Protein Targeting and Function: Ubiquitin Modifications Jim Collawn October 17 th, 2006.
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Transcript of Protein Targeting and Function: Ubiquitin Modifications Jim Collawn October 17 th, 2006.
Protein Targeting and Function:Ubiquitin Modifications
Jim CollawnOctober 17th, 2006
References
1. Hicke and Dunn (2003) Regulation of membrane protein transport by ubiquitin and ub-binding proteins. Annu. Rev. Cell Dev. Biol. 19: 141-172.
2. Minireview: Regulation of Epithelial Na+ Channel Trafficking. Endocrinology 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 molecules
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
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 proteinsAPF-1 identified as ubiquitin
Protein Adduct Protein Degradation Ubiquitin Found Function Found
Avram Hershko – Nobel Lecture
HershkoCiechanover (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
APF-1 Protein Ubiquitin-Proteosomal Pathway
1978Hershko
“APF factor”
2004
Ubiquitinylation – Proteosome Degradation
E3 determines protein substrate
E1-Ub~1000 aa, Cysteine-636 Ub attachment site
E2: TransferUb Conj
Conserved cysteineActive “thio ester”
E3: LigaseSpecificw/E2
Usually do not conjugateHold complex together
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)
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
UbUb
Conjugation – Type BondsMulti-Enzyme Process
E1
Ub RGG
C
ATP ADP
E1 CUb RGG
High-energythioester bond
E2 C
E2 CUb RGG
High-energythioester bond
Protein
Protein
E2 CUb RGG
E3K
GGR
UbIsopeptide bonds
Protein
UbUb
Ub
USP
Ubiquitin AA Sequence
MQIFVKTLTG KTITLEVEPS DTIENVKAKI
QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN
IQKESTLHLV LRLRGG
48
63
6
Roles of Ubiquitination
Different Types of Ubiquitin Tags
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
Modulalar Ub-binding Domains
Lipid binding preference: ENTH domain: PI-4,5-P (PM) FYVE domain: PI-3-P
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)
Posttranslational Modification (ubiquitination) and Protein Sorting
ESCRT Proteins and the MVB
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)
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)
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 receptorEGFRMacrophage colony-stimulating factorPDGFRHepatocyte growth factor receptorTransforming growth factor receptor2-adrenergic receptor
Immune response:IL-2 receptorT cell receptorCXCR4IgG receptor (Fcg receptor II)Pre-T cell receptorB7-2
Transmembrane Proteins Regulated by Ub-dependent Sorting
In metazoans:Neurotransmission: Ion channels:AMPA glutamate receptors ENaCGlycine receptors ClC-5
Cell-cell contacts: Immune molecules E-cadherin downregulated by
viruses:Occludin MHC class I
B7-2Developmental patterning: ICAM-1Delta CD4NotchRoundabout
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 family
HECT domain E3
HomologousE6-APC terminus
Substrate specificity
Ubiquitin E3 Enzymes
E6-AP: papilloma virus E6 oncoprotein – associate protein
RING finger E3
Really Interesting New Gene
Ubiquitin E3 Enzymes
MultipleE3 RING Ligases
Complex RING
E2 Ub Transfer vsE3 Ub Transfer
E3 – Substrate Recognition (RING E3)
Cbl protein
Internalization of EGFR, CSF-1, Hepatocyte Growth FR
Ubiquitin Conjugation Deubiquitination
UbUb
UbUb
Ub
Protein
UbUb
Ub
UbUb
UbUbUb
Protein
Ub
USP E1
E2
E3
De-ubiquitination
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 DiseasesChromatin Structure/transcription
Largest family enzymes in Ub system
Poly-Ub Chains
Ub
K
K
Ub
UbUb
Ub
UbUb
Ub
Ub
K48 Linkage
K63 Linkage
K63
K48
Peters, J.M. 1998Ubiquitin and the Biology of the Cell
Signal to proteosome
K48, Ub4
Cell Signaling
K63
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
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
Nedd4 and other Ubiquitin ligases important for protein transport
ENaC Subunits
Regulation of ENaC Surface Stability
Regulation of ENaC Surface Stability
Ub-like Proteins
• SUMO-1 (sentrin, smt-3)• 1996 – covalent modification – RanGAP1• RanGAP1 nearly quantitative modified• Cytosolic RanGAP1 to nuclear pore• Activate shuttling factor
Ubiquitin-like Proteins:
Ubiquitin Superfold and Ubiquitons
Ub – blueSUMO-1 – greenNEDD8 - red
UB αβ roll suprfold
SUMO• 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
Competition/RegulationSUMO
Reactive Oxygen Species: Oxidizes reactive thiols on SUMO enzymes
Uba1/Aos1- S – S – Ubc9
Thus: SUMO can not attach and proteins not Sumoylated
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