targeting and Function,References,Hicke and Dunn (2019) Regulation of membrane protein transport by ubiquitin and ub-binding proteins. Annu. Rev. Cell Dev. Biol. 19: 141-172. 2. Minireview: Regulation of Epi
2、thelial Na+ Channel Trafficking. Endocrinology 146: 5079-5085.,ReferencesHicke and Dunn (2019,Outline,History and Evolution of Ub-pathwaySites where ubiquitination regulates protein trafficking/functionRegulation of sodium channel stability at the cell surface (ENaC)Other ubiquitin-like molecules,Ou
3、tlineHistory and Evolution o,Cellular Protein Degradation,Lysosomal Nonspecific Endocytosis Foreign proteins Energy favorable to degrade proteins Non-lysosomal Specificity, requires ATP Conditions of stress Ubiquitin-proteosomal pathway 26S proteosome Role in cellular processes/signaling,Cellular Pr
4、otein Degradation L,Non-lysosomal Protein Degradation,1977 Etlinger and Goldberg (PNAS) Protein degradation in reticulocytes But - no lysosomes ATP dependent1980 Wilkinson et al (JBC) Identify protein degradation system in reticulocyte Two fractions (ATP-dependent Proteolytic Factor; APF)APF-1 fract
5、ion conjugates to proteinsAPF-1 identified as ubiquitin,Protein Adduct Protein Degradation Ubiquitin FoundFunction Found,Non-lysosomal Protein Degradat,Avram Hershko Nobel LectureHershkoCiechanover (grad student 76-81; major credit)Irwin Rose (Fox Chase Cancer Center)“The Ubiquitin System for Protei
6、n Degradation and some of its Roles in the Control of the Cell Cycle”December 8, 2019,Avram Hershko Nobel Lecture,APF-1 Protein Ubiquitin-Proteosomal Pathway,1978Hershko,“APF factor”,2019,APF-1 Protein Ubiquitin-Prot,Ubiquitinylation Proteosome Degradation,E3 determines protein substrate,Ubiquitinyl
7、ation Proteosome,E1-Ub1000 aa, Cysteine-636 Ub attachment site,E2:TransferUb ConjConserved cysteineActive “thio ester”,E3:LigaseSpecificw/E2Usually do not conjugateHold complex together,E1-UbE2:TransferE3:Ligase,19S and 20S Proteasome Subunits Characteristics,20S SubunitBarrelContains 6 proteolytic
8、sites2x Tryptic2x Chymotryptic2x Peptidylglutamyl- peptidase Linearized protein required,19S SubunitBase and LidContains subunits with known and unknown functionsTetra-Ub (K48) recognitionDeubiquitination activityProtein unfolding activity (Chaperone function),19S and 20S Proteasome Subuni,Ubiquitin
9、,76 Amino AcidsCompact, globular superfold protein, 9 kDHighly conserved (3 aa difference from yeast to man)Expressed in EVERY eukaryotic cellC-terminus extends away from proteinPost-translational modification on proteinsAttaches on Lysine residuesFunctions:Marks protein for proteasomal degradationC
10、an “activate” protein trigger cellular effectEndocytosisModify the activity of protein transport machinery,Ubiquitin76 Amino Acids,Conjugation Type BondsMulti-Enzyme Process,Protein,Protein,E2,C,Ub,RGG,E3,K,GGR,Ub,Isopeptide bonds,Protein,Ub,Ub,Ub,USP,UbUbConjugation Type BondsM,Ubiquitin AA Sequenc
11、e,MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRGG,48,63,6,Ubiquitin AA SequenceMQIFVKTLT,Roles of Ubiquitination,Roles of Ubiquitination,Different Types of Ubiquitin Tags,Different Types of Ubiquitin T,Modulalar Ub-binding Domains (endocytic proteins):,Ubiquitin a
12、ssociated (UBA) or interacting motif (UIM) UVE (ub conjugating enzyme E2 variant CUE These domains bind to Ile44 on Ub Examples Espsin (Eps15-interacting protein) Eps15 (epidermal growth factor receptor substrate 15,Modulalar Ub-binding Domains,Modulalar Ub-binding Domains,Lipid binding preference:
13、ENTH domain: PI-4,5-P (PM) FYVE domain: PI-3-P,Modulalar Ub-binding DomainsLi,Endocytic Machinery that is monoubiquitinated,CIN85 (Cbl-interacting protein of 85 kDa)required for activated growth factor receptors Epsins Eps15 Hrs Rabex Numb (binds to Eps15 and AP-2) and neg regulates Notch (promotes
14、Notch endocytosis),Endocytic Machinery that is mo,Posttranslational Modification (ubiquitination) and Protein Sorting,Posttranslational Modification,ESCRT Proteins and the MVB,ESCRT Proteins and the MVB,Ubiquitin and Protein Sorting,In yeast, Ub is used for endocytosis and delivery into internal ves
15、icles of the MVB In higher eukaryotes, it is used for the same purpose for regulated transport For regulated internalization and/or lysosomal targeting, an example is the signal transducing receptors: G protein-coupled receptors (GPCRs)b2-adrenergic and CXCR4 (MVB only) Receptor tyrosine kinases (RT
16、Ks),Ubiquitin and Protein Sorting,Ub Signal for Internalization and Sorting into the MVB,In yeast, Ub is used for endocytosis and delivery into internal vesicles of the MVB In higher eukaryotes, it is used for the same purpose for regulated transport For regulated internalization and/or lysosomal ta
17、rgeting, an example is the signal transducing receptors: G protein-coupled receptors (GPCRs)b2-adrenergic and CXCR4 (MVB only) Receptor tyrosine kinases (RTKs),Ub Signal for Internalization,Transmembrane Proteins Regulated by Ub-dependent Sorting,In yeast: Ste2, Ste3, Ste6, Uracil permease, Carboxyp
18、eptidase S, etcIn metazoans:Regulation of growth/differentiation:Growth hormone receptorEGFRMacrophage colony-stimulating factorPDGFRHepatocyte growth factor receptorTransforming growth factor b receptorb2-adrenergic receptorImmune response:IL-2 receptorT cell receptorCXCR4IgG receptor (Fcg receptor
19、 II)Pre-T cell receptorB7-2,Transmembrane Proteins Regulat,Transmembrane Proteins Regulated by Ub-dependent Sorting,In metazoans:Neurotransmission:Ion channels:AMPA glutamate receptorsENaCGlycine receptorsClC-5Cell-cell contacts:Immune molecules E-cadherindownregulated by viruses:OccludinMHC class I
20、B7-2Developmental patterning:ICAM-1DeltaCD4NotchRoundabout,Transmembrane Proteins Regulat,E3 Ligases,Bridging factors: E2 + substrate Belong to three families Homologous E6AP Carboxy TerminusHECT (Rsp5/Nedd4) Really Interesting New GeneRING; major family (Cbl) Homology (U-box) proteinsUFD2; small fa
21、mily,E3 Ligases Bridging factors: E,HECT domain E3,HomologousE6-APC terminus,Substrate specificity,Ubiquitin E3 Enzymes,E6-AP: papilloma virus E6 oncoprotein associate protein,HECT domain E3HomologousSubstr,RING finger E3,Really Interesting New Gene,Ubiquitin E3 Enzymes,RING finger E3Really Interest
22、i,MultipleE3 RING Ligases,Complex RING,MultipleComplex RING,E2 Ub Transfer vsE3 Ub Transfer,E2 Ub Transfer,E3 Substrate Recognition (RING E3),Cbl protein,Internalization of EGFR, CSF-1, Hepatocyte Growth FR,E3 Substrate Recognition (RI,Ubiquitin Conjugation Deubiquitination,Ub,Ub,Ub,Ub,Ub,Ub,Ub,Prot
23、ein,Ub,USP,E1,E2,E3,De-ubiquitination,Ubiquitin Conjugation,Deubiquitinating Activity,Ub C-terminal hydrolase familyUCH“Cysteine protease” Ub-specific processing protease UBP“Cysteine protease” Others Ub reutilized,Biological Function,Proteosome regulation,Cell GrowthDevelopmentOncogenesisNeuronal D
24、iseasesChromatin Structure/transcription,Largest family enzymes in Ub system,Deubiquitinating ActivityUb C-,Poly-Ub Chains,Ub,K,K,Ub,Ub,Ub,Ub,K48 Linkage,K63 Linkage,K63,K48,Peters, J.M. 2019Ubiquitin and the Biology of the Cell,Signal to proteosomeK48, Ub4,Cell SignalingK63,Poly-Ub ChainsUb K,ENaC
25、function,Major ion channel that controls salt and fluid resorption in the kidney Mutations in the PPXY motif cause accumulations of channels at the cell surface and result in Liddles syndrome, and inherited form of hypertension,ENaC function Major ion chann,ENac surface Stability,Nedd 4 (HECT ligase
26、)-negatively regulates ENaC surface stability Nedd4 WW domains bind PPXY motif of ENaC subunits Nedd4 also interacts with serum and glucocorticoid-regulated kinase (SGK) SGK contains two PPXY motifs that bind to Nedd4 WW domains SGK-dependent Nedd4 P inhibits the Nedd4-ENaC interaction therefore, Ne
27、dd4 P increases ENaC at the cell surface,ENac surface Stability Nedd 4,Nedd4 and other Ubiquitin ligases important for protein transport,Nedd4 and other Ubiquitin liga,ENaC Subunits,ENaC Subunits,Regulation of ENaC Surface Stability,Regulation of ENaC Surface Sta,Regulation of ENaC Surface Stability
28、,Regulation of ENaC Surface Sta,Ub-like Proteins,SUMO-1 (sentrin, smt-3)2019 covalent modification RanGAP1RanGAP1 nearly quantitative modifiedCytosolic RanGAP1 to nuclear poreActivate shuttling factor,Ub-like ProteinsSUMO-1 (sentr,Ubiquitin-like Proteins:,Ubiquitin-like Proteins:,Ubiquitin Superfold
29、 and Ubiquitons,Ub blueSUMO-1 greenNEDD8 - red,UB roll suprfold,Ubiquitin Superfold and Ubiqui,SUMO,SUMOSUMO-1 & SUMO-2/3Shared characteristicsC-terminal -GG essential for conjugationAffix to lysine residues in targetNOT directly associated with proteasomal degradation,SUMOSUMO,Competition/Regulatio
30、nSUMO,Reactive Oxygen Species: Oxidizes reactive thiols on SUMO enzymes,Uba1/Aos1- S S Ubc9Thus: SUMO can not attach and proteins not Sumoylated,Competition/RegulationReactive,Examples of SUMO function,RanGAPIkBc-Junp53 and mdm2,Causes nuclear translocationBlocks Ub-conjugation site, prevents degradationInhibits transcriptional activityBlocks mdm2 self-ubiquitination, prevents degradationSUMO-p53 in DNA binding domain apoptotic activity,PROTEIN,SUMO Effect,Examples of SUMO functionRanGA,
