Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P62988 (Ubiquitin)
 4,326 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

TRAF6 is a signal transducer in the NF-kappaB pathway that activates IkappaB kinase (IKK) in response to proinflammatory cytokines. We have purified a heterodimeric protein complex that links TRAF6 to IKK activation. Peptide mass fingerprinting analysis reveals that this complex is composed of the ubiquitin conjugating enzyme Ubc13 and the Ubc-like protein Uev1A. We find that TRAF6, a RING domain protein, functions together with Ubc13/Uev1A to catalyze the synthesis of unique polyubiquitin chains linked through lysine-63 (K63) of ubiquitin. Blockade of this polyubiquitin chain synthesis, but not inhibition of the proteasome, prevents the activation of IKK by TRAF6. These results unveil a new regulatory function for ubiquitin, in which IKK is activated through the assembly of K63-linked polyubiquitin chains.
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PMID:Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. 2941 May 30

Polyubiquitin chains, in which the C-terminus and a lysine side chain of successive ubiquitin molecules are linked by an isopeptide bond, function to target substrate proteins for degradation by the 26S proteasome. Chains of at least four ubiquitin moieties appear to be required for efficient recognition by the 26S proteasome, although the conformations of the polyubiquitin chains recognized by the proteasome or by other enzymes involved in ubiquitin metabolism are currently unknown. A new crystal form of tetraubiquitin, which has two possible chain connectivities that are indistinguishable in the crystal, is reported. In one possible connectivity, the tetraubiquitin chain is extended and packs closely against the antiparallel neighbor chain in the crystal to conceal a hydrophobic surface implicated in 26S proteasome recognition. In the second possibility, the tetraubiqutitin forms a closed compact structure, in which that same hydrophobic surface is buried. Both of these conformations are quite unlike the structure of tetraubiquitin that was previously determined in a different crystal form [Cook et al. (1994), J. Mol. Biol. 236, 601--609]. The new structure suggests that polyubiquitin chains may possess a substantially greater degree of conformational flexibility than has previously been appreciated.
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PMID:Structure of a new crystal form of tetraubiquitin. 1117 99

Although polyubiquitin chains linked through Lys(29) of ubiquitin have been implicated in the targeting of certain substrates to proteasomes, the signaling properties of these chains are poorly understood. We previously described a ubiquitin-protein isopeptide ligase (E3) from erythroid cells that assembles polyubiquitin chains through either Lys(29) or Lys(48) of ubiquitin (Mastrandrea, L. D., You, J., Niles, E. G., and Pickart, C. M. (1999) J. Biol. Chem. 274, 27299-27306). Here we describe the purification of this E3 based on its affinity for a linear fusion of ubiquitin to the ubiquitin-conjugating enzyme UbcH5A. Among five major polypeptides in the affinity column eluate, the activity of interest was assigned to the product of a previously cloned human cDNA known as KIAA10 (Nomura, N., Miyajima, N., Sazuka, T., Tanaka, A., Kawarabayasi, Y., Sato, S., Nagase, T., Seki, N., Ishikawa, K., and Tabata, S. (1994) DNA Res. 1, 27-35). The KIAA10 protein is a member of the HECT (homologous to E6-AP carboxyl terminus) domain family of E3s. These E3s share a conserved C-terminal (HECT) domain that functions in the catalysis of ubiquitination, while their divergent N-terminal domains function in cognate substrate binding (Huibregtse, J. M., Scheffner, M., Beaudenon, S., and Howley, P. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2563-2567). Recombinant KIAA10 catalyzed the assembly of both Lys(29)- and Lys(48)-linked polyubiquitin chains. Surprisingly, the C-terminal 428 residues of KIAA10 were both necessary and sufficient for this activity, suggesting that the ability to assemble polyubiquitin chains may be a general property of HECT domains. The N-terminal domain of KIAA10 interacted in vitro with purified 26 S proteasomes and with the isolated S2/Rpn1 subunit of the proteasome's 19 S regulatory complex, suggesting that the N-terminal domains of HECT E3s may function in proteasome binding as well as substrate binding.
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PMID:A HECT domain E3 enzyme assembles novel polyubiquitin chains. 1127 95

Polyubiquitin chains assembled through lysine 48 (Lys-48) of ubiquitin act as a signal for substrate proteolysis by 26 S proteasomes, whereas chains assembled through Lys-63 play a mechanistically undefined role in post-replicative DNA repair. We showed previously that the products of the UBC13 and MMS2 genes function in error-free post-replicative DNA repair in the yeast Saccharomyces cerevisiae and form a complex that assembles Lys-63-linked polyubiquitin chains in vitro. Here we confirm that the Mms2.Ubc13 complex functions as a high affinity heterodimer in the chain assembly reaction in vitro and report the results of a kinetic characterization of the polyubiquitin chain assembly reaction. To test whether a Lys-63-linked polyubiquitin chain can signal degradation, we conjugated Lys-63-linked tetra-ubiquitin to a model substrate of 26 S proteasomes. Although the noncanonical chain effectively signaled substrate degradation, the results of new genetic epistasis studies agree with previous genetic data in suggesting that the proteolytic activity of proteasomes is not required for error-free post-replicative repair.
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PMID:In vitro assembly and recognition of Lys-63 polyubiquitin chains. 1136 80

TRAF6 is a signal transducer that activates IkappaB kinase (IKK) and Jun amino-terminal kinase (JNK) in response to pro-inflammatory mediators such as interleukin-1 (IL-1) and lipopolysaccharides (LPS). IKK activation by TRAF6 requires two intermediary factors, TRAF6-regulated IKK activator 1 (TRIKA1) and TRIKA2 (ref. 5). TRIKA1 is a dimeric ubiquitin-conjugating enzyme complex composed of Ubc13 and Uev1A (or the functionally equivalent Mms2). This Ubc complex, together with TRAF6, catalyses the formation of a Lys 63 (K63)-linked polyubiquitin chain that mediates IKK activation through a unique proteasome-independent mechanism. Here we report the purification and identification of TRIKA2, which is composed of TAK1, TAB1 and TAB2, a protein kinase complex previously implicated in IKK activation through an unknown mechanism. We find that the TAK1 kinase complex phosphorylates and activates IKK in a manner that depends on TRAF6 and Ubc13-Uev1A. Moreover, the activity of TAK1 to phosphorylate MKK6, which activates the JNK-p38 kinase pathway, is directly regulated by K63-linked polyubiquitination. We also provide evidence that TRAF6 is conjugated by the K63 polyubiquitin chains. These results indicate that ubiquitination has an important regulatory role in stress response pathways, including those of IKK and JNK.
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PMID:TAK1 is a ubiquitin-dependent kinase of MKK and IKK. 2941 May 30

The ubiquitin conjugating enzyme complex Mms2-Ubc13 plays a key role in post-replicative DNA repair in yeast and the NF-kappaB signal transduction pathway in humans. This complex assembles novel polyubiquitin chains onto yet uncharacterized protein targets. Here we report the crystal structure of a complex between hMms2 (Uev1) and hUbc13 at 1.85 A resolution and a structure of free hMms2 at 1.9 A resolution. These structures reveal that the hMms2 monomer undergoes a localized conformational change upon interaction with hUbc13. The nature of the interface provides a physical basis for the preference of Mms2 for Ubc13 as a partner over a variety of other structurally similar ubiquitin-conjugating enzymes. The structure of the hMms2-hUbc13 complex provides the conceptual foundation for understanding the mechanism of Lys 63 multiubiquitin chain assembly and for its interactions with the RING finger proteins Rad5 and Traf6.
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PMID:Crystal structure of the human ubiquitin conjugating enzyme complex, hMms2-hUbc13. 1147 44

The N-end rule relates the amino terminus to the rate of degradation through the ubiquitin/26 S proteasome pathway. Proteins bearing basic (type 1) or large hydrophobic (type 2) amino termini are assumed to be targeted through this pathway by their higher affinity for binding to the responsible E3 ligase compared with proteins bearing other residues (type 3). Paradoxically, a significant fraction of eukaryotic protein degradation occurs through the N-end rule pathway, although the majority of cellular proteins are type 3 substrates. We have exploited specific interactions between ubiquitin carrier proteins (E2/Ubc) and their cognate E3 ligases to purify for the first time the mammalian N-end rule ligase E3alpha/Ubr1 to near homogeneity. In vitro studies show that E3alpha forms lysine 48-linked polyubiquitin degradation signals on type 1-3 substrates and is absolutely dependent on Ubc2/Rad6 orthologs. Biochemically defined kinetic studies show that the basis of N-end rule specificity is a k(cat) rather than the K(m) effect originally proposed, since all three substrate classes show similar binding affinities (K(m) approximately 5 microm) but V(max) values that are 100- and 50-fold greater for type 1 and 2 versus type 3 model substrates, respectively. In addition, the N-end rule dipeptides lysylalanine and phenylalanylalanine are general noncompetitive inhibitors for E3alpha-catalyzed ubiquitination of type 1-3 substrates rather than type-specific competitive inhibitors as predicted. These observations are consistent with a model in which the N-end rule effect reflects substrate binding-induced transitions in E3alpha to a catalytically competent conformer, the equilibrium for which depends on the identity of the amino terminus or the presence of basic or hydrophobic surface features. The model reconciles conflicts between specific predictions and empirical observations relating N-end rule targeting in addition to explicating the efficacy of selected dipeptides as potent in vivo inhibitors of this pathway.
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PMID:N-end rule specificity within the ubiquitin/proteasome pathway is not an affinity effect. 1149 6

Lysine 48-linked polyubiquitin chains are the principle signal for targeting proteins for degradation by the 26 S proteasome. Here we report that the conjugation of Nedd8 to ROC1-CUL1, a subcomplex of the SCF-ROC1 E3 ubiquitin ligase, selectively stimulates Cdc34-catalyzed lysine 48-linked multiubiquitin chain assembly. We have further demonstrated that separate regions within the human Cdc34 C-terminal tail are responsible for multiubiquitin chain assembly and for physical interactions with the Nedd8-conjugated ROC1-CUL1 to assemble extensive ubiquitin polymers. Structural comparisons between Nedd8 and ubiquitin reveal that six charged residues (Lys4, Glu12, Glu14, Arg25, Glu28, and Glu31) are uniquely present on the surface of Nedd8. Replacement of each of the six residues with the corresponding amino acid in ubiquitin decreases the ability of Nedd8 to activate the ubiquitin ligase activity of ROC1-CUL1. Moreover, maintenance of the proper charges at amino acid positions 14 and 25 are necessary for retaining wild type levels of activity, whereas introduction of the opposite charges at these positions abolishes the Nedd8 activation function. These results suggest that Nedd8 charged surface residues mediate the activation of ROC1-CUL1 to specifically support Cdc34-catalyzed ubiquitin polymerization.
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PMID:The Nedd8-conjugated ROC1-CUL1 core ubiquitin ligase utilizes Nedd8 charged surface residues for efficient polyubiquitin chain assembly catalyzed by Cdc34. 1167 91

Ubiquitin (Ub) regulates important cellular processes through covalent attachment to its substrates. The fate of a substrate depends on the number of ubiquitin moieties conjugated, as well as the lysine linkage of Ub-Ub conjugation. The major function of Ub is to regulate the in vivo half-life of its substrates. Once a multi-Ub chain is attached to a substrate, it must be shielded from deubiquitylating enzymes for the 26 S proteasome to recognize it. Molecular mechanisms of the postubiquitylation processes are poorly understood. Here, we have characterized a family of proteins that preferentially binds ubiquitylated substrates and multi-Ub chains through a motif termed the ubiquitin-associated domain (UBA). Our in vivo genetic analysis demonstrates that such interactions require specific lysine residues of Ub that are important for Ub chain formation. We show that Saccharomyces cerevisiae cells lacking two of these UBA proteins, Dsk2 and Rad23, are deficient in protein degradation mediated by the UFD pathway and that the intact UBA motif of Dsk2 is essential for its function in proteolysis. Dsk2 and Rad23 can form a complex(es), suggesting that they cooperate to recognize a subset of multi-Ub chains and deliver the Ub-tagged substrates to the proteasome. Our results suggest a molecular mechanism for differentiation of substrate fates, depending on the precise nature of the mono-Ub or multi-Ub lysine linkage, and provide a foundation to further investigate postubiquitylation events.
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PMID:Recognition of specific ubiquitin conjugates is important for the proteolytic functions of the ubiquitin-associated domain proteins Dsk2 and Rad23. 1180 21

The RING finger of BRCA1 confers ubiquitin ligase activity that is markedly enhanced when complexed with another RING-containing protein, BARD1, and is required for the function of this tumor suppressor protein in protecting genomic integrity. Here, we report that co-expression of BRCA1-(1-639) and BARD1 in bacteria can assemble a potent ubiquitin ligase activity. Purified BRCA1-(1-639)*BARD1 stimulated the Ubc5c-mediated monoubiquitination of histone H2A/H2AX in vitro, suggesting a possible role for BRCA1*BARD1 in modifying chromatin structure. Moreover, the truncated BRCA1*BARD1 complex exhibited efficient autoubiquitination activity in vitro capable of assembling non-lysine 48-linked polyubiquitin chains on both BRCA1-(1-639) and BARD1. When co-expressed in cells by transient transfection, the recombinant BRCA1-(1-300).BARD1 complex was found to be associated with polyubiquitin chains, suggesting that BRCA1-(1-300)*BARD1 was ubiquitinated in vivo as well. These results raise the possibility that BRCA1*BARD1 acts to assemble non-lysine 48-linked polyubiquitin chains that may serve as part of a signaling platform required for coordinating DNA repair-related events.
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PMID:Autoubiquitination of the BRCA1*BARD1 RING ubiquitin ligase. 1192 91


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