1、Protein Targeting and Function:Ubiquitin ModificationsJim CollawnOctober 17th, 2019Protein Targeting and FunctionReferencesHicke 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 Epithel

2、ial Na+ Channel Trafficking. Endocrinology 146: 5079-5085.ReferencesHicke and Dunn (2019OutlineHistory and Evolution of Ub-pathwaySites where ubiquitination regulates protein trafficking/functionRegulation of sodium channel stability at the cell surface (ENaC)Other ubiquitin-like moleculesOutlineHis

3、tory and Evolution oCellular 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/signalingCellular Protein Degr

4、adation LNon-lysosomal Protein Degradation1977 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 fraction conjugat

5、es to proteinsAPF-1 identified as ubiquitin Protein Adduct Protein Degradation Ubiquitin FoundFunction FoundNon-lysosomal Protein DegradatAvram Hershko Nobel LectureHershkoCiechanover (grad student 76-81; major credit)Irwin Rose (Fox Chase Cancer Center)“The Ubiquitin System for Protein Degradation

6、and some of its Roles in the Control of the Cell Cycle”December 8, 2019Avram Hershko Nobel LectureAPF-1 Protein Ubiquitin-Proteosomal Pathway1978Hershko“APF factor”2019APF-1 Protein Ubiquitin-ProtUbiquitinylation Proteosome DegradationE3 determines protein substrateUbiquitinylation Proteosome E1-Ub1

7、000 aa, Cysteine-636 Ub attachment siteE2:TransferUb ConjConserved cysteineActive “thio ester”E3:LigaseSpecificw/E2Usually do not conjugateHold complex togetherE1-UbE2:TransferE3:Ligase19S and 20S Proteasome Subunits Characteristics20S SubunitBarrelContains 6 proteolytic sites2x Tryptic2x Chymotrypt

8、ic2x Peptidylglutamyl- peptidase Linearized protein required19S SubunitBase and LidContains subunits with known and unknown functionsTetra-Ub (K48) recognitionDeubiquitination activityProtein unfolding activity (Chaperone function)19S and 20S Proteasome SubuniUbiquitin76 Amino AcidsCompact, globular

9、 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 degradationCan “activate” protein trigger ce

10、llular effectEndocytosisModify the activity of protein transport machineryUbiquitin76 Amino AcidsUbUbConjugation Type BondsMulti-Enzyme Process E1 UbRGGCATPADP E1 CUbRGGHigh-energythioester bond E2 C E2 CUbRGGHigh-energythioester bondProteinProtein E2 CUbRGGE3KGGRUbIsopeptide bonds ProteinUbUbUbUSPU

11、bUbConjugation Type BondsMUbiquitin AA SequenceMQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRGG48636Ubiquitin AA SequenceMQIFVKTLTRoles of UbiquitinationRoles of UbiquitinationDifferent Types of Ubiquitin TagsDifferent Types of Ubiquitin TModulalar Ub-binding Domai

12、ns (endocytic proteins): Ubiquitin associated (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 15Modulalar Ub-binding Domains Modulalar Ub-binding

13、 Domains Lipid binding preference: ENTH domain: PI-4,5-P (PM) FYVE domain: PI-3-PModulalar Ub-binding DomainsLiEndocytic 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) a

14、nd neg regulates Notch (promotes Notch endocytosis)Endocytic Machinery that is moPosttranslational Modification (ubiquitination) and Protein SortingPosttranslational ModificationESCRT Proteins and the MVBESCRT Proteins and the MVBUbiquitin and Protein Sorting In yeast, Ub is used for endocytosis and

15、 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 targeting, an example is the signal transducing receptors: G protein-coupled receptors (GPCRs)b2-adrenergic and CXCR4 (MVB only) Re

16、ceptor tyrosine kinases (RTKs)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 internali

17、zation 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 (RTKs)Ub Signal for Internalization Transmembrane Proteins Regulated by Ub-dependent SortingIn yeast: Ste2, Ste3, Ste6, U

18、racil permease, Carboxypeptidase 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 receptorCXCR4Ig

19、G receptor (Fcg receptor II)Pre-T cell receptorB7-2Transmembrane Proteins RegulatTransmembrane Proteins Regulated by Ub-dependent SortingIn metazoans:Neurotransmission:Ion channels:AMPA glutamate receptorsENaCGlycine receptorsClC-5Cell-cell contacts:Immune molecules E-cadherindownregulated by viruse

20、s:OccludinMHC class IB7-2Developmental patterning:ICAM-1DeltaCD4NotchRoundaboutTransmembrane Proteins RegulatE3 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) pro

21、teinsUFD2; small familyE3 Ligases Bridging factors: EHECT domain E3HomologousE6-APC terminusSubstrate specificityUbiquitin E3 EnzymesE6-AP: papilloma virus E6 oncoprotein associate proteinHECT domain E3HomologousSubstrRING finger E3Really Interesting New GeneUbiquitin E3 EnzymesRING finger E3Really

22、InterestiMultipleE3 RING LigasesComplex RINGMultipleComplex RINGE2 Ub Transfer vsE3 Ub Transfer E2 Ub TransferE3 Substrate Recognition (RING E3) Cbl protein Internalization of EGFR, CSF-1, Hepatocyte Growth FRE3 Substrate Recognition (RIUbiquitin Conjugation DeubiquitinationUbUbUbUbUbProteinUbUbUbUb

23、UbUbUbUbProteinUb USP E1 E2 E3De-ubiquitinationUbiquitin Conjugation Deubiquitinating ActivityUb C-terminal hydrolase familyUCH“Cysteine protease” Ub-specific processing protease UBP“Cysteine protease” Others Ub reutilized Biological FunctionProteosome regulationCell GrowthDevelopmentOncogenesisNeur

24、onal DiseasesChromatin Structure/transcriptionLargest family enzymes in Ub systemDeubiquitinating ActivityUb C-Poly-Ub ChainsUb K KUbUbUbUbUbUbUbUbK48 LinkageK63 LinkageK63K48Peters, J.M. 2019Ubiquitin and the Biology of the CellSignal to proteosomeK48, Ub4Cell SignalingK63Poly-Ub ChainsUb KENaC fun

25、ction 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 hypertensionENaC function Major ion channENac surface Stability Nedd 4 (HECT ligase)-neg

26、atively 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, Nedd4 P

27、 increases ENaC at the cell surfaceENac surface Stability Nedd 4Nedd4 and other Ubiquitin ligases important for protein transportNedd4 and other Ubiquitin ligaENaC SubunitsENaC SubunitsRegulation of ENaC Surface StabilityRegulation of ENaC Surface StaRegulation of ENaC Surface StabilityRegulation of ENaC Surface StaUb-like ProteinsSUMO-1 (sentrin, smt-3)2019 covalent modification RanGAP1RanGAP1 nearly quantitative modifiedCytosolic RanG