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Query: UNIPROT:P62988 (Ubiquitin)
 4,326 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A pulse treatment of embryos of Norway spruce with cytokinin suppresses germinative development and induces the coordinate formation of adventitious buds from subepidermal cell layers. To analyse the patterns of gene expression associated with germination and the alterations induced by the bud induction treatment, we have isolated cDNA clones corresponding to genes that are differentially expressed in cytokinin-treated and untreated in vitro germinating embryos. One category of 14 clones hybridized to transcripts that were abundant specifically during germination. The expression of 8 of these genes was reduced by the bud induction treatment. Four clones, including one identified as a histone H2A gene, recognized transcripts that showed an increased abundance in bud-induced versus in vitro germinating embryos. A second category of 13 clones hybridized to transcripts that increased in abundance during post-germinative development of the seedling. Among these a subset of 8 clones, including an alpha-tubulin clone, corresponds to genes suppressed by the bud induction treatment, whereas 5 clones, including a gene with sequence similarity to polyubiquitin, were unaffected by the treatment. One clone hybridized to a message abundant in the seed, during early germination as well as in the vegetative bud, and showed 60% partial sequence identity to a barley (1----3)-beta-glucanase gene. Genes expressed exclusively in bud-induced or in vitro germinating embryos were not found. The results show that a major difference in gene expression between treated and untreated embryos is related to the shift from extensive cell proliferation to elongation and differentiation that occurs at the transition from germination to post-germinative development, and which is suppressed in the bud-induced embryos.
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PMID:Differential gene expression during germination and after the induction of adventitious bud formation in Norway spruce embryos. 137 81

A ubiquitin hydrolase that removes ubiquitin from a multi-ubiquitinated protein has been purified 600-fold from Saccharomyces cerevisiae. Four different ubiquitin-protein conjugates were assayed as substrates during the purification procedure. Enzymic activities that removed ubiquitin from ubiquitinated histone H2A, a ubiquitin-ubiquitin dimer and a ubiquitin-ribosomal fusion protein were separated during the purification from an activity that removed a single ubiquitin molecule linked by an isopeptide bond to a ubiquitinated protein. The size of the native enzyme was 160 kDa, based on its sedimentation in a sucrose gradient, and the subunit molecular mass was estimated to be 160 kDa, based on a profile of proteins eluted in different fractions by thiol-affinity chromatography. The partially purified hydrolase was not inhibited by a variety of protease inhibitors, except for thiol-blocking reagents. The natural substrate for this enzyme may be the polyubiquitin chain containing ubiquitin molecules bound to each other in isopeptide bonds, with one of them linked to a lysine residue of a protein targeted for intracellular proteolysis.
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PMID:Partial purification and substrate specificity of a ubiquitin hydrolase from Saccharomyces cerevisiae. 184 17

Ubiquitin, a small 76-amino acid protein which is highly conserved in eukaryotic cells, occurs in several forms other than the free polypeptide. Among these are protein conjugates in which ubiquitin is covalently linked in lysylpeptide bond to lysl residues of other proteins and fusion proteins in which the amino-terminal domain is the precise ubiquitin sequence. Ubiquitin plays a role in cellular proteolytic degradation and in chromatin structure and has been postulated to be involved in the induction of a set of proteins which function during the cellular response to various kinds of environmental stress. We have measured the various forms of ubiquitin in cultures of chicken embryo fibroblasts under normal growth conditions and after treatment with a thermal or chemical stress. Levels of free ubiquitin fell slightly, ubiquitin conjugate levels rose shortly after stress began, and both then increased substantially as one of the cell's ubiquitin-encoding genes was activated by stress. The level of a protein synthesized as the carboxyl-terminal domain of one ubiquitin fusion protein was unchanged by a heat stress. The most dramatic effect was seen in the rapid disappearance of the ubiquitinated form of histone H2A, one of the major ubiquitin conjugates in cells in the interphase portion of their growth cycle. A significant rise in protein turnover was detected as a result of the stress, but occurred only when cells were removed from the stress condition. These results suggest that ubiquitin plays an important role both during and after stress, but fails to support hypotheses for ubiquitin and proteolysis in the activation of stress genes.
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PMID:Ubiquitin in stressed chicken embryo fibroblasts. 282 67

Murine erythroleukemia cells are useful for studying the regulation of erythroid differentiation since these malignant pronormoblasts differentiate to orthochromatic normoblasts when treated with a variety of inducing agents. Changes in chromatin proteins have been described following inducer exposure. The significance of these changes, which are greatest in terminally differentiated cells remains unknown. Ubiquitin is a highly conserved 8.5 kilodalton peptide that is covalently linked to up to 10% of histone H2A. We demonstrate that following exposure of MEL cells to inducers of differentiation, a transient increase in ubiquitination of H2A occurs. This change is coincident with the onset of differentiation. This result suggests that ubiquitination of H2A may have a role in the nuclear changes necessary for erythroleukemic cell differentiation.
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PMID:A transient increase in histone H2A ubiquitination is coincident with the onset of erythroleukemic cell differentiation. 283 26

Ubiquitin, a 76 residue protein, occurs in eukaryotic cells either free or covalently joined via its carboxyl terminus to epsilon-amino groups of lysine residues in a wide variety of protein species. Previous work has shown that ubiquitin-protein conjugates are preferred substrates in vitro for a non-lysosomal ATP-dependent proteolytic pathway, suggesting that ubiquitin may function as a signal for attack by proteinases specific for ubiquitin-protein conjugates. One strategy to define the potential significance of the ubiquitin-dependent proteolytic pathway is to identify conditional mutants in the pathway. ts85 is a mouse derived cell-cycle mutant which has been shown to lose uH2A, a specific ubiquitin-histone H2A conjugate, at the nonpermissive temperature. We show that the loss of uH2A from ts85 cells is due to reduced ubiquitin-protein conjugation. We further show that the reduced conjugation is due to the specific thermolability of ubiquitin activating enzyme, E1, one of the three enzymic components of the ubiquitin-protein ligase system. We therefore proceeded to test whether the degradation of short-lived proteins is also temperature-sensitive in ts85 cells. Indeed, while more than 70% of the prelabeled abnormal (amino acid analog-containing) proteins or puromycyl peptides are degraded within 4 hours at the permissive temperature in the mutant (ts85), wild type (FM3A), and revertant (ts85R-MN3) cells, less than 15% of these proteins are degraded in ts85 cells at the nonpermissive temperature. In contrast, the rate of degradation of these proteins does not change significantly in either wild-type or revertant cells between permissive and nonpermissive temperatures. Degradation of normal short-lived proteins is also specifically temperature-sensitive in ts85 cells. Immunochemical analysis shows a strong and specific reduction in ubiquitin-protein conjugate levels in vivo at the nonpermissive temperature in ts85 cells. Taken together, our in vitro and in vivo findings with ts85 cells demonstrate that the degradation of the bulk of short-lived proteins in this higher eukaryotic cell is accomplished through a ubiquitin-mediated pathway.
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PMID:Mammalian cell cycle mutant defective in intracellular protein degradation and ubiquitin-protein conjugation. 299 83

Ubiquitin was radioiodinated and introduced into HeLa cells by the erythrocyte-mediated fusion procedure. Fractionation of injected HeLa cells and subsequent NaDodSO4/polyacrylamide gel electrophoresis showed that HeLa nuclei contained two major labeled proteins: ubiquitin and the histone H2A-ubiquitin conjugate, protein A24. HeLa cytosol contained ubiquitin and a series of ubiquitin-protein conjugates of diverse molecular weights. When injected HeLa cells were treated with phenylhydrazine to denature the cotransferred hemoglobin, a series of prominent ubiquitin-globin conjugates appeared. The identity of these conjugates was established by microinjection experiments in which both proteins were labeled. At low doses of phenylhydrazine, the intracellular concentration of globin-ubiquitin conjugates was proportional to the rate of hemoglobin degradation. This result, together with the observation that ubiquitin conjugation to globin is markedly enhanced by phenylhydrazine-induced denaturation of hemoglobin, provides support for the hypothesis that the covalent attachment of ubiquitin to proteins signals proteolysis.
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PMID:Conjugation of ubiquitin to denatured hemoglobin is proportional to the rate of hemoglobin degradation in HeLa cells. 631 May 49

Ubiquitin-conjugating enzymes (E2s) play a key role in ubiquitin-mediated proteolysis by catalysing the conjugation of ubiquitin to protein substrates. We have previously reported the cDNA cloning of a 14 kDa conjugating enzyme [E2(14)k; Wing, Dumas and Banville (1992) J. Biol. Chem. 267, 6495-6501] that efficiently supported ubiquitination and protein degradation in reticulocyte extracts. Surprisingly, the structure of this E2 was markedly more similar to the Saccharomyces cerevisiae DNA repair gene RAD6, than to the S. cerevisiae UBC4/UBC5 genes which are required for the degradation of short-lived proteins and support much of the ubiquitination of yeast proteins. This suggested that mammalian homologues of UBC4/UBC5 remained to be identified. Using oligonucleotides derived from the S. cerevisiae UBC4 sequence as primers in a PCR reaction with rat muscle cDNA as a template, a 390 bp DNA fragment was amplified which predicted an amino acid sequence that was 83% identical to yeast UBC4. Screening a rat testes cDNA library identified a family of cDNAs which predicted two very similar proteins with basic pIs and molecular masses of approx. 16,700 Da. Isoform 2E was expressed in Escherichia coli and purified to homogeneity. It supported ubiquitination to reticulocyte and testis proteins more rapidly in vitro and produced larger conjugates than E2(14)k. Examination of RNA from different tissues indicated that this type of E2 was expressed in a broad spectrum of tissues but at particularly high levels in the testis. Fractionation of a testis extract by anion-exchange chromatography identified several putative ubiquitin protein ligase activities with which this E2 could interact in promoting conjugation of ubiquitin to proteins. One of these activities supported conjugation of ubiquitin to histone H2A, a substrate degraded in the ubiquitin system by a non-N-end rule mechanism. This paper reports the first cloning of a apparent mammalian homologue of S. cerevisiae UBC4/UBC5. Its high expression in testis and ability to efficiently support conjugation to testis proteins suggest that this family of E2s may play a role in the proteolysis that occurs during spermatogenesis.
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PMID:Molecular cloning, expression and characterization of a ubiquitin conjugation enzyme (E2(17)kB) highly expressed in rat testis. 782 19

Ubiquitin-carrier proteins (E2s, ubiquitin-conjugating enzymes, UBCs) participate in proteolysis by catalyzing transfer of activated ubiquitin to the protein substrates, which are bound to specific ubiquitin-protein ligases (E3s). Yeast UBC2 (RAD6) and the mammalian E2(14kDa) bind to the ligase that recognizes and is involved in the degradation of certain free amino-terminal substrates ("N-end rule" substrates). As such proteins are rather scarce, the role of these E2s in general proteolysis is probably limited. Here, we report the purification and characterization of a novel 18-kDa species of E2 from rabbit reticulocytes. Unlike most members of the E2 family, this enzyme does not adsorb to anion exchange resin in neutral pH, and it is purified from the unadsorbed material (Fraction 1). Thus, it is designated E2-F1. Like all members of the E2 family, it generates a thiol ester with ubiquitin that serves as an intermediate in the conjugation reaction. Sequence analysis revealed a significant homology to many known species of E2s. The enzyme generates multiply ubiquitinated proteins in the presence of an E3 that has not been characterized yet. Most importantly, the ubiquitination via this E2 leads to the degradation of certain non-"N-end rule" substrates such as glyceraldehyde-3-phosphate dehydrogenase (Val at the NH2 terminus) and to the ubiquitination and degradation of certain N-alpha-acetylated proteins such as histone H2A, actin, and alpha-crystallin. The enzyme is also involved in the conjugation and degradation of the tumor suppressor protein p53.
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PMID:Purification and characterization of a novel species of ubiquitin-carrier protein, E2, that is involved in degradation of non-"N-end rule" protein substrates. 814 44

Lens cells must remove obsolete or damaged proteins produced during development, maturation and aging to maintain lens transparency. In reticulocytes removal of abnormal or obsolete proteins is thought to involve a ubiquitin-dependent proteolytic pathway. Two hallmarks of ubiquitin (Ub) dependent proteolysis have previously been demonstrated in lens cell or tissue supernatants: (1) the presence of ubiquitin conjugates, and (2) ATP-dependent proteolysis. Nevertheless, conclusive proof was lacking of a requirement for ubiquitination of substrate proteins for proteolysis. Here we show that in bovine lens epithelial cell (BLEC) supernatant, ATP-dependent proteolysis is also ubiquitin-dependent. Ubiquitin-activating enzyme (E1), the first enzyme in the cascade of ubiquitin ligation, was purified over 3000-fold from a rabbit reticulocyte lysate using Ubiquitin-Sepharose, and showed ATP-PPi exchange activity. Antiserum to E1 was prepared in goats and affinity-purified on Protein G-Sepharose. Western blot analysis revealed that both the goat antiserum and purified antibody (anti-E1(IgG)) recognize specifically E1. Anti-E1(IgG) inhibits 86% of the ATP-dependent degradation of labeled histone H2A in reticulocyte lysate and 75% of the ATP-dependent degradation in BLEC. Upon reconstitution with purified E1, 100% and 80% of proteolysis was restored in reticulocytes and BLEC supernatant, respectively. This confirms that there is a ubiquitin-dependent proteolytic system in lens.
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PMID:Bovine lens epithelial cells have a ubiquitin-dependent proteolysis system. 838 Mar 40

Degradation of a protein via the ubiquitin system involves two discrete steps, signaling by covalent conjugation of multiple moieties of ubiquitin and degradation of the tagged substrate. Conjugation is catalyzed via a three-step mechanism that involves three distinct enzymes that act successively: E1, E2, and E3. The first two enzymes catalyze activation of ubiquitin and transfer of the activated moiety to E3, respectively. E3, to which the substrate is specifically bound, catalyzes formation of a polyubiquitin chain that is anchored to the targeted protein. The polyubiquitin-tagged protein is degraded by the 26 S proteasome, and free and reutilizable ubiquitin is released. In addition to the three conjugating enzymes, targeting of certain proteins requires association with ancillary proteins and/or post-translational modification(s). Using a specific antibody to deplete cell extract from the molecular chaperone Hsc70, we demonstrate that this protein is required for the degradation of actin, alpha-crystallin, glyceraldehyde-3-phosphate dehydrogenase, alpha-lactalbumin, and histone H2A. In contrast, the degradation of bovine serum albumin, lysozyme, and oxidized RNase A is Hsc70-independent. Mechanistic analysis revealed that the chaperone is required for the conjugation reaction; however, it does not substitute for E3. Involvement of the chaperone in the proteolytic process requires complex formation with the substrate. Formation of this complex appears to be essential in the proteolytic process. In addition, the proper function of the chaperone in the proteolytic process requires the presence of K+, which allows rapid cycles of dissociation and association of the complex. The chaperone may act by binding to the substrate and unfolding it to expose a ubiquitin ligase-binding site. In addition, it can also act directly on the ubiquitination machinery.
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PMID:Ubiquitin-dependent degradation of certain protein substrates in vitro requires the molecular chaperone Hsc70. 908 24


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