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)

The ubiquitin-proteasome pathway (UPP) regulates critical cell processes, including the cell cycle, cytokine-induced gene expression, differentiation, and cell death. Recently we demonstrated that this pathway responds to oxidative stress in mammalian cells and proposed that activities of ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes (E2s) are regulated by cellular redox status (i.e., GSSG:GSH ratio). To test this hypothesis, we altered the GSSG:GSH ratio in retinal pigment epithelial cells with the thiol-specific oxidant, diamide, and assessed activities of the UPP. Treatment of cells with diamide resulted in a dose-dependent increase in the GSSG:GSH ratio resulting from loss of GSH and a coincident increase in GSSG. Increases in the GSSG:GSH ratio from 0.02 in untreated cells to > or = 0.5 in diamide-treated cells were accompanied by dose-dependent reductions in the levels of endogenous Ub-protein conjugates, endogenous E1-ubiquitin thiol esters, and de novo ubiquitin-conjugating activity. As determined by the ability to form E1-ubiquitin and E2s-ubiquitin thiol esters, E1 and E2s were both inhibited by elevated GSSG:GSH ratios. Inhibition of E1 was associated with the formation of E1-protein mixed disulfides. Activities of E1 and E2s gradually recovered to preoxidation levels, coincident with gradual recovery of the GSSG:GSH ratio. These data support S-thiolation/dethiolation as a mechanism regulating E1 and E2 activities in response to oxidant insult. Ubiquitin-dependent proteolytic capacity was regulated by the GSSG:GSH ratio in a manner consistent with altered ubiquitin-conjugating activity. However, ubiquitin-independent proteolysis was unaffected by changes in the GSSG:GSH ratio. Potential adaptive and pathological consequences of redox regulation of UPP activities are discussed.
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PMID:Redox regulation of ubiquitin-conjugating enzymes: mechanistic insights using the thiol-specific oxidant diamide. 957 83

Ubiquitin is often implicated as a specific tag for protein degradation via the ubiquitin system although only a limited number of physiological proteins have been shown to be degraded in their native tissues via this pathway in vivo. Ubiquitin may also, however, have other functions of a regulatory nature (non-catabolic ubiquitylation). The ubiquitylation of calmodulin appears to fall into this category. Ubiquitin is linked to free calmodulin in the presence of the second messenger Ca2+ by the enzyme ubiquitin-calmodulin ligase (uCaM synthetase: EC 6.3.2.21) and there is no evidence that this step is followed by degradation of calmodulin via the ATP-dependent 26-S protease. Due to a lack of natural substrates and sufficient tissue material, only a few components of the ubiquitin system have been obtained in truly homogeneous form from reticulocytes. We therefore decided to attempt this for the calmodulin ligase. The enzymic components of the uCaM synthetase system copurified over several steps and could be highly enriched by a novel sample displacement technique on an ion-exchange resin. A fractionation of the synthetase components by affinity chromatography on ubiquitin-Sepharose and calmodulin-Sepharose yielded two essentially inactive components: a ubiquitin-Sepharose binding fraction (uCaM Syn-F1) and a calmodulin-Sepharose binding fraction (uCaM Syn-F2). The full activity of uCaM synthetase can be reconstituted when these two fractions are reunited. uCaM Syn-F1 could then be separated from all other enzymes of ubiquitin metabolism and, employing the second component with the natural substrate calmodulin, could be purified over 3500-fold to homogeneity. The ability to catalyze its own thiol labile ubiquitylation identified it as a member of the ubiquitin-activating enzyme family (E1). The homogeneous preparation contained a single protein of molecular mass 213 +/- 21 kDa (mean +/- SEM) as determined by gel filtration. The molecular mass of the monomer was determined by electrospray ion mass spectrometry to 112,140 +/- 47 Da (mean +/- SD). N-terminal sequence analysis (20 amino acids) led to a single N-terminal peptide beginning at residue 57 of the known rabbit cDNA sequence. No ragged N-terminus was detected, as would be expected by the action of an aminopeptidase or other peptidases of low specificity. The monomer molecular mass calculated from the cDNA sequence (Arg57-Arg1058) is 111,975 Da, characterizing this enzyme from reticulocytes as a homodimer of 224 kDa.
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PMID:The ubiquityl-calmodulin synthetase system from rabbit reticulocytes: isolation of the ubiquitin-binding first component, a ubiquitin-activating enzyme. 971 91

Ubiquitin-calmodulin ligase (uCaM synthetase: EC 6.3.2.21), which has been detected in all tissues so far examined, catalyzes the Ca2+-dependent reversible synthesis of ubiquityl-calmodulin which is not directed to degradation by the ATP-dependent 26-S protease [Laub, M. & Jennissen, H. P. (1997) Biochim. Biophys. Acta 1357, 173-191]. As has been shown in the preceding paper in this journal, the uCaM synthetase holosystem can be separated into two essential protein components: uCaM Syn-F1, a ubiquitin-binding protein belonging to the ubiquitin-activating enzyme family (E1) and uCaM Syn-F2 which bestows the reaction specificity leading to the covalent modification of calmodulin with ubiquitin. UCaM Syn-F2, which binds to calmodulin-Sepharose in a Ca2+-dependent manner, has been purified over 3500-fold in seven steps from rabbit reticulocytes and has a native molecular mass of approximately 620 kDa. It binds calmodulin with a Km of 5 microM and to uCaM Syn-F1, i.e. ubiquitin-activating enzyme (E1), with a Km of 3 nM. The maximal specific activity obtained in enriched uCaM Syn-F2 is 6-8 pkat/mg. The pH optimum of uCaM synthetase lies at pH 8.5. In kinetic experiments the Km values for 125I-ubiquitin and ATP/Mg2+ were determined to be 8 microM and 16 nM, respectively, for the uCaM synthetase holosystem. The existence of a third separable protein component of uCaM synthetase, as is the case in E1, E2, E3 systems, is very unlikely since affinity chromatography on calmodulin-Sepharose, two ion-exchange chromatography steps and finally a gel-filtration step failed to indicate any additional protein component essential for synthetase activity. We therefore propose a two-component model for uCaM synthetase. This model is also supported by simple hyperbolic velocity curves in kinetic experiments based on the variation of these two components. The data suggests that uCaM Syn-F2 is neither an E2 nor an E3 but evidently combines the properties of both, making the Ca2+-dependent uCaM synthetase the member of a group of two-component ubiquitin ligase systems.
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PMID:The ubiquityl-calmodulin synthetase system from rabbit reticulocytes: isolation of the calmodulin-binding second component and enzymatic properties. 971 92

Ubiquitin-conjugating enzymes (Ubc) are involved in ubiquitination of proteins in the protein degradation pathway of eukaryotic cells. Ubc transfers the ubiquitin (Ub) molecules to target proteins by forming a thioester bond between their active site cysteine residue and the C-terminal glycine residue of ubiquitin. Here, we report on the NMR assignment and secondary structure of class I human ubiquitin-conjugating enzyme 2b (HsUbc2b). Chemical shift perturbation studies allowed us to map the contact area and binding interface between ubiquitin and HsUbc2b by1H-15N HSQC NMR spectroscopy. The serine mutant of the active site Cys88 of HsUbc2b was employed to obtain a relatively stable covalent ubiquitin complex of HsUbc2b(C88S). Changes in chemical shifts of amide protons and nitrogen atoms induced by the formation of the covalent complex were measured by preparing two segmentally labeled complexes with either ubiquitin or HsUbc2b(C88S)15N-labeled. In ubiquitin, the interaction is primarily sensed by the C-terminal segment Val70 - Gly76, and residues Lys48 and Gln49. The surface area on ubiquitin, as defined by these residues, overlaps partially with the presumed binding site with ubiquitin-activating enzyme (E1). In HsUbc2b, most of the affected residues cluster in the vicinity of the active site, namely, around the active site Cys88 itself, the second alpha-helix, and the flexible loop which connects helices alpha2 and alpha3 and which is adjacent to the active site. An additional site on HsUbc2b for a weak interaction with ubiquitin could be detected in a titration study where the two proteins were not covalently linked. This site is located on the backside of HsUbc2b opposite to the active site and is part of the beta-sheet. The covalent and non-covalent interaction sites are clearly separated on the HsUbc2b surface, while no such clear-cut segregation of the interaction area was observed on ubiquitin.
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PMID:Characterization of the binding interface between ubiquitin and class I human ubiquitin-conjugating enzyme 2b by multidimensional heteronuclear NMR spectroscopy in solution. 1038 68

Ubiquitination of receptor protein-tyrosine kinases (RPTKs) terminates signaling by marking active receptors for degradation. c-Cbl, an adapter protein for RPTKs, positively regulates RPTK ubiquitination in a manner dependent on its variant SRC homology 2 (SH2) and RING finger domains. Ubiquitin-protein ligases (or E3s) are the components of ubiquitination pathways that recognize target substrates and promote their ligation to ubiquitin. The c-Cbl protein acted as an E3 that can recognize tyrosine-phosphorylated substrates, such as the activated platelet-derived growth factor receptor, through its SH2 domain and that recruits and allosterically activates an E2 ubiquitin-conjugating enzyme through its RING domain. These results reveal an SH2-containing protein that functions as a ubiquitin-protein ligase and thus provide a distinct mechanism for substrate targeting in the ubiquitin system.
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PMID:The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase. 1057 87

Alzheimer's disease (AD) is characterized by the presence of neurofibrillary tangles (NFT), senile plaques, and cerebrovascular deposits of amyloid-beta. Ubiquitin has also been shown to be present in some of the inclusions characteristic of this disease. To obtain further insight into the role played by the ubiquitin pathway in AD, we investigated the capacity of postmortem samples of cerebral cortex from normal and AD patients to form high-molecular-weight ubiquitin-protein conjugates. Activity of the ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes (E2) involved in the ubiquitin pathway was also determined. In normal samples, the amount of high-molecular-weight ubiquitin-protein conjugates (HMW-UbPC) in cytosol increased with incubation time, whereas, in samples of AD cases, these were almost undetectable. The addition of an adult rat fraction, enriched in ubiquitinating enzymes, restored the capacity of AD brain cytosolic fraction to form conjugates. The trypsin-like proteolytic activity of the 26S proteasome was found to be decreased in AD cytosol brain. Assay of the activity of E1 and E2 by thiol-ester formation revealed a significant decrease in AD samples. Moreover, Western blotting using a specific antibody against E1 showed a dramatic drop of this enzyme in the cytosolic fraction, whereas normal levels were found in the particulate fraction, suggesting a possible delocalization of the enzyme. Our results suggest that a failure in the ubiquitination enzymatic system in brain cytosol may contribute to fibrillar pathology in AD.
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PMID:Defective ubiquitination of cerebral proteins in Alzheimer's disease. 1102 Feb 23

Selective protein degradation by the ubiquitin-proteosome pathway has recently emerged as a powerful regulatory mechanism in a wide variety of cellular processes. Ubiquitin conjugation requires the sequential activity of three enzymes or protein complexes called the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2), and the ubiquitin-protein ligase (E3). In most eukaryotes, there are a small number of similar E1 isoforms without apparent functional specificity. The specific selection of target proteins is accomplished by the E2 and E3 proteins. One of the best-characterized families of E3s are the SCF complexes. The SCF is composed of a cullin (Cdc53), SKP1, RBX1 and one member of a large family of proteins called F-box proteins. The function of the F-box protein is to interact with target proteins. In some cases, the stability of the F-box protein may regulate activity of the SCF complex. In addition, post-translational modification of the cullin subunit by the ubiquitin-like protein RUB/NEDD8 appears to regulate SCF function. In plants, the SCF has so far been implicated in floral development, circadian clock, and response to the plant growth regulators auxin and jasmonic acid.
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PMID:F-box proteins and protein degradation: an emerging theme in cellular regulation. 1111 56

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


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