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Protein Targeting and Function: Ubiquitin Modifications Jim Collawn October 17 th, 2006.

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Presentation on theme: "Protein Targeting and Function: Ubiquitin Modifications Jim Collawn October 17 th, 2006."— Presentation transcript:

1 Protein Targeting and Function: Ubiquitin Modifications Jim Collawn October 17 th, 2006

2 References 1.Hicke and Dunn (2003) Regulation of membrane protein transport by ubiquitin and ub-binding proteins. Annu. Rev. Cell Dev. Biol. 19: Minireview: Regulation of Epithelial Na+ Channel Trafficking. Endocrinology 146:

3 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 molecules

4 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

5 Non-lysosomal Protein Degradation 1977 – Etlinger and Goldberg (PNAS) Protein degradation in reticulocytes But no lysosomes ATP dependent 1980 – Wilkinson et al (JBC) Identify protein degradation system in reticulocyte Two fractions – (ATP-dependent Proteolytic Factor; APF) APF-1 fraction conjugates to proteins APF-1 identified as ubiquitin Protein Adduct  Protein Degradation Ubiquitin FoundFunction Found

6 Avram Hershko – Nobel Lecture Hershko Ciechanover (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, 2004

7 APF-1 Protein  Ubiquitin-Proteosomal Pathway 1978 Hershko “APF factor” 2004

8 Ubiquitinylation – Proteosome Degradation E3 determines protein substrate

9 E1-Ub ~1000 aa, Cysteine-636 Ub attachment site E2:Transfer Ub Conj Conserved cysteine Active “thio ester” E3:Ligase Specific w/E2 Usually do not conjugate Hold complex together

10 19S and 20S Proteasome Subunits Characteristics 20S Subunit –Barrel –Contains 6 proteolytic sites 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)

11 Ubiquitin –76 Amino Acids Compact, globular 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 cellular effect Endocytosis Modify the activity of protein transport machinery

12 Ub Conjugation – Type Bonds Multi-Enzyme Process E1 Ub RGG C ATPADP E1 C Ub RGG High-energy thioester bond E2 C C Ub RGG High-energy thioester bond Protein E2 C Ub RGG E3 K GGRGGR Ub Isopeptide bonds Protein Ub USP

13 Ubiquitin AA Sequence MQIFV K TLTG KTITLEVEPS DTIENVKA K I QDKEGIPPDQ QRLIFAG K QL EDGRTLSDYN IQ K ESTLHLV LRLR GG

14 Roles of Ubiquitination

15 Different Types of Ubiquitin Tags

16 Modulalar Ub-binding Domains (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 15

17 Modulalar Ub-binding Domains Lipid binding preference: ENTH domain: PI-4,5-P (PM) FYVE domain: PI-3-P

18 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 Notch endocytosis)

19 Posttranslational Modification (ubiquitination) and Protein Sorting

20 ESCRT Proteins and the MVB

21 Ubiquitin and Protein Sorting 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, an example is the signal transducing receptors: G protein-coupled receptors (GPCRs)  2 -adrenergic and CXCR4 (MVB only) Receptor tyrosine kinases (RTKs)

22 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, an example is the signal transducing receptors: G protein-coupled receptors (GPCRs)  2 -adrenergic and CXCR4 (MVB only) Receptor tyrosine kinases (RTKs)

23 Transmembrane Proteins Regulated by Ub-dependent Sorting In yeast: Ste2, Ste3, Ste6, Uracil permease, Carboxypeptidase S, etc In metazoans: Regulation of growth/differentiation: Growth hormone receptor EGFR Macrophage colony-stimulating factor PDGFR Hepatocyte growth factor receptor Transforming growth factor  receptor  2 -adrenergic receptor Immune response: IL-2 receptor T cell receptor CXCR4 IgG receptor (Fcg receptor II) Pre-T cell receptor B7-2

24 Transmembrane Proteins Regulated by Ub-dependent Sorting In metazoans: Neurotransmission:Ion channels: AMPA glutamate receptorsENaC Glycine receptorsClC-5 Cell-cell contacts:Immune molecules E-cadherindownregulated by viruses: OccludinMHC class I B7-2 Developmental patterning:ICAM-1 DeltaCD4 Notch Roundabout

25 E3 Ligases Bridging factors: E2 + substrate Belong to three families Homologous E6AP Carboxy Terminus HECT (Rsp5/Nedd4) Really Interesting New Gene RING; major family (Cbl) Homology (U-box) proteins UFD2; small family

26 HECT domain E3 Homologous E6-AP C terminus Substrate specificity Ubiquitin E3 Enzymes E6-AP: papilloma virus E6 oncoprotein – associate protein

27 RING finger E3 Really Interesting New Gene Ubiquitin E3 Enzymes

28 Multiple E3 RING Ligases Complex RING

29 E2 Ub Transfer vs E3 Ub Transfer

30 E3 – Substrate Recognition (RING E3) Cbl protein Internalization of EGFR, CSF-1, Hepatocyte Growth FR

31 Ubiquitin Conjugation Deubiquitination Ub Protein Ub Protein Ub USP E1 E2 E3 De-ubiquitination

32 Deubiquitinating Activity Ub C-terminal hydrolase family UCH “Cysteine protease” Ub-specific processing protease UBP “Cysteine protease” Others Ub reutilized Biological Function Proteosome regulation Cell Growth Development Oncogenesis Neuronal Diseases Chromatin Structure/transcription Largest family enzymes in Ub system

33 Poly-Ub Chains Ub K K K48 Linkage K63 Linkage K63 K48 Peters, J.M Ubiquitin and the Biology of the Cell Signal to proteosome K48, Ub 4 Cell Signaling K63

34 ENaC 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 Liddle’s syndrome, and inherited form of hypertension

35 ENac surface Stability Nedd 4 (HECT ligase) - 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, Nedd4 P increases ENaC at the cell surface

36 Nedd4 and other Ubiquitin ligases important for protein transport

37 ENaC Subunits

38 Regulation of ENaC Surface Stability

39

40 Ub-like Proteins SUMO-1 (sentrin, smt-3) 1996 – covalent modification – RanGAP1 RanGAP1 nearly quantitative modified Cytosolic RanGAP1 to nuclear pore Activate shuttling factor

41 Ubiquitin-like Proteins:

42 Ubiquitin Superfold and Ubiquitons Ub – blue SUMO-1 – green NEDD8 - red UB αβ roll suprfold

43 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 degradation

44 Competition/Regulation SUMO Reactive Oxygen Species: Oxidizes reactive thiols on SUMO enzymes Uba1/Aos1- S – S – Ubc9 Thus: SUMO can not attach and proteins not Sumoylated

45 Examples of SUMO function RanGAP IkB c-Jun p53 and mdm2 Causes nuclear translocation Blocks Ub-conjugation site, prevents degradation Inhibits transcriptional activity Blocks mdm2 self-ubiquitination, prevents degradation SUMO-p53 in DNA binding domain   apoptotic activity PROTEIN SUMO Effect


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