1、Protein Targeting and Function:Ubiquitin ModificationsJim CollawnOctober 17th,2019References1.Hicke and Dunn(2019)Regulation of membrane protein transport by ubiquitin and ub-binding proteins.Annu.Rev.Cell Dev.Biol.19:141-172.2.2.Minireview:Regulation of Epithelial Na+Channel Trafficking.Endocrinolo
2、gy 146:5079-5085.Outline History and Evolution of Ub-pathway Sites where ubiquitination regulates protein trafficking/function Regulation of sodium channel stability at the cell surface(ENaC)Other ubiquitin-like moleculesCellular Protein Degradation Lysosomal Nonspecific Endocytosis Foreign proteins
3、 Energy favorable to degrade proteins Non-lysosomal Specificity,requires ATP Conditions of stress Ubiquitin-proteosomal pathway 26S proteosome Role in cellular processes/signalingNon-lysosomal Protein Degradation1977 Etlinger and Goldberg(PNAS)Protein degradation in reticulocytes But-no lysosomes AT
4、P dependent1980 Wilkinson et al(JBC)Identify protein degradation system in reticulocyte Two fractions (ATP-dependent Proteolytic Factor;APF)APF-1 fraction conjugates to proteinsAPF-1 identified as ubiquitin Protein Adduct Protein Degradation Ubiquitin FoundFunction FoundAvram Hershko Nobel LectureHe
5、rshkoCiechanover(grad student 76-81;major credit)Irwin Rose(Fox Chase Cancer Center)“The Ubiquitin System for Protein Degradation and some of its Roles in the Control of the Cell Cycle”December 8,2019APF-1 Protein Ubiquitin-Proteosomal Pathway1978Hershko“APF factor”2019Ubiquitinylation Proteosome De
6、gradationE3 determines protein substrateE1-Ub1000 aa,Cysteine-636 Ub attachment siteE2:TransferUb ConjConserved cysteineActive“thio ester”E3:LigaseSpecificw/E2Usually do not conjugateHold complex together19S and 20S Proteasome Subunits Characteristics 20S Subunit Barrel Contains 6 proteolytic sites
7、2x Tryptic 2x Chymotryptic 2x Peptidylglutamyl-peptidase Linearized protein required 19S Subunit Base and Lid Contains subunits with known and unknown functions Tetra-Ub(K48)recognition Deubiquitination activity Protein unfolding activity(Chaperone function)Ubiquitin 76 Amino Acids Compact,globular
8、superfold protein,9 kD Highly conserved(3 aa difference from yeast to man)Expressed in EVERY eukaryotic cell C-terminus extends away from protein Post-translational modification on proteins Attaches on Lysine residues Functions:Marks protein for proteasomal degradation Can“activate”protein trigger c
9、ellular effect Endocytosis Modify the activity of protein transport machineryUbUbConjugation Type BondsMulti-Enzyme Process E1 UbRGGCATPADP E1 CUbRGGHigh-energythioester bond E2 C E2 CUbRGGHigh-energythioester bondProteinProtein E2 CUbRGGE3KGGRUbIsopeptide bonds ProteinUbUbUbUSPUbiquitin AA Sequence
10、MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRGG48636Roles of UbiquitinationDifferent Types of Ubiquitin TagsModulalar Ub-binding Domains (endocytic proteins):Ubiquitin associated(UBA)or interacting motif(UIM)UVE(ub conjugating enzyme E2 variant CUE These domains b
11、ind to Ile44 on Ub Examples Espsin(Eps15-interacting protein)Eps15(epidermal growth factor receptor substrate 15Modulalar Ub-binding Domains Lipid binding preference:ENTH domain:PI-4,5-P(PM)FYVE domain:PI-3-PEndocytic Machinery that is monoubiquitinated CIN85(Cbl-interacting protein of 85 kDa)requir
12、ed for activated growth factor receptors Epsins Eps15 Hrs Rabex Numb(binds to Eps15 and AP-2)and neg regulates Notch(promotes Notch endocytosis)Posttranslational Modification(ubiquitination)and Protein SortingESCRT Proteins and the MVBUbiquitin and Protein Sorting In yeast,Ub is used for endocytosis
13、 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 targeting,an example is the signal transducing receptors:G protein-coupled receptors(GPCRs)b2-adrenergic and CXCR4(MVB only)Rece
14、ptor tyrosine kinases(RTKs)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 targeting,a
15、n example is the signal transducing receptors:G protein-coupled receptors(GPCRs)b2-adrenergic and CXCR4(MVB only)Receptor tyrosine kinases(RTKs)Transmembrane Proteins Regulated by Ub-dependent SortingIn yeast:Ste2,Ste3,Ste6,Uracil permease,Carboxypeptidase S,etcIn metazoans:Regulation of growth/diff
16、erentiation:Growth hormone receptorEGFRMacrophage colony-stimulating factorPDGFRHepatocyte growth factor receptorTransforming growth factor b receptorb2-adrenergic receptorImmune response:IL-2 receptorT cell receptorCXCR4IgG receptor(Fcg receptor II)Pre-T cell receptorB7-2Transmembrane Proteins Regu
17、lated by Ub-dependent SortingIn metazoans:Neurotransmission:Ion channels:AMPA glutamate receptorsENaCGlycine receptorsClC-5Cell-cell contacts:Immune molecules E-cadherindownregulated by viruses:OccludinMHC class IB7-2Developmental patterning:ICAM-1DeltaCD4NotchRoundaboutE3 Ligases Bridging factors:E
18、2+substrate Belong to three families Homologous E6AP Carboxy TerminusHECT(Rsp5/Nedd4)Really Interesting New GeneRING;major family(Cbl)Homology(U-box)proteinsUFD2;small familyHECT domain E3HomologousE6-APC terminusSubstrate specificityUbiquitin E3 EnzymesE6-AP:papilloma virus E6 oncoprotein associate
19、 proteinRING finger E3Really Interesting New GeneUbiquitin E3 EnzymesMultipleE3 RING LigasesComplex RINGE2 Ub Transfer vsE3 Ub Transfer E3 Substrate Recognition(RING E3)Cbl protein Internalization of EGFR,CSF-1,Hepatocyte Growth FRUbiquitin Conjugation DeubiquitinationUbUbUbUbUbProteinUbUbUbUbUbUbUb
20、UbProteinUb USP E1 E2 E3De-ubiquitinationDeubiquitinating ActivityUb C-terminal hydrolase familyUCH“Cysteine protease”Ub-specific processing protease UBP“Cysteine protease”Others Ub reutilized Biological FunctionProteosome regulationCell GrowthDevelopmentOncogenesisNeuronal DiseasesChromatin Structu
21、re/transcriptionLargest family enzymes in Ub systemPoly-Ub ChainsUb K KUbUbUbUbUbUbUbUbK48 LinkageK63 LinkageK63K48Peters,J.M.2019Ubiquitin and the Biology of the CellSignal to proteosomeK48,Ub4Cell SignalingK63ENaC function Major ion channel that controls salt and fluid resorption in the kidney Mut
22、ations in the PPXY motif cause accumulations of channels at the cell surface and result in Liddles syndrome,and inherited form of hypertensionENac surface Stability Nedd 4(HECT ligase)-negatively regulates ENaC surface stability Nedd4 WW domains bind PPXY motif of ENaC subunits Nedd4 also interacts
23、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 increases ENaC at the cell surfaceNedd4 and other Ubiquitin ligases important for protein transportENaC SubunitsRe
24、gulation of ENaC Surface StabilityRegulation of ENaC Surface StabilityUb-like Proteins SUMO-1 (sentrin,smt-3)2019 covalent modification RanGAP1 RanGAP1 nearly quantitative modified Cytosolic RanGAP1 to nuclear pore Activate shuttling factorUbiquitin-like Proteins:Ubiquitin Superfold and UbiquitonsUb
25、 blueSUMO-1 greenNEDD8-redUB roll suprfoldSUMO SUMO SUMO-1&SUMO-2/3 Shared characteristics C-terminal-GG essential for conjugation Affix to lysine residues in target NOT directly associated with proteasomal degradationCompetition/RegulationSUMOReactive Oxygen Species:Oxidizes reactive thiols on SUMO
26、 enzymesUba1/Aos1-S S Ubc9Thus:SUMO can not attach and proteins not SumoylatedExamples of SUMO function RanGAP IkB c-Jun p53 and mdm2 Causes nuclear translocation Blocks Ub-conjugation site,prevents degradation Inhibits transcriptional activityBlocks mdm2 self-ubiquitination,prevents degradationSUMO-p53 in DNA binding domain apoptotic activityPROTEINSUMO Effect
