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

The degradation of many proteins requires their prior attachment to ubiquitin. Proteolytic substrates are characteristically multiubiquitinated through the formation of ubiquitin-ubiquitin linkages. Lys-48 of ubiquitin can serve as a linkage site in the formation of such chains and is required for the degradation of some substrates of this pathway in vitro. We have characterized the recessive and dominant effects of a Lys-48-to-Arg mutant of ubiquitin (UbK48R) in Saccharomyces cerevisiae. Although UbK48R is expected to terminate the growth of Lys-48 multiubiquitin chains and thus to exert a dominant negative effect on protein turnover, overproduction of UbK48R in wild-type cells results in only a weak inhibition of protein turnover, apparently because the mutant ubiquitin can be removed from multiubiquitin chains. Surprisingly, expression of UbK48R complements several phenotypes of polyubiquitin gene (UB14) deletion mutants. However, UbK48R cannot serve as a sole source of ubiquitin in S. cerevisiae, as evidenced by its inability to rescue the growth of ubi1 ubi2 ubi3 ubi4 quadruple mutants. When provided solely with UbK48R, cells undergo cell cycle arrest with a terminal phenotype characterized by replicated DNA, mitotic spindles, and two-lobed nuclei. Under these conditions, degradation of amino acid analog-containing proteins is severely inhibited. Thus, multiubiquitin chains containing Lys-48 linkages play a critical role in protein degradation in vivo.
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PMID:Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant. 803 26

A Ubiquitin-like peptide was accidentally isolated from rat bladder by using 5% acetic acid wash while we were isolating antibacterial peptides. The purified molecule was obtained by reverse phase high performance liquid chromatography. Gas phase microsequence analysis indicated the N-terminal sequences of the molecule as follows: MET-GLN-ILE-PHE-VAL-LYS-THR-LEU-THR-GLY-LYS-THR-ILE-THR-LEU- GLU-VAL-GLU-PRO-SER-ASP-THR-ILE-GLU-ASN, which is homologous to human ubiquitin. Ubiquitin plays a role in the differentiation of pre-B lymphocytes, Thus, it is suggested from the findings of this molecule and the endogenous antibacterial polypeptides in mucosa or mucosal epithelium that mucosal epithelium also might be one of immune cells or immunity-associated cells, which may secrete effector molecules directly to kill adherent microbes and produce regulating factors in mucosal immune response.
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PMID:[Rat bladder ubiquitin-like molecule: isolation, purification and N-terminal sequencing]. 824 87

We examined ubiquitination and ubiquitin-mediated degradation of glycated protein by rabbit reticulocyte lysate fraction II (ubiquitin-free preparation). Non-glycated lysozyme and three glycated lysozyme preparations with different glucose binding ratios were used as substrates. Glycation sites of the lysozyme were mostly the epsilon-NH2 group of lysine residues, since modification at the alpha-NH2 group of the amino terminal was not detectable. Ubiquitin was conjugated with three glycated lysozyme preparations, which contained 1.4, 2.8 and 4.5 mol glucose per mol, by fraction II supplemented with hemin. The extent of formed conjugates was reduced 81, 72 and 56% of those of ubiquitin-non-glycated lysozyme conjugates, respectively. Additionally, ubiquitin-mediated degradation of the resultant conjugates was reduced and their respective rates were 97, 56 and 19% of that of the non-glycated lysozyme. These results indicated that both ubiquitin conjugation and ubiquitin-mediated degradation of the lysozyme were inhibited by nonenzymatic glucose binding to the lysozyme.
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PMID:Inhibitory effect of nonenzymatic glycation on ubiquitination and ubiquitin-mediated degradation of lysozyme. 838 87

Ubiquitin conjugation is a signal for degradation of eukaryotic proteins by the 26S protease. Conjugation of a homopolymeric multiubiquitin chain to a substrate lysine residue results in 10-fold faster degradation than does conjugation of monoubiquitin, but the molecular basis of enhanced targeting by chains is unknown. We show that ubiquitin residues L8, I44, and V70 are critical for targeting. Mutation of pairs of these residues to alanine had little effect on attachment of ubiquitin to substrates but severely inhibited degradation of the resulting conjugates. The same mutations blocked the binding of chains to a specific subunit (S5a) of the regulatory complex of the 26S protease. The side chains implicated in this binding--L8, I44, and V70--form repeating patches on the chain surface. Thus, hydrophobic interactions between these patches and S5a apparently contribute to enhanced proteolytic targeting by multiubiquitin chains.
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PMID:Surface hydrophobic residues of multiubiquitin chains essential for proteolytic targeting. 857 Jun 49

A novel member of the ubiquitin carrier protein family, designated E2EPF, has been cloned by our laboratory and expressed in a bacterial system in an active form. Ubiquitin carrier proteins, or E2s, catalyze one step in a multistep process that leads to the covalent conjugation of ubiquitin to substrate proteins. In this paper, we show that recombinant E2EPF catalyzes auto/multiubiquitination, the conjugation of multiple ubiquitin molecules to itself. Multiubiquitination has been shown previously to be required for targeting of a substrate protein for rapid degradation. Using a rabbit reticulocyte lysate system, E2EPF was shown to support the degradation of a model substrate in an ATP- and ubiquitin-dependent fashion. In contrast to a previous study which showed that selective protein degradation in one system is dependent upon multiubiquitination via the lysine 48 residue of ubiquitin, multiubiquitination, and proteolytic targeting by E2EPF was shown here to be independent of the lysine 48 multiubiquitin linkage. This functional characterization of E2EPF revealed a combination of features that distinguishes this enzyme from all previously characterized members of the ubiquitin carrier protein family. These results also suggest several possible autoregulatory models for E2EPF involving auto- and multiubiquitination.
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PMID:Characterization of a novel keratinocyte ubiquitin carrier protein. 857 60

Covalent conjugation of ubiquitin to intracellular proteins is a signal for degradation by the 26S protease. Conjugation is usually accomplished by the sequential action of activating (E1), conjugating (E2), and ligase (E3) enzymes. Each of these enzymes forms a covalent thiol ester with ubiquitin as part of its catalytic cycle. In most cases, the apparent role of the ubiquitin conjugating enzyme (E2) is to transfer ubiquitin from the E1 active site to the E3 active site. Ubiquitin is then delivered from E3 to the substrate lysine residue. An unusually large, reticulocyte-specific enzyme, known as E2-230K, is unique among the large family of E2 enzymes is being susceptible to inhibition by inorganic arsenite [Klemperer et al. (1989) Biochemistry 28, 6035-6041]. We show that phenylarsenoxides potently inhibit E2-230K, apparently by binding to vicinal Cys residues of the enzyme: bound aminophenylarsenoxide partially protects the enzyme against inactivation by N-ethylmalemide (NEM), and prior enzyme inactivation with NEM blocks enzyme binding to immobilized phenylarsenoxide. Studies on the mechanistic basis of inhibition showed that a concentration of (aminophenyl)arsenoxide that produced complete inhibition of steady-state turnover had no effect on the turnover of the preformed E2-ubiquitin adduct. However, when the enzyme was preincubated with this concentration of inhibitor prior to initiation of adduct formation, the level of E2-associated ubiquitin was reduced by 60%. These results are consistent with a model in which two Cys residues of the enzyme sequentially form thiol esters with ubiquitin and the second of these Cys residues is bound to arsenic in the enzyme-inhibitor complex. In this model, E2-230K functions as an E2-E3 hybrid.
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PMID:Mechanism of ubiquitin conjugating enzyme E2-230K: catalysis involving a thiol relay? 863 98

Isopeptidase T (IPaseT) can hydrolyze isopeptide bonds of polyubiquitin (polyUb) chains, simple C-terminal derivatives of Ub, and certain peptides. We recently reported that IPaseT is regulated by ubiquitin (Ub); while submicromolar Ub activates, higher concentrations inhibit this enzyme [Stein et al. (1995) Biochemistry 34, 12616]. To explain these observations, we proposed a model for IPaseT involving two binding sites for Ub. According to the model, the two sites are adjacent to one another and are the extended active site that binds two Ub moieties of a polyUb chain. The "activation site" binds the Ub that donates Lys to the isopeptide bond. The "inhibition site" is adjacent and binds the Ub that donates the C-terminal Gly to the isopeptide bond. We now report that the interaction of IPaseT with the C-terminal aldehyde of Ub (Ub-H) is also modulated by Ub. In the absence of Ub, Ub-H inhibits IPaseT with a Ki of 2.3 nM, while at 0.6 microM Ub, where the "activation site" is occupied, Ki is less than 0.1 nM. At high Ub concentrations, where both the "activation" and "inhibition" sites are occupied, IPaseT cannot bind Ub-H. We also determined the kinetics of inhibition of IPaseT by Ub-H. In the absence of Ub, a two-step mechanism is followed. In the first step, Ub-H slowly combines with IPaseT to form a relatively weak complex (K1 = 260 nM) that slowly isomerizes to the final, stable complex that accumulates in the steady-state (k2 = 2 x 10(-3) s-1; k-2 = 0.02 x 10(-3) s-1). In contrast, Ub-activated IPaseT is inhibited by Ub-H through a three-step process. In the first step, Ub-H rapidly combines with IPaseT to form a complex (K1 = 10 nM) that slowly isomerizes to a second, more stable complex (k2 = 18 x 10(-3) s-1; k-2 = 1.5 x 10(-3) s-1). In the third step, the second complex converts to the final complex (k3 = 1.5 x 10(-3) s-1; k-3 < 0.2 x 10(-3) s-1). To unify the results of this study with our previous results on catalysis, we propose that binding of Ub either to catalytic transition states or to tetrahedral inhibition intermediates liberates more free energy than binding of Ub to the reactant state of IPaseT and that IPaseT can utilize this binding energy to stabilize both of these tetrahedral species. The overall effect is a Ub-induced increase in catalytic efficiency or inhibitory potency.
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PMID:Kinetic studies on the inhibition of isopeptidase T by ubiquitin aldehyde. 884 Nov 33

We have recently reported that the yeast plasma membrane uracil permease undergoes cell-surface ubiquitination, which is dependent on the Npi1/Rsp5 ubiquitin-protein ligase. Ubiquitination of this permease, like that of some other transporters and receptors, signals endocytosis of the protein, leading to its subsequent vacuolar degradation. This process does not involve the proteasome, which binds and degrades ubiquitin-protein conjugates carrying Lys48-linked ubiquitin chains. The data presented here show that ubiquitination and endocytosis of uracil permease are impaired in yeast cells lacking the Doa4p ubiquitin-isopeptidase. Both processes were rescued by overexpression of wild-type ubiquitin. Mutant ubiquitins carrying Lys-->Arg mutations at Lys29 and Lys48 restored normal permease ubiquitination. In contrast, a ubiquitin mutated at Lys63 did not restore permease polyubiquitination. Ubiquitin-permease conjugates are therefore extended through the Lys63 of ubiquitin. When polyubiquitination through Lys63 is blocked, the permease still undergoes endocytosis, but at a reduced rate. We have thus identified a natural target of Lys63-linked ubiquitin chains. We have also shown that monoubiquitination is sufficient to induce permease endocytosis, but that Lys63-linked ubiquitin chains appear to stimulate this process.
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PMID:Ubiquitin lys63 is involved in ubiquitination of a yeast plasma membrane protein. 931 43

The Caenorhabditis elegans ubiquitin-conjugating enzyme UBC-1 is distinct from other RAD6 homologues in possessing a C-terminal tail 40 amino acid residues long [Leggett, Jones and Candido (1995) DNA Cell Biol. 14, 883-891]. Such extensions from the core catalytic domain have been found in a subset of known conjugating enzymes, where they have been shown to have diverse roles including target recognition, membrane attachment and sporulation. In the present study we used mutagenesis in vitro to examine the role of the tail in specific aspects of UBC-1 structure and activity. Cross-linking experiments with purified recombinant UBC-1 reveal that it forms dimers and probably tetramers. The acidic tail of UBC-1 has an important role in this interaction because deletions of the tail significantly decrease, but do not abolish, this self-association. Ubiquitin conjugation assays show that, in addition to accepting a thiol-bound ubiquitin at its active site, UBC-1 is stably mono-ubiquitinated. Deletion analysis and site-directed mutagenesis localize the site of ubiquitination to Lys-162 in the tail. These findings demonstrate that the C-terminal tail of UBC-1 is important both for its quaternary structure and post-translational modification in vitro.
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PMID:Biochemical characterization of Caenorhabditis elegans UBC-1: self-association and auto-ubiquitination of a RAD6-like ubiquitin-conjugating enzyme in vitro. 935 1

The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a type I anchored integral membrane phosphoprotein with two independent functions. First, it regulates virus release from a post-endoplasmic reticulum (ER) compartment by an ion channel activity mediated by its transmembrane anchor. Second, it induces the selective down regulation of host cell receptor proteins (CD4 and major histocompatibility complex class I molecules) in a process involving its phosphorylated cytoplasmic tail. In the present work, we show that the Vpu-induced proteolysis of nascent CD4 can be completely blocked by peptide aldehydes that act as competitive inhibitors of proteasome function and also by lactacystin, which blocks proteasome activity by covalently binding to the catalytic beta subunits of proteasomes. The sensitivity of Vpu-induced CD4 degradation to proteasome inhibitors paralleled the inhibition of proteasome degradation of a model ubiquitinated substrate. Characterization of CD4-associated oligosaccharides indicated that CD4 rescued from Vpu-induced degradation by proteasome inhibitors is exported from the ER to the Golgi complex. This finding suggests that retranslocation of CD4 from the ER to the cytosol may be coupled to its proteasomal degradation. CD4 degradation mediated by Vpu does not require the ER chaperone calnexin and is dependent on an intact ubiquitin-conjugating system. This was demonstrated by inhibition of CD4 degradation (i) in cells expressing a thermally inactivated form of the ubiquitin-activating enzyme E1 or (ii) following expression of a mutant form of ubiquitin (Lys48 mutated to Arg48) known to compromise ubiquitin targeting by interfering with the formation of polyubiquitin complexes. CD4 degradation was also prevented by altering the four Lys residues in its cytosolic domain to Arg, suggesting a role for ubiquitination of one or more of these residues in the process of degradation. The results clearly demonstrate a role for the cytosolic ubiquitin-proteasome pathway in the process of Vpu-induced CD4 degradation. In contrast to other viral proteins (human cytomegalovirus US2 and US11), however, whose translocation of host ER molecules into the cytosol occurs in the presence of proteasome inhibitors, Vpu-targeted CD4 remains in the ER in a transport-competent form when proteasome activity is blocked.
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PMID:CD4 glycoprotein degradation induced by human immunodeficiency virus type 1 Vpu protein requires the function of proteasomes and the ubiquitin-conjugating pathway. 949 87


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