Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

XIAP is a potent suppressor of apoptosis that directly inhibits specific members of the caspase family of cysteine proteases. Here we demonstrate a novel role for XIAP in the control of intracellular copper levels. XIAP was found to interact with MURR1, a factor recently implicated in copper homeostasis. XIAP binds to MURR1 in a manner that is distinct from that utilized by XIAP to bind caspases, and consistent with this, MURR1 did not affect the antiapoptotic properties of XIAP. However, cells and tissues derived from Xiap-deficient mice were found to contain reduced copper levels, while suppression of MURR1 resulted in increased intracellular copper in cultured cells. Consistent with these opposing effects, XIAP was observed to negatively regulate MURR1 protein levels by the formation of K48 polyubiquitin chains on MURR1 that promote its degradation. These findings represent the first described phenotypic alteration in Xiap-deficient mice and demonstrate that XIAP can function through MURR1 to regulate copper homeostasis.
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PMID:A novel role for XIAP in copper homeostasis through regulation of MURR1. 1468 66

Ubiquitination regulates the stability and/or activity of numerous cellular proteins. The corollary is that de-ubiquitinating enzymes, which 'trim' polyubiquitin chains from specific substrate proteins, play key roles in controlling fundamental cellular activities. Ubiquitin is essential at several stages during the activation of NF-kappaB (nuclear factor kappaB), a central co-ordinator of inflammation and other immune processes. Ubiquitination is known to cause degradation of the inhibitory molecule IkappaBalpha (inhibitor of kappaB). In addition, activation of TRAF (tumour-necrosis-factor-receptor-associated factor) and IKKgamma (IkappaB kinase gamma)/NEMO (NF-kappaB essential modifier) signal adaptors relies on their modification with 'nonclassical' forms of polyubiquitin chains. Ubiquitin also plays a key role in determining cell fate by modulating the stability of numerous pro-apoptotic or anti-apoptotic proteins. The zinc-finger protein A20 has dual functions in inhibiting NF-kappaB activation and suppressing apoptosis. The molecular mechanisms of these anti-inflammatory and cytoprotective effects are unknown. Here we demonstrate that A20 is a de-ubiquitinating enzyme. It contains an N-terminal catalytic domain that belongs to the ovarian-tumour superfamily of cysteine proteases. A20 cleaved ubiquitin monomers from branched polyubiquitin chains linked through Lys48 or Lys63 and bound covalently to a thiol-group-reactive, ubiquitin-derived probe. Mutation of a conserved cysteine residue in the catalytic site (Cys103) abolished these activities. A20 did not have a global effect on ubiquitinated cellular proteins, which indicates that its activity is target-specific. The biological significance of the catalytic domain is unknown.
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PMID:Zinc-finger protein A20, a regulator of inflammation and cell survival, has de-ubiquitinating activity. 1474 87

Ubiquitin is a small polypeptide that is conjugated to proteins and commonly serves as a degradation signal. The attachment of ubiquitin (Ub) to a substrate proceeds through a multi-enzyme cascade involving an activating enzyme (E1), a conjugating enzyme (E2), and a protein ligase (E3). We previously demonstrated that a murine E2, UbcM2, is imported into nuclei by the transport receptor importin-11. We now show that the import mechanism for UbcM2 and two other human class III E2s (UbcH6 and UBE2E2) uniquely requires the covalent attachment of Ub to the active site cysteine of these enzymes. This coupling of E2 activation and transport arises from the selective interaction of importin-11 with the Ub-loaded forms of these enzymes. Together, these findings reveal that Ub charging can function as a nuclear import trigger, and identify a novel link between E2 regulation and karyopherin-mediated transport.
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PMID:Ubiquitin charging of human class III ubiquitin-conjugating enzymes triggers their nuclear import. 1554 18

Ubiquitin-mediated proteolysis plays a central role in controlling intracellular levels of essential regulatory molecules such as p53, cyclins, myc, BRCA1, HIF-1alpha, etc. The Kruppel-like factor 5 (KLF5) transcription factor regulates biological processes involved in carcinogenesis, angiogenesis, and smooth muscle cell differentiation. In carcinogenesis, KLF5's role has been indicated by frequent genetic deletion as well as functional studies. Here we show that KLF5 is an unstable protein with a short half-life. Destruction of KLF5 was prevented by each of the proteasome-specific inhibitors tested but not by an inhibitor for trypsin-like proteases and cysteine proteases or by a lysosome inhibitor in epithelial cells. Furthermore, KLF5 underwent ubiquitination, and deletion of a 56-amino-acid sequence adjacent to a known transactivation domain of KLF5 significantly reduced its ubiquitination and degradation. Interestingly, cancer cells appeared to be more active in KLF5 degradation than untransformed epithelial cells, yet their proteasome activity was not higher. These results suggest that KLF5 protein is degraded at least in part through ubiquitination-proteasome pathway, which may have become hyperactive for KLF5 in cancer cells.
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PMID:Ubiquitin-proteasome degradation of KLF5 transcription factor in cancer and untransformed epithelial cells. 1573 97

Degradation of misfolded and damaged proteins by the 26 S proteasome requires the substrate to be tagged with a polyubiquitin chain. Assembly of polyubiquitin chains and subsequent substrate labeling potentially involves three enzymes, an E1, E2, and E3. E2 proteins are key enzymes and form a thioester intermediate through their catalytic cysteine with the C-terminal glycine (Gly76) of ubiquitin. This thioester intermediate is easily hydrolyzed in vitro and has eluded structural characterization. To overcome this, we have engineered a novel ubiquitin-E2 disulfide-linked complex by mutating Gly76 to Cys76 in ubiquitin. Reaction of Ubc1, an E2 from Saccharomyces cerevisiae, with this mutant ubiquitin resulted in an ubiquitin-E2 disulfide that could be purified and was stable for several weeks. Chemical shift perturbation analysis of the disulfide ubiquitin-Ubc1 complex by NMR spectroscopy reveals an ubiquitin-Ubc1 interface similar to that for the ubiquitin-E2 thioester. In addition to the typical E2 catalytic domain, Ubc1 contains an ubiquitin-associated (UBA) domain, and we have utilized NMR spectroscopy to demonstrate that in this disulfide complex the UBA domain is freely accessible to non-covalently bind a second molecule of ubiquitin. The ability of the Ubc1 to bind two ubiquitin molecules suggests that the UBA domain does not interact with the thioester-bound ubiquitin during polyubiquitin chain formation. Thus, construction of this novel ubiquitin-E2 disulfide provides a method to characterize structurally the first step in polyubiquitin chain assembly by Ubc1 and its related class II enzymes.
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PMID:Ubiquitin manipulation by an E2 conjugating enzyme using a novel covalent intermediate. 1601 32

Ubc13, a ubiquitin-conjugating enzyme (Ubc), requires the presence of a Ubc variant (Uev) for polyubiquitination. Uevs, although resembling Ubc in sequence and structure, lack the active site cysteine residue and are catalytically inactive. The yeast Uev (Mms2) incites noncanonical Lys63-linked polyubiquitination by Ubc13, whereas the increased diversity of Uevs in higher eukaryotes suggests an unexpected complication in ubiquitination. In this study, we demonstrate that divergent activities of mammalian Ubc13 rely on its pairing with either of two Uevs, Uev1A or Mms2. Structurally, we demonstrate that Mms2 and Uev1A differentially modulate the length of Ubc13-mediated Lys63-linked polyubiquitin chains. Functionally, we describe that Ubc13-Mms2 is required for DNA damage repair but not nuclear factor kappaB (NF-kappaB) activation, whereas Ubc13-Uev1A is involved in NF-kappaB activation but not DNA repair. Our finding suggests a novel regulatory mechanism in which different Uevs direct Ubcs to diverse cellular processes through physical interaction and alternative polyubiquitination.
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PMID:Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants Mms2 and Uev1A. 1612 84

Individual ubiquitin (Ub)-protein ligases (E3s) cooperate with specific Ub-conjugating enzymes (E2s) to modify cognate substrates with polyubiquitin chains. E3s belonging to the Really Interesting New Gene (RING) and Homologous to E6-Associated Protein (E6AP) C-Terminus (HECT) domain families utilize distinct molecular mechanisms. In particular, HECT E3s, but not RING E3s, form a thiol ester with Ub before transferring Ub to the substrate lysine. Here we report that different HECT domain E3s can employ distinct mechanisms of polyubiquitin chain synthesis. We show that E6AP builds up a K48-linked chain on its HECT cysteine residue, while KIAA10 builds up K48- and K29-linked chains as free entities. A small region near the N-terminus of the conserved HECT domain helps to bring about this functional distinction. Thus, a given HECT domain can specify both the linkage of a polyubiquitin chain and the mechanism of its assembly.
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PMID:Different HECT domain ubiquitin ligases employ distinct mechanisms of polyubiquitin chain synthesis. 1634 Oct 92

Ubiquitin-conjugating enzymes (E2s or Ubcs) are essential components in the ubiquitination apparatus. These enzymes accept ubiquitin from an E1 enzyme and then, usually with the aid of an E3 enzyme, donate the ubiquitin to the target protein. The function of E2 relies critically on the chemistry of its active site cysteine residue since this residue must form a thioester bond with the carboxyl terminus of ubiquitin. Despite the plethora of structural information that is available, there has been a notable dearth of information regarding the chemical basis of E2 function. Toward filling this large void in our understanding of E2 function, we have examined the pK(a) of the active site cysteine using a combination of experimental and theoretical approaches. We find, remarkably, that the pK(a) of the active site cysteine residue is elevated by approximately 2 pH units above that of a free cysteine. We have identified residues that contribute to the increase in this pK(a). On the basis of experimental values obtained with three different E2 proteins, we believe this to be a general and important characteristic of E2 protein chemistry. Sequence comparison suggests that the electrostatic environment is maintained not through strict residue conservation but through different combinations of residues near the active site. We propose that the elevated pK(a) is a regulatory mechanism that prevents the highly exposed cysteine residue in free E2 from reacting promiscuously with electron deficient chemical moieties in the cell.
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PMID:The active site cysteine of ubiquitin-conjugating enzymes has a significantly elevated pKa: functional implications. 1634 31

Mutations in the parkin gene, encoding an E3 ubiquitin-protein ligase, are a frequent cause of autosomal recessive parkinsonism and are also involved in sporadic Parkinson's disease. Loss of Parkin function is thought to compromise the polyubiquitylation and proteasomal degradation of specific substrates, leading to their deleterious accumulation. Several studies have analyzed the effects of parkin gene mutations on the biochemical properties of the protein. However, the absence of a cell-free system for studying intrinsic Parkin activity has limited the interpretation of these studies. Here we describe the biochemical characterization of Parkin and 10 pathogenic variants carrying amino-acid substitutions throughout the sequence. Mutations in the RING fingers or the ubiquitin-like domain decreased the solubility of the protein in detergent and increased its tendency to form visible aggregates. None of the mutations studied compromised the binding of Parkin to a series of known protein partners/substrates. Moreover, only two variants with substitutions of conserved cysteine residues of the second RING finger were inactive in a purely in vitro ubiquitylation assay, demonstrating that loss of ligase activity is a minor pathogenic mechanism. Interestingly, in this in vitro assay, Parkin catalyzed the linkage of single ubiquitin molecules only, whereas the ubiquitin-protein ligases CHIP and Mdm2 promoted the formation of polyubiquitin chains. Similarly, in mammalian cells Parkin promoted the multimonoubiquitylation of its substrate p38, rather than its polyubiquitylation. Thus, Parkin may mediate polyubiquitylation or proteasome-independent monoubiquitylation depending on the protein context. The discovery of monoubiquitylated Parkin species in cells hints at a novel post-translational modification potentially involved in the regulation of Parkin function.
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PMID:Biochemical analysis of Parkinson's disease-causing variants of Parkin, an E3 ubiquitin-protein ligase with monoubiquitylation capacity. 1671

Ubiquitin (Ub)-fold proteins are rapidly emerging as an important class of eukaryotic modifiers, which often exert their influence by post-translational addition to other intracellular proteins. Despite assuming a common beta-grasp three-dimensional structure, their functions are highly diverse because of distinct surface features and targets and include tagging proteins for selective breakdown, nuclear import, autophagic recycling, vesicular trafficking, polarized morphogenesis, and the stress response. Here we describe a novel family of Membrane-anchored Ub-fold (MUB) proteins that are present in animals, filamentous fungi, and plants. Extending from the C terminus of the Ub-fold is typically a cysteine-containing CAAX (where A indicates aliphatic amino acid) sequence that can direct the attachment of either a 15-carbon farnesyl or a 20-carbon geranylgeranyl moiety in vitro. Modified forms of several MUBs were detected in transgenic Arabidopsis thaliana, suggesting that these MUBs are prenylated in vivo. Both cell fractionation and confocal microscopic analyses of Arabidopsis plants expressing GFP-MUB fusions showed that the modified forms are membrane-anchored with a significant enrichment on the plasma membrane. This plasma membrane location was blocked in vivo in prenyltransferase mutants and by mevinolin, which inhibits the synthesis of prenyl groups. In addition to the five MUBs with CAAX boxes, Arabidopsis has one MUB variant with a cysteine-rich C terminus distinct from the CAAX box that is also membrane-anchored, possibly through the attachment of a long chain acyl group. Although the physiological role(s) of MUBs remain unknown, the discovery of these prenylated forms further expands the diversity and potential functions of Ub-fold proteins in eukaryotic biology.
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PMID:MUBs, a family of ubiquitin-fold proteins that are plasma membrane-anchored by prenylation. 1683 69


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