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)

Both p53 and its repressor Mdm2 are subject to ubiquitination and proteasomal degradation. We show that knockdown of the deubiquitinating enzyme USP5 (isopeptidase T) results in an increase in the level and transcriptional activity of p53. Suppression of USP5 stabilizes p53, whereas it has little or no effect on the stability of Mdm2. This provides a mechanism for transcriptional activation of p53. USP5 knockdown interferes with the degradation of ubiquitinated p53 rather than attenuating p53 ubiquitination. In vitro studies have shown that a preferred substrate for USP5 is unanchored polyubiquitin. Consistent with this, we observed for the first time in a mammalian system that USP5 makes a major contribution to Lys-48-linked polyubiquitin disassembly and that suppression of USP5 results in the accumulation of unanchored polyubiquitin chains. Ectopic expression of a C-terminal mutant of ubiquitin (G75A/G76A), which also causes the accumulation of free polyubiquitin, recapitulates the effects of USP5 knockdown on the p53 pathway. We propose a model in which p53 is selectively stabilized because the unanchored polyubiquitin that accumulates after USP5 knockdown is able to compete with ubiquitinated p53 but not with Mdm2 for proteasomal recognition. This raises the possibility that there are significant differences in proteasomal recognition of p53 and Mdm2. These differences could be exploited therapeutically. Our study reveals a novel mechanism for regulation of p53 and identifies USP5 as a potential target for p53 activating therapeutic agents for the treatment of cancer.
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PMID:Suppression of the deubiquitinating enzyme USP5 causes the accumulation of unanchored polyubiquitin and the activation of p53. 1909 88

An unusual deubiquitinating (DUB) activity exists in HeLa cell extracts that is highly specific for cleaving K63-linked but not K48-linked polyubiquitin chains. The activity is insensitive to both N-ethyl-maleimide and ubiquitin aldehyde, indicating that it lacks an active site cysteine residue, and gel filtration experiments show that it resides in a high molecular weight (approximately 600 kDa) complex. Using a biochemical approach, we found that the K63-specific DUB activity co-fractionated through seven chromatographic steps with three multisubunit complexes: the 19S (PA700) portion of the 26S proteasome, the COP9 signalosome (CSN) and a novel complex that includes the JAMM/MPN+ domain-containing protein Brcc36. When we analysed the individual complexes, we found that the activity was intrinsic to PA700 and the Brcc36 isopeptidase complex (BRISC), but that the CSN-associated activity was due entirely to an interaction with Brcc36. None of the complexes cleave K6, K11, K29, K48 or alpha-linked polyubiquitin, but they do cleave K63 linkages within mixed-linkage chains. Our results suggest that specificity for K63-linked polyubiquitin is a common property of the JAMM/MPN+ family of DUBs.
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PMID:K63-specific deubiquitination by two JAMM/MPN+ complexes: BRISC-associated Brcc36 and proteasomal Poh1. 1929 98

BRISC (Brcc36-containing isopeptidase complex) is a four-subunit deubiquitinating (DUB) enzyme that has a catalytic subunit, called Brcc36, that is a member of the JAMM/MPN(+) family of zinc metalloproteases. A notable feature of BRISC is its high specificity for cleaving Lys(63)-linked polyubiquitin. Here, we show that BRISC selectivity is not due to preferential binding to Lys(63)-linked polyubiquitin but is instead dictated by how the substrate isopeptide linkage is oriented within the enzyme active site. BRISC possesses a high affinity binding site for the ubiquitin hydrophobic surface patch that accounts for the bulk of the affinity between enzyme and substrate. Although BRISC can interact with either subunit of a diubiquitin conjugate, substrate cleavage occurs only when BRISC is bound to the hydrophobic patch of the distal (i.e. the "S1") ubiquitin at a ubiquitin-ubiquitin cleavage site. The importance of the Lys(63)-linked proximal (S1') ubiquitin was underscored by our finding that BRISC could not cleave the isopeptide bond joining a ubiquitin to a non-ubiquitin substrate. Finally, we also show that Abro1, another BRISC subunit, binds directly to Brcc36 and that the Brcc36-Abro1 heterodimer includes a minimal complex with Lys(63)-specific DUB activity.
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PMID:Specificity of the BRISC deubiquitinating enzyme is not due to selective binding to Lys63-linked polyubiquitin. 2003 57

DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) degradation involves ubiquitin modification and efficient proteasomal targeting of the nascent misfolded protein. We show that a deubiquitinating enzyme, ubiquitin C-terminal hydrolase-L1 (UCH-L1), is highly expressed in cystic fibrosis (CF) airway epithelial cells in vitro and in vivo. We hypothesized that the elevation in UCH-L1 in CF cells represents a cellular adaptation to counterbalance excessive proteasomal degradation. The bronchial epithelial cell lines IB3-1 (CF, high UCH-L1 expression) and S9 (non-CF, low UCH-L1 expression) were transiently transfected with wild type (WT) or DeltaF508 CFTR, WT UCH-L1 or small interfering RNA-UCH-L1, and a variety of ubiquitin mutants. We observed a positive correlation between UCH-L1 expression and steady state levels of WT- or DeltaF508-CFTR, and this stabilizing effect was confined to the early stages of CFTR synthesis. Immunolocalization of UCH-L1 by confocal microscopy revealed a partial co-localization with a ribosomal subunit and the endoplasmic reticulum. The UCH-L1-associated increase in CFTR levels was correlated with an increase in ubiquitinated CFTR (CFTR-Ub). Co-transfection with mutant ubiquitins and treatment with proteasome inhibitors suggested that UCH-L1 was reducing the proteasomal targeting of CFTR during synthesis by shortening conjugated polyubiquitin chains. Although not sufficient by itself to rescue mutant CFTR therapeutically, the elevation of UCH-L1 and its effect on CFTR processing provides insight into its potential roles in CF and other diseases.
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PMID:Ubiquitin C-terminal hydrolase-L1 protects cystic fibrosis transmembrane conductance regulator from early stages of proteasomal degradation. 2014 97

The ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a deubiquitinating enzyme that catalyses the hydrolysis of polyubiquitin precursors and small ubiquitin adducts. UCH-L1 has been detected in a variety of malignant and metastatic tumours but its biological function in these cells is unknown. We have previously shown that UCH-L1 is highly expressed in Burkitt's lymphoma (BL) and is up-regulated upon infection of B lymphocytes with Epstein-Barr virus (EBV). Here we show that knockdown of UCH-L1 by RNAi inhibits the proliferation of BL cells in suspension and semisolid agar and activates strong LFA-1-dependent homotypic adhesion. Induction of cell adhesion correlated with cation-induced binding to ICAM-1, clustering of LFA-1 into lipid rafts and constitutive activation of the Rap1 and Rac1 GTPases. Expression of a catalytically active UCH-L1 promoted the proliferation of a UCH-L1-negative EBV transformed lymphoblastoid cell line (LCL) and inhibited cell adhesion, whereas a catalytic mutant had no effect, confirming the requirement of UCH-L1 enzymatic activity for the regulation of these phenotypes. Our results identify UCH-L1 as a new player in the signalling pathways that promote the proliferation and invasive capacity of malignant B cells.
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PMID:The ubiquitin C-terminal hydrolase UCH-L1 regulates B-cell proliferation and integrin activation. 2018 92

Abro1 (also known as KIAA0157) is a scaffold protein that recruits polypeptides to assemble the BRISC (BRCC36-containing isopeptidase complex) deubiquitinating (DUB) enzyme. The four subunits of BRISC enzyme include Abro1, NBA1, BRE, and BRCC36 proteins. The DUB activity of the BRISC enzyme is exclusively directed against Lys63-linked polyubiquitin that does not have a proteolytic role but regulates protein function. In this report, we identified Abro1 as a specific interactor of THAP5, a zinc finger transcription factor that is involved in G2/M control and apoptosis. Abro1 was predominantly expressed in the heart and its protein level was regulated following experimentally induced myocardial ischemia/reperfusion (MI/R) injury. Furthermore, in patients with coronary artery disease (CAD), there was a dramatic increase in Abro1 protein level in the myocardial infarction (MI) area. Increase in Abro1 leads to a significant reduction in Lys63-linked ubiquitination of specific protein targets. Reducing the Abro1 protein level exacerbated cellular damage and cell death of cardiomyocytes due to MI/R injury. Additionally, overexpression of Abro1 in a heterologous system provided significant protection against oxidative stress-induced apoptosis. In conclusion, our results demonstrate that Abro1 protein level substantially increases in myocardial injury and coronary artery disease and this up-regulation is part of a novel cardioprotective mechanism. In addition, our data suggest a potential new link between Lys63-specific ubiquitination, its modulation by the BRISC DUB enzyme, and the development and progression of heart disease.
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PMID:Regulation of Abro1/KIAA0157 during myocardial infarction and cell death reveals a novel cardioprotective mechanism for Lys63-specific deubiquitination. 2119 82

Ubiquitin carboxy-terminal hydrolase L5 (UCHL5) is a proteasome-associated deubiquitinating enzyme, which, along with RPN11 and USP14, is known to carry out deubiquitination on proteasome. As a member of the ubiquitin carboxy-terminal hydrolase (UCH) family, UCHL5 is unusual because, unlike UCHL1 and UCHL3, it can process polyubiquitin chain. However, it does so only when it is bound to the proteasome; in its free form, it is capable of releasing only relatively small leaving groups from the C-terminus of ubiquitin. Such a behavior might suggest at least two catalytically distinct forms of the enzyme, an apo form incapable of chain processing activity, and a proteasome-induced activated form capable of cleaving polyubiquitin chain. Through the crystal structure analysis of two truncated constructs representing the catalytic domain (UCH domain) of this enzyme, we were able to visualize a state of this enzyme that we interpret as its inactive form, because the catalytic cysteine appears to be in an unproductive orientation. While this work was in progress, the structure of a different construct representing the UCH domain was reported; however, in that work the structure reported was that of an inactive mutant [catalytic Cys to Ala; Nishio K et al. (2009) Biochem Biophys Res Commun 390, 855-860], which precluded the observation that we are reporting here. Additionally, our structures reveal conformationally dynamic parts of the enzyme that may play a role in the structural transition to the more active form.
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PMID:Crystal structure of the catalytic domain of UCHL5, a proteasome-associated human deubiquitinating enzyme, reveals an unproductive form of the enzyme. 2199 38

Ubiquitin-tagged substrates are degraded by the 26S proteasome, which is a multisubunit complex comprising a proteolytic 20S core particle capped by 19S regulatory particles. The approval of bortezomib for the treatment of multiple myeloma validated the 20S core particle as an anticancer drug target. Here we describe the small molecule b-AP15 as a previously unidentified class of proteasome inhibitor that abrogates the deubiquitinating activity of the 19S regulatory particle. b-AP15 inhibited the activity of two 19S regulatory-particle-associated deubiquitinases, ubiquitin C-terminal hydrolase 5 (UCHL5) and ubiquitin-specific peptidase 14 (USP14), resulting in accumulation of polyubiquitin. b-AP15 induced tumor cell apoptosis that was insensitive to TP53 status and overexpression of the apoptosis inhibitor BCL2. We show that treatment with b-AP15 inhibited tumor progression in four different in vivo solid tumor models and inhibited organ infiltration in an acute myeloid leukemia model. Our results show that the deubiquitinating activity of the 19S regulatory particle is a new anticancer drug target.
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PMID:Inhibition of proteasome deubiquitinating activity as a new cancer therapy. 2221 77

The diverse influences of ubiquitin, mediated by its post-translational covalent modification of other proteins, have been extensively investigated. However, more recently roles for unanchored (nonsubstrate linked) polyubiquitin chains have also been proposed. Here we describe the use of ubiquitin-binding domains to affinity purify endogenous unanchored polyubiquitin chains and their subsequent characterization by mass spectrometry (MS). Using the A20 Znf domain of the ubiquitin receptor ZNF216 we isolated a protein from skeletal muscle shown by a combination of nanoLC-MS and LC-MS/MS to represent an unmodified and unanchored K48-linked ubiquitin dimer. Selective purification of unanchored polyubiquitin chains using the Znf UBP (BUZ) domain of USP5/isopeptidase-T allowed the isolation of K48 and K11-linked ubiquitin dimers, as well as revealing longer chains containing as many as 15 ubiquitin moieties, which include the K48 linkage. Top-down nanoLC-MS/MS of the A20 Znf-purified ubiquitin dimer generated diagnostic ions consistent with the presence of the K48 linkage, illustrating for the first time the potential of this approach to probe connectivity within endogenous polyubiquitin modifications. As well as providing initial proteomic insights into the molecular composition of endogenous unanchored polyubiquitin chains, this work also represents the first definition of polyubiquitin chain length in vivo.
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PMID:Insights into the molecular composition of endogenous unanchored polyubiquitin chains. 2226 64

Human ubiquitin-specific cysteine protease 5 (USP5, also known as ISOT and isopeptidase T), an 835-residue multidomain enzyme, recycles ubiquitin by hydrolyzing isopeptide bonds in a variety of unanchored polyubiquitin substrates. Activation of the enzyme's hydrolytic activity toward ubiquitin-AMC (7-amino-4-methylcoumarin), a fluorogenic substrate, by the addition of free, unanchored monoubiquitin suggested an allosteric mechanism of activation by the ZnF-UBP domain (residues 163-291), which binds the substrate's unanchored diglycine carboxyl tail. By determining the structure of full-length USP5, we discovered the existence of a cryptic ZnF-UBP domain (residues 1-156), which was tightly bound to the catalytic core and was indispensable for catalytic activity. In contrast, the previously characterized ZnF-UBP domain did not contribute directly to the active site; a paucity of interactions suggested flexibility between these two domains consistent with an ability by the enzyme to hydrolyze a variety of different polyubiquitin chain linkages. Deletion of the known ZnF-UBP domain did not significantly affect rate of hydrolysis of ubiquitin-AMC and suggested that it is likely associated mainly with substrate targeting and specificity. Together, our findings show that USP5 uses multiple ZnF-UBP domains for substrate targeting and core catalytic function.
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PMID:Two ZnF-UBP domains in isopeptidase T (USP5). 2228 93


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