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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)

We have constructed interspecific somatic cell hybrids between a temperature-sensitive (ts) mutant cell line of mouse FM3A cells, ts85, that has a heat-labile ubiquitin-activating enzyme (E1) and a human diploid fibroblast cell line, IMR-90. A hybrid clone that could grow stably at a nonpermissive temperature (39 degrees C) was obtained. Segregation of the hybrid cells at a permissive temperature (33 degrees C) gave rise to temperature-sensitive clones. The electrophoresis of extracted histones and karyotype analysis of the segregants revealed a close correlation of the ability to grow at 39 degrees C, the presence of uH2A (ubiquitin-H2A semihistone) at 39 degrees C, and the presence of the human X chromosome. One of the hybrid clones that could grow at the nonpermissive temperature contained the X chromosome as the only human chromosome. The sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern of affinity-purified E1 showed that this hybrid clone contained both human and mouse type E1. Thus we conclude that the functional gene for human E1 is located on the X chromosome.
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PMID:Human ubiquitin-activating enzyme (E1): compensation for heat-labile mouse E1 and its gene localization on the X chromosome. 184 93

The RAD6 gene from the yeast Saccharomyces cerevisiae encodes a ubiquitin carrier protein (E2) required for a variety of cellular processes including DNA repair, induced mutagenesis, and sporulation. Here we identify an E2 from a higher plant, wheat, that is similar to RAD6 with respect to both structure and in vitro substrate specificity. The protein was purified from wheat germ by a combination of ubiquitin covalent affinity chromatography and anion-exchange HPLC and has an apparent molecular mass of 23 kDa [referred to as E2(23 kDa)]. E2(23 kDa) was capable of binding ubiquitin by means of a thiol ester linkage in an ATP-dependent and ubiquitin-activating enzyme-dependent reaction. In the presence of a variety of target proteins, E2(23 kDa), like the RAD6 gene product, formed covalent ubiquitin-protein conjugates in vitro only with histones in a ubiquitin protein ligase-independent reaction. E2(23 kDa) recognized both core and linker histones with an apparent order of preference of H2A greater than or equal to H1 greater than H2B greater than H3 greater than H4. This E2 protein was approximately 17-fold more effective at conjugating ubiquitin to histones than three other purified wheat germ E2 proteins tested. Mouse anti-E2(23 kDa) antibodies were used to isolate E2(23 kDa) DNA sequences from a wheat cDNA expression library. Antibody-positive clones were confirmed by amino acid identity of the sequence deduced from the cDNA to the peptide sequence of an E2(23 kDa) tryptic fragment. Protein expressed in Escherichia coli by the E2(23 kDa) cDNA was capable of both thiol ester adduct formation and conjugation of ubiquitin to histones. Analysis of the E2(23 kDa) cDNA shows that it encodes a protein with considerable amino acid sequence similarity to the yeast RAD6 gene product. Similarities exist at the amino terminus, the region surrounding the putative ubiquitin binding site, and at the carboxyl terminus, which is unusually acidic. Based on both the structural and enzymatic similarities to the RAD6 gene product, E2(23 kDa) may represent the first DNA repair enzyme identified in higher plants.
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PMID:A ubiquitin carrier protein from wheat germ is structurally and functionally similar to the yeast DNA repair enzyme encoded by RAD6. 255 33

The covalent ligation of the 8.6-kDa protein ubiquitin to histones within transcriptionally poised regions is believed to participate in the localized regulation of chromatin structure. This unique post-translational modification is thought to be distinct from similar cytosolic reactions in requiring ubiquitin-activating enzyme (E1) and one or more putative ubiquitin carrier proteins (E2) but not isopeptide ligase (E3). Apparently homogeneous preparations of the E2 isozymes were tested for their ability to catalyze the E3-independent conjugation of ubiquitin to linker histone H1 and core histones H2A, H2B, H3, and H4 in the presence of catalytic amounts of E1. Significant rates of nonprocessive core histone monoubiquitination were catalyzed by the E2(14kDa), E2(20kDa), and E2(32kDa) isozymes but not by either E2(17kDa) or E2(24kDa). The former three E2 isozymes also supported slow rates of direct multiple ubiquitination to secondary ligation sites on the histones. Rate studies for the monoubiquitination of H2A and H2B revealed that: 1) E2(14kDa) catalyzed a second order reaction with respect to histone concentration; 2) E2(32kDa)-mediated ligation proceeded by hyperbolic kinetics, yielding Km values of 2.8 and 12 microM for H2A and H2B, respectively; and 3) E2(20kDa) exhibited complex kinetics composed of both second order and hyperbolic pathways, the latter having Km values of 0.83 and 1.5 microM for H2A and H2B, respectively. Pulse-chase kinetics suggested that both ubiquitin thiol esters formed to E2(20kDa) were catalytically competent in H2A ligation. The active E2 isozymes also catalyzed the processive multiple ubiquitination of calf thymus H1. Other rate studies determined that Kd values for binding of the active E2 species to E1 ternary complex were 0.1 nM for E2(14kDa), 0.4 nM for E2(32kDa), and 3.6 nM for E2(20kDa). The data indicate that E2(20kDa) and E2(32kDa) are specific but mechanistically distinct ligation enzymes responsible for the conjugation of ubiquitin to nucleosomal proteins.
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PMID:Functional diversity among putative E2 isozymes in the mechanism of ubiquitin-histone ligation. 284 16

A prerequisite for the selective degradation of intracellular proteins by the ubiquitin-dependent proteolytic pathway is the attachment of a chain of ubiquitin monomers to the targeted protein. In these multiubiquitin chains, the carboxyl-terminal glycine 76 of ubiquitin is linked via an isopeptide bond to the epsilon-amino group of lysine 48 in the adjacent ubiquitin. It remains to be determined whether these chains are preassembled and attached en masse to the target, are made by sequential conjugation of ubiquitin monomers to ubiquitins already linked to the protein, or both. Using the 20-kDa ubiquitin-conjugating enzyme TaUBC7 from wheat, we have generated free, glycine 76-->lysine 48-linked multiubiquitin chains (Ubqn), and have individually purified Ubqn species (n < or = 6) by anion-exchange, high pressure liquid chromatography. The migration of these chains during SDS-polyacrylamide gel electrophoresis was indistinguishable from those of major ubiquitin immunoreactive proteins in cell lysates from a variety of eukaryotes suggesting that free, multiubiquitin chains are abundant in vivo. One of these chain members (Ubq2) was purified from wheat and was demonstrated via amino acid sequence analysis of tryptic fragments to consist of two ubiquitin monomers joined via a glycine 76-->lysine 48 linkage. We also show in vitro that purified Ubq2 and Ubq4 are competent in activation by ubiquitin-activating enzyme (E1), in transfer to E2s, and in ubiquitin-protein ligase (E3)-independent conjugation to other Ubqn species and to histones H2A/B. These data demonstrate that multiubiquitin chains exist as free, functional structures in vivo and support the possibility that at least a portion of free ubiquitin is preassembled into multiubiquitin chains prior to its attachment to proteolytic substrates.
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PMID:Multiubiquitin chains linked through lysine 48 are abundant in vivo and are competent intermediates in the ubiquitin proteolytic pathway. 822 36

Eukaryotes regulate gene expression and other nuclear processes through the posttranslational modification of histones. In S. cerevisiae, the mono-ubiquitylation of histone H2B on lysine 123 (H2B K123ub) affects nucleosome stability, broadly influences gene expression and other DNA-templated processes, and is a prerequisite for additional conserved histone modifications that are associated with active transcription, namely the methylation of lysine residues in H3. While the enzymes that promote these chromatin marks are known, regions of the nucleosome required for the recruitment of these enzymes are undefined. To identify histone residues required for H2B K123ub, we exploited a functional interaction between the ubiquitin-protein ligase, Rkr1/Ltn1, and H2B K123ub in S. cerevisiae. Specifically, we performed a synthetic lethal screen with cells lacking RKR1 and a comprehensive library of H2A and H2B residue substitutions, and identified H2A residues that are required for H2B K123ub. Many of these residues map to the nucleosome acidic patch. The substitutions in the acidic patch confer varying histone modification defects downstream of H2B K123ub, indicating that this region contributes differentially to multiple histone modifications. Interestingly, substitutions in the acidic patch result in decreased recruitment of H2B K123ub machinery to active genes and defects in transcription elongation and termination. Together, our findings reveal a role for the nucleosome acidic patch in recruitment of histone modification machinery and maintenance of transcriptional integrity.
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PMID:The Nucleosome Acidic Patch Regulates the H2B K123 Monoubiquitylation Cascade and Transcription Elongation in Saccharomyces cerevisiae. 2624 81