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: EC:6.3.2.19 (ubiquitin-protein ligase)
799 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ubiquitin-mediated proteolysis of securin and mitotic cyclins is essential for exit from mitosis. The final step in ubiquitination of these and other proteins is catalysed by the anaphase-promoting complex (APC), a multi-subunit ubiquitin-protein ligase (E3). Little is known about the molecular reaction resulting in APC-dependent substrate ubiquitination or the role of individual APC subunits in the reaction. Using a well-defined in vitro system, we show that highly purified APC from Saccharomyces cerevisiae ubiquitinates a model cyclin substrate in a processive manner. Analysis of mutant APC lacking the Doc1/Apc10 subunit (APC(doc1 Delta)) indicates that Doc1 is required for processivity. The specific molecular defect in APC(doc1 Delta) is identified by a large increase in apparent K(M) for the cyclin substrate relative to the wild-type enzyme. This suggests that Doc1 stimulates processivity by limiting substrate dissociation. Addition of recombinant Doc1 to APC(doc1 Delta) fully restores enzyme function. Doc1-related domains are found in mechanistically distinct ubiquitin-ligase enzymes and may generally stimulate ubiquitination by contributing to substrate-enzyme affinity.
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PMID:The Doc1 subunit is a processivity factor for the anaphase-promoting complex. 1240 45

Ubiquitin-mediated proteolysis of cell cycle regulators is a major element of the cell cycle control. The anaphase-promoting complex (APC/C) is a large multisubunit ubiquitin-protein ligase required for the ubiquitination and degradation of G1 and mitotic checkpoint regulators. APC/C-dependent proteolysis regulates cyclin levels in G1, and triggers the separation of sister chromatids at the metaphase-anaphase transition and the destruction of mitotic cyclins at the end of mitosis. Furthermore, it was recently shown that APC/C regulates the degradation of crucial regulators of signal transduction pathways. We report here gene alterations in several components of this complex in human colon cancer cells, including APC6/CDC16 and APC8/CDC23 which are known to be key function elements. The experimental expression of a truncation mutant of APC8/CDC23 subunit (CDC23DeltaTPR) leads to abnormal levels of APC/C targets such as cyclin B1 and disturbs the cell cycle progression of colon epithelial cells through mitosis. Overall, these data support the hypothesis of a deleterious role of these mutations during colorectal carcinogenesis.
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PMID:Alterations of anaphase-promoting complex genes in human colon cancer cells. 1262 11

Ubiquitin-protein ligases (E3s) determine the substrate specificity of ubiquitylation and, until recently, had been classified into two families, the HECT and RING-finger families. The U-box is a domain of approximately 70 amino acids that is present in proteins from yeast to humans. The prototype U-box protein, yeast Ufd2, was identified as a ubiquitin chain assembly factor (E4) that cooperates with a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and an E3 to catalyze the formation of a ubiquitin chain on artificial substrates. We recently showed that mammalian U-box proteins, in conjunction with an E1 and an E2, mediate polyubiquitylation in the absence of a HECT type or RING-finger type E3. U-box proteins have thus been defined as a third family of E3s. We here review recent progress in the characterization of U-box proteins and of their role in the quality control system that underlies the cellular stress response to the intracellular accumulation of abnormal proteins.
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PMID:U-box proteins as a new family of ubiquitin ligases. 1264 16

We previously showed that lens epithelial cells have a fully functional ubiquitin-proteasome pathway (UPP) and that ubiquitin-conjugating activity is up-regulated in response to oxidative stress. In this study we assessed the protein levels and activities of different components of the UPP in lens fibers. Calf lenses were dissected into four different regions: epithelial layer, outer cortex, inner cortex and nucleus. Relative levels of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (E2s), endogenous ubiquitin conjugates, 19S and 20S proteasome subunits were determined by Western blotting. The activities of E1 and E2 were determined by thiol ester assays and the activities of the proteasome and isopeptidases were determined using ubiquitinated alpha-lactalbumin as a substrate. This work demonstrates that lens fibers, including those in the nuclear region, contain most, if not all, of the components for the UPP. Ubiquitin conjugation activity, proteasome activity and isopeptidase activity were also detected in all layers of the lens. The reduced ubiquitin conjugation activity in the inner regions of the lens appeared to be due to a decline in levels of a specific family of E2s, Ubc4 or Ubc5, which were shown to be the rate-limiting enzymes for the formation of high mass conjugates in the lens. Supplementation of Ubc4 or Ubc5 can partially restore the ubiquitin conjugation activity in the inner regions of the lens. Since Ubc4 and Ubc5 are involved in selectively ubiquitinating damaged or abnormal proteins, the decline in levels and activities of these E2s may be responsible for the accumulation of abnormal proteins in inner regions of the lens.
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PMID:Lens fibers have a fully functional ubiquitin-proteasome pathway. 1269 26

Procaspase-3 (p32) is processed by upstream caspases to p12 and p20 subunits, which heterodimerize. Concomitant with formation of the active heterotetramer, p20 is autoprocessed to p17. Treatment of HL-60 cells with lactacystin, a selective inhibitor of the proteasome, exponentially increased caspase-3-like hydrolytic activity and induced apoptosis but had little or no effect on the activity of upstream caspase-8, caspase-9, or granzyme B. Lactacystin treatment decreased the p32 zymogen and evoked the accumulation of the p17 and p12 subunits. Treatment of transfected human retinoblast 911 cells with a proteasome inhibitor evoked the accumulation of epitope-tagged p12, p17, and p20 but had no effect on p32 zymogen. This result suggests that caspase-3 subunits, in contrast to the zymogen, are unstable because of degradation by the ubiquitin-proteasome system. Ubiquitin conjugates of p12 and p17 accumulated in cells that were cotransfected with p12 and a caspase inactive mutant of p17. Substitution of arginine for all eight lysines of p12 almost abolished its ubiquitination. Any single lysine or lysine pair was sufficient for p12 ubiquitination. Lactacystin treatment of HL-60 cells induced proteolytic processing of the X-linked inhibitor of apoptosis (XIAP) and decreased full-length XIAP, which is known to have ubiquitin-protein ligase activity for active caspase-3. These findings indicate that caspase-3 subunits can be degraded by the ubiquitin-proteasome system and suggest that lactacystin induces apoptosis in part by disabling the ubiquitin-protein ligase function of XIAP and by stabilizing active caspase-3 subunits.
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PMID:Preservation of caspase-3 subunits from degradation contributes to apoptosis evoked by lactacystin: any single lysine or lysine pair of the small subunit is sufficient for ubiquitination. 1286 38

Quality control of intracellular proteins is essential for cellular homeostasis. Molecular chaperones recognize and contribute to the refolding of misfolded or unfolded proteins, whereas the ubiquitin-proteasome system mediates the degradation of such abnormal proteins. Ubiquitin-protein ligases (E3s) determine the substrate specificity for ubiquitylation and have been classified into HECT and RING-finger families. More recently, however, U-box proteins, which contain a domain (the U box) of about 70 amino acids that is conserved from yeast to humans, have been identified as a new type of E3. The prototype U-box protein, yeast Ufd2, was identified as a ubiquitin chain assembly factor (E4) that cooperates with a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and an E3 to catalyze the formation of a ubiquitin chain on artificial substrates. Yeast Ufd2 is functionally implicated in cell survival under stressful conditions. This review addresses recent progress in characterization of the role of E3 enzymes, especially that of U-box proteins, in quality control of intracellular proteins.
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PMID:Ubiquitylation as a quality control system for intracellular proteins. 1294 64

In skeletal muscle, as in any mammalian tissue, protein levels are dictated by relative rates of protein synthesis and breakdown. Recent studies have shown that the ubiquitin-proteasome-dependent proteolytic pathway is mainly responsible for the breakdown of myofibrillar proteins. In this pathway proteins that are to be degraded are first tagged with a polyubiquitin degradation signal. Ubiquitination is performed by the ubiquitin-activating enzyme, ubiquitin-conjugating enzymes and ubiquitin-protein ligases, which are responsible for the recognition of specific substrates. Polyubiquitinated protein substrates are then specifically recognised and degraded by the 26S proteasome. The present review focuses on: (1) the mechanisms of ubiquitination-deubiquitination that make the system highly selective; (2) the mechanisms of proteolysis in skeletal muscle. In particular, the role of the system in the remodelling of skeletal muscle during exercise and disuse and in recovery or regeneration that prevails during post-atrophic conditions is reviewed.
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PMID:The role of ubiquitin-proteasome-dependent proteolysis in the remodelling of skeletal muscle. 1529 55

Modification of cellular proteins with a small protein called ubiquitin has profound effects on their activities. Ubiquitin is covalently attached to lysine residues of acceptor proteins through the concerted action of E1 ubiquitin-activating enzyme, E2 ubiquitin-carrier proteins and E3 ligases. Mammalian cells contain a large number of E3 ligases, which determine the specificity of ubiquitination reactions. Recent studies have revealed that ubiquitination can be reversed by deubiquitinating enzymes that release ubiquitin monomers from modified proteins. Signalling networks that control inflammation are tightly regulated by a multitude of ubiquitination and deubiquitination reactions. This article begins by summarising current understanding of these pathways at a molecular level, and then focuses on the importance of ubiquitination and deubiquitination during the regulation of the pro-inflammatory transcription factor NF-kappaB. Finally, the potential for ubiquitin modifications to be targeted by novel classes of anti-inflammatory drugs is discussed.
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PMID:Regulation of pro-inflammatory signalling networks by ubiquitin: identification of novel targets for anti-inflammatory drugs. 1596 57

Selective protein degradation by the 26 S proteasome usually requires a polyubiquitin chain attached to the protein substrate by three classes of enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). This reaction can produce different polyubiquitin chains that, depending on size and linkage type, can provide distinct intracellular signals. Interestingly, polyubiquitination is sometimes regulated by additional conjugation factors, called E4s (polyubiquitin chain conjugation factors). Yeast UFD2 (ubiquitin fusion degradation protein-2), the first E4 to be described, binds to the ubiquitin moieties of preformed conjugates and catalyses ubiquitin-chain elongation together with E1, E2, and E3. Recent studies have illustrated that the E4 enzyme UFD2 co-operates with an orchestra of ubiquitin-binding factors in an escort pathway to transfer and deliver polyubiquitinated substrates to the 26 S proteasome. Here we propose a model in which E4-dependent polyubiquitination pathways are modulated by different ubiquitin-binding proteins, using ataxin-3 as an example.
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PMID:Orchestra for assembly and fate of polyubiquitin chains. 1625 Aug 94

Ubiquitin-conjugating enzymes (E2s) collaborate with the ubiquitin-activating enzyme (E1) and ubiquitin ligases (E3s) to attach ubiquitin to target proteins. RING-containing E3s simultaneously bind to E2s and substrates, bringing them into close proximity and thus facilitating ubiquitination. We show herein that, although the E3-binding site on the human E2 UbcH5b is distant from its active site, two RING-type minimal E3 modules lacking substrate-binding functions greatly stimulate the rate of ubiquitin release from the UbcH5b-ubiquitin thioester. Using statistical coupling analysis and mutagenesis, we identify and characterize clusters of coevolving and functionally linked residues within UbcH5b that span its E3-binding and active sites. Several UbcH5b mutants are defective in their stimulation by E3s despite their abilities to bind to these E3s, to form ubiquitin thioesters, and to release ubiquitin at a basal rate. One such mutation, I37A, is distant from both the active site and the E3-binding site of UbcH5b. Our studies reveal structural determinants for communication between distal functional sites of E2s and suggest that RING-type E3s activate E2s allosterically.
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PMID:Mechanistic insight into the allosteric activation of a ubiquitin-conjugating enzyme by RING-type ubiquitin ligases. 1636 95


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