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)

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

Dynamin is a GTP-binding protein whose oligomerization-dependent assembly around the necks of lipid vesicles mediates their scission from parent membranes. Dynamin is thus directly involved in the regulation of endocytosis. Sumoylation is a post-translational protein modification whereby the ubiquitin-like modifier Sumo is covalently attached to lysine residues on target proteins by a process requiring the concerted action of an activating enzyme (ubiquitin-activating enzyme), a conjugating enzyme (ubiquitin carrier protein), and a ligating enzyme (ubiquitin-protein isopeptide ligase). Here, we show that dynamin interacts with Sumo-1, Ubc9, and PIAS-1, all of which are members of the sumoylation machinery. Ubc9 and PIAS-1 are known ubiquitin carrier protein and ubiquitin-protein isopeptide ligase enzymes, respectively, for the process of sumoylation. We have identified the coiled-coil GTPase effector domain (GED) of dynamin as the site on dynamin that interacts with Sumo-1, Ubc9, and PIAS-1. Although we saw no evidence of covalent Sumo-1 attachment to dynamin, Sumo-1 and Ubc9 are shown here to inhibit the lipid-dependent oligomerization of dynamin. Expression of Sumo-1 and Ubc9 in mammalian cells down-regulated the dynamin-mediated endocytosis of transferrin, whereas dynamin-independent fluid-phase uptake was not affected. Furthermore, using high resolution NMR spectroscopy, we have identified amino acid residues on Sumo-1 that directly interact with the GED of dynamin. The results suggest that the GED of dynamin may serve as a scaffold that concentrates the sumoylation machinery in the vicinity of potential acceptor proteins.
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PMID:Dynamin interacts with members of the sumoylation machinery. 1512 15

The degradation of ethanol-inducible cytochrome P450 2E1 (CYP2E1) and phenobarbital-inducible cytochrome P450 2B1 (CYP2B1) expressed in tetracycline (Tc)-inducible HeLa cell lines was characterized. A steady-state pulse-chase analysis was used to determine a half-life of 3.8 h for CYP2E1 while the half-life of CYP2B1 was 2.3-fold greater in the same cell line. In contrast, NADPH cytochrome P450 reductase which is constitutively expressed in Tc-HeLa cells had a half-life of about 30 h. Lactacystin and other selective proteasome inhibitors including N-benzyloxycarbonyl-leucyl-leucyl-leucinal (MG132) and N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-norvalinal (MG115) significantly inhibited both CYP2E1 and CYP2B1 degradation. The turnover of CYP2E1 was slightly inhibited by calpain inhibitors while CYP2B1 turnover was not altered. Inhibitors of lysosomal proteolysis had no effect on the degradation of either protein. Treatment of cells with brefeldin A did not alter the degradation of either P450 which suggested the degradation occurred in the endoplasmic reticulum (ER). Even in the presence of proteasome inhibitors high molecular weight ubiquitin conjugates were not observed. Mutagenesis of two putative ubiquitination sites (Lys 317 and 324) did not alter the degradation of CYP2E1. The role of ubiquitination in the degradation of CYP2E1 was also examined in a Chinese hamster mutant cell line E36ts20 that contains a thermolabile ubiquitin-activating enzyme (E1). The turnover of CYP2E1 was not significantly different at the nonpermissive temperature in the ts20 when compared to the control E36 cells. Furthermore, the addition of the hsp90 inhibitors geldanamycin, herbimycin, and radicicol had no effect on the turnover of CYP2E1, differentiating the degradation of CYP2E1 from other substrates for proteasome-dependent degradation.
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PMID:Proteasome-dependent degradation of cytochromes P450 2E1 and 2B1 expressed in tetracycline-regulated HeLa cells. 1536 48

RING domains are found in a large number of eukaryotic proteins. Most function as E3 ubiquitin-protein ligases, catalyzing the terminal step in the ubiquitination process. Structurally, these domains have been characterized as binding two zinc ions in a stable cross-brace motif. The tumorigenic human gamma-herpesvirus Kaposi's sarcoma-associated herpesvirus encodes a ubiquitin-protein ligase termed K3, which functions as an immune evasion molecule by ubiquitinating major histocompatibility complex class I. K3 possesses at its N terminus a domain related to cellular RING domains but with an altered zinc ligand arrangement. This domain was initially characterized as a plant homeodomain, a structure not previously known to function as an E3. Here, it is conclusively demonstrated that the K3 N-terminal domain is a variant member of the RING domain family and not a plant homeodomain. The domain is found to interact with the cellular ubiquitin-conjugating enzymes UbcH5A to -C and UbcH13, which dock to the equivalent surface as on classical cellular RING domains. Interaction with UbcH13 suggests a possible role for K3 in catalyzing Lys(63)-linked ubiquitination.
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PMID:Solution structure of the Kaposi's sarcoma-associated herpesvirus K3 N-terminal domain reveals a Novel E2-binding C4HC3-type RING domain. 1546 11

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

Conjugation of ubiquitin and other Class 1 ubiquitin-like polypeptides to specific protein targets serves diverse regulatory functions in eukaryotes. The obligatory first step of conjugation requires ATP-coupled activation of the ubiquitin-like protein by members of a superfamily of evolutionarily related enzymes. Kinetic and equilibrium studies of the human ubiquitin-activating enzyme (HsUba1a) reveal that mutations within the ATP.Mg(2+) binding site have remarkably pleiotropic effects on the catalytic phenotype of the enzyme. Mutation of Asp(576) or Lys(528) results in dramatically impaired binding affinities for ATP.Mg(2+), a shift from ordered to random addition in co-substrate binding, and a significantly reduced rate of ternary complex formation that shifts the rate-limiting step to ubiquitin adenylate formation. Mutations at neither position affect the affinity of HsUbc2b binding; however, differences in k(cat) values determined from ternary complex formation versus HsUbc2b transthiolation suggest that binding of the E2 enhances the rate of bound ubiquitin adenylate formation. These results confirm that Asp(576) and Lys(528) are important for ATP.Mg(2+) binding but are essential catalytic groups for ubiquitin adenylate transition state stabilization. The latter mechanistic effect explicates the observed loss-of-function phenotype associated with mutation of residues paralogous to Asp(576) within the activating enzymes for other ubiquitin-like proteins.
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PMID:Pleiotropic effects of ATP.Mg2+ binding in the catalytic cycle of ubiquitin-activating enzyme. 1659 81

We have developed a system for producing biotinylated recombinant proteins in mammalian cells. The expression construct consists of an inducible tetracycline response element (TRE) that drives expression of a bicistronic cassette comprising a biotin acceptor peptide (BioTag) fused to either terminus of the target protein, the gene for Escherichia coli biotin ligase (BirA), and an intervening internal ribosome entry site (IRES). By either transient or stable transfection of Chinese hamster ovary (CHO) Tet-On cells, we successfully expressed, detected, and immobilized biotinylated human Itch, a pleiotropic multi-domain ubiquitin-protein ligase, as well as Gla-RTK, a putative vitamin K-dependent receptor tyrosine kinase. The biotinylation of recombinant Itch in transiently transfected CHO Tet-On cells required biotin supplementation and coexpression of BirA, occurred quantitatively and specifically on the lysine residue of the BioTag, and enabled detection of Itch by Western blot in as little as 10ng of total lysate protein. Stably selected clones were rapidly pre-screened for doxycycline (dox)-inducible BirA expression by ELISA, and subsequently screened for dox-inducible expression of biotinylated Itch. Biotinylated Gla-RTK was detectable in as little as 5ng of total lysate protein from transiently transfected CHO Tet-On cells, and exhibited pronounced tyrosine phosphorylation. In stable clones however, constitutive phosphorylation was prevented by reducing the expression level of Gla-RTK through the titration of dox. These results demonstrate the utility of this system for the expression of 'difficult' proteins, particularly those that are cytotoxic or those that may require lower expression levels to ensure appropriate post-translational modification.
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PMID:A versatile system for site-specific enzymatic biotinylation and regulated expression of proteins in cultured mammalian cells. 1708 93

Proper transcription by RNA polymerase II is dependent on the modification state of the chromatin template. The Paf1 complex is associated with RNA polymerase II during transcription elongation and is required for several histone modifications that mark active genes. To uncover additional factors that regulate chromatin or transcription, we performed a genetic screen for mutations that cause lethality in the absence of the Paf1 complex component Rtf1. Our results have led to the discovery of a previously unstudied gene, RKR1. Strains lacking RKR1 exhibit phenotypes associated with defects in transcription and chromatin function. These phenotypes include inositol auxotrophy, impaired telomeric silencing, and synthetic lethality with mutations in SPT10, a gene that encodes a putative histone acetyltransferase. In addition, deletion of RKR1 causes severe genetic interactions with mutations that prevent histone H2B lysine 123 ubiquitylation or histone H3 lysine 4 methylation. RKR1 encodes a conserved nuclear protein with a functionally important RING domain at its carboxy terminus. In vitro experiments indicate that Rkr1 possesses ubiquitin-protein ligase activity. Taken together, our results identify a new participant in a protein ubiquitylation pathway within the nucleus that acts to modulate chromatin function and transcription.
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PMID:Identification of Rkr1, a nuclear RING domain protein with functional connections to chromatin modification in Saccharomyces cerevisiae. 1728 62

The conjugation of proteins with ubiquitin plays numerous regulatory roles through both proteasomal-dependent and nonproteasomal-dependent functions. Alterations in ubiquitylation are observed in a wide range of pathologic conditions, including numerous malignancies. For this reason, there is great interest in targeting the ubiquitin-proteasome system in cancer. Several classes of proteasome inhibitors, which block degradation of ubiquitylated proteins, are widely used in research, and one, Bortezomib, is now in clinical use. Despite the well-defined and central role of the ubiquitin-activating enzyme (E1), no cell permeable inhibitors of E1 have been identified. Such inhibitors should, in principle, block all functions of ubiquitylation. We now report 4[4-(5-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester (PYR-41) as the first such inhibitor. Unexpectedly, in addition to blocking ubiquitylation, PYR-41 increased total sumoylation in cells. The molecular basis for this is unknown; however, increased sumoylation was also observed in cells harboring temperature-sensitive E1. Functionally, PYR-41 attenuates cytokine-mediated nuclear factor-kappaB activation. This correlates with inhibition of nonproteasomal (Lys-63) ubiquitylation of TRAF6, which is essential to IkappaB kinase activation. PYR-41 also prevents the downstream ubiquitylation and proteasomal degradation of IkappaBalpha. Furthermore, PYR-41 inhibits degradation of p53 and activates the transcriptional activity of this tumor suppressor. Consistent with this, it differentially kills transformed p53-expressing cells. Thus, PYR-41 and related pyrazones provide proof of principle for the capacity to differentially kill transformed cells, suggesting the potential for E1 inhibitors as therapeutics in cancer. These inhibitors can also be valuable tools for studying ubiquitylation.
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PMID:Inhibitors of ubiquitin-activating enzyme (E1), a new class of potential cancer therapeutics. 1790 57

Hepatitis C virus (HCV) F protein is encoded by the +1 reading frame of the viral genome. It overlaps with the core protein coding sequence, and multiple mechanisms for its expression have been proposed. The full-length F protein that is synthesized by translational ribosomal frameshift at codons 9 to 11 of the core protein sequence is a labile protein. By using a combination of genetic, biochemical, and cell biological approaches, we demonstrate that this HCV F protein can bind to the proteasome subunit protein alpha3, which reduces the F-protein level in cells in a dose-dependent manner. Deletion-mapping analysis identified amino acids 40 to 60 of the F protein as the alpha3-binding domain. This alpha3-binding domain of the F protein together with its upstream sequence could significantly destabilize the green fluorescent protein, an otherwise stable protein. Further analyses using an F-protein mutant lacking lysine and a cell line that contained a temperature-sensitive E1 ubiquitin-activating enzyme indicated that the degradation of the F protein was ubiquitin independent. Based on these observations as well as the observation that the F protein could be degraded directly by the 20S proteasome in vitro, we propose that the full-length HCV F protein as well as the F protein initiating from codon 26 is degraded by an ubiquitin-independent pathway that is mediated by the proteasome subunit alpha3. The ability of the F protein to bind to alpha3 raises the possibility that the HCV F protein may regulate protein degradation in cells.
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PMID:Ubiquitin-independent degradation of hepatitis C virus F protein. 1897 Dec 67


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