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)

Several genes, including RPS4X (ribosomal protein subunit 4), ZFX (zinc finger on the X chromosome), and UBE1 (ubiquitin-activating enzyme), have been shown to be expressed from the inactive X chromosome of cultured human cells. By contrast, these genes are subject to X-chromosome inactivation in tissues from adult mice. We have now examined the inactivation status of these genes in cultured mouse cells to determine whether the differences in X-chromosome inactivation between species is due to an intrinsic difference between human and mouse X-chromosome genes or whether it is a function of gene reactivation in cell culture per se. The expression of three mouse X-chromosome genes, Rps4, Zfx, and Ube1 was examined by reverse transcriptase polymerase chain reaction (RT-PCR) in heterozygous cultured cells from a cross of a laboratory mouse by Mus spretus, which were selected to uniformly express the X chromosome from the laboratory mouse parent. No expression of the M. spretus alleles of these genes was observed in the cell line (Hobmski), which is consistent with the patterns of expression previously observed in mouse in vivo and indicates that these genes remain stably inactivated in an immortalized mouse cell line. By cytogenetic and RT-PCR analyses the Hobmski cell line was shown to retain a late-replicating X chromosome from M. spretus, which expressed the M. spretus allele of the X (inactive) specific transcript (Xist). The Hobmski cell line will be a useful resource for studying the features that maintain X-chromosome genes inactive.
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PMID:Maintenance of X inactivation of the Rps4, Zfx, and Ube1 genes in a mouse in vitro system. 768 8

E6-AP is a 100-kDa cellular protein that interacts with the E6 protein of the cancer-associated human papillomavirus types 16 and 18. The E6/E6-AP complex binds to and targets the p53 tumor-suppressor protein for ubiquitin-mediated proteolysis. E6-AP is an E3 ubiquitin-protein ligase which accepts ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the ubiquitin to targeted substrates. The amino acid sequence of E6-AP shows similarity to a number of protein sequences over an approximately 350-aa region corresponding to the carboxyl termini of both E6-AP and the E6-AP-related proteins. Of particular note is a conserved cysteine residue within the last 32-34 aa, which in E6-AP is likely to be the site of ubiquitin thioester formation. Two of the E6-AP-related proteins, a rat 100-kDa protein and a yeast 95-kDa protein (RSP5), both of previously unknown function, are shown here to form thioesters with ubiquitin. Mutation of the conserved cysteine residue of these proteins destroys their ability to accept ubiquitin. These data strongly suggest that the rat 100-kDa protein and RSP5, as well as the other E6-AP-related proteins, belong to a class of functionally related E3 ubiquitin-protein ligases, defined by a domain homologous to the E6-AP carboxyl terminus (hect domain).
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PMID:A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. 776 80

The E6 protein of the high-risk human papillomaviruses inactivates the tumor suppressor protein p53 by stimulating its ubiquitinylation and subsequent degradation. Ubiquitinylation is a multistep process involving a ubiquitin-activating enzyme, one of many distinct ubiquitin-conjugating enzymes, and in certain cases, a ubiquitin ligase. In human papillomavirus-infected cells, E6 and the E6-associated protein are thought to act as a ubiquitin-protein ligase in the ubiquitinylation of p53. Here we describe the cloning of a human ubiquitin-conjugating enzyme that specifically ubiquitinylates E6-associated protein. Furthermore, we define the biochemical pathway of p53 ubiquitinylation and demonstrate that in vivo inhibition of various components in the pathway leads to an inhibition of E6-stimulated p53 degradation.
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PMID:Reconstitution of p53-ubiquitinylation reactions from purified components: the role of human ubiquitin-conjugating enzyme UBC4 and E6-associated protein (E6AP). 772 50

Cyclin B is degraded at the onset of anaphase by a ubiquitin-dependent proteolytic system. We have fractionated mitotic Xenopus egg extracts to identify components required for this process. We find that UBC4 and at least one other ubiquitin-conjugating enzyme can support cyclin B ubiquitination. The mitotic specificity of cyclin ubiquitination is determined by a 20S complex that contains homologs of budding yeast CDC16 and CDC27. Because these proteins are required for anaphase in yeast and mammalian cells, we refer to this complex as the anaphase-promoting complex (APC). CDC27 antibodies deplete APC activity, while immunopurified CDC27 complexes are sufficient to complement either interphase extracts or a mixture of recombinant UBC4 and the ubiquitin-activating enzyme E1. These results suggest that APC functions as a regulated ubiquitin-protein ligase that targets cyclin B for destruction in mitosis.
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PMID:A 20S complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B. 773 80

A mouse cell mutant, ts85, containing the temperature-sensitive ubiquitin-activating enzyme was arrested in G2 phase at the non-permissive temperature. In the arrested cells, azure C, a nucleolus-specific stain, revealed a U-shaped or ring-shaped arrangement of nucleolar lobes with an unstained region in the center. Silver staining of the nucleolar organizer region (NOR) and fluorescence in situ hybridization (FISH) with rDNA both gave signals in azure C-positive regions. Electron microscopic examination revealed a cloud of unidentified electron-dense particles (diameter approximately 70 nm) in the azure C-negative center space. When the arrested cells were released into M-phase, we observed the association of NOR-bearing chromosomes with a pulverization-like abnormality. FISH with rDNA and NOR silver staining demonstrated that the pulverization-like abnormality was restricted to NORs. The frequent occurrence of persistent nucleolar material in prophase and prometaphase of the stressed cells after release indicated a delayed dissociation of the nucleolus that brought about the abnormal chromosomes in M-phase. ts85 cells transfected with the mouse E1 cDNA recovered growth at the non-permissive temperature and no longer showed abnormal nucleolar morphology. It seems that the ubiquitin system plays a role in the dissolution of the nucleolus, possibly involving the NOR-bearing chromosomes.
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PMID:Abnormal integrity of the nucleolus associated with cell cycle arrest owing to the temperature-sensitive ubiquitin-activating enzyme E1. 774 60

Cell cycle progression in eukaryotes is controlled by the p34cdc2/CDC28 protein kinase and its short-lived, phase-specific regulatory subunits called cyclins. In Xenopus oocytes, degradation of M-phase (B-type) cyclins is required for exit from mitosis and is mediated by the ubiquitin-dependent proteolytic system. Here we show that B-type-cyclin degradation in yeast involves an essential nuclear ubiquitin-conjugating enzyme, UBC9. Repression of UBC9 synthesis prevents cell cycle progression at the G2 or early M phase, causing the accumulation of large budded cells with a single nucleus, a short spindle and replicated DNA. In ubc9 mutants both CLB5, an S-phase cyclin, and CLB2, an M-phase cyclin, are stabilized. In wild-type cells the CLB5 protein is unstable throughout the cell cycle, whereas CLB2 turnover occurs only at a specific cell-cycle stage. Thus distinct degradation signals or regulated interaction with the ubiquitin-protein ligase system may determine the cell-cycle specificity of cyclin proteolysis.
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PMID:Role of a ubiquitin-conjugating enzyme in degradation of S- and M-phase cyclins. 780 43

Ubiquitination of proteins involves the concerted action of the E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzymes and E3 ubiquitin-protein ligases. It has been proposed that E3s function as 'docking proteins', specifically binding substrate proteins and specific E2s, and that ubiquitin is then transferred directly from E2s to substrates. We show here that formation of a ubiquitin thioester on E6-AP, an E3 involved in the human papillomavirus E6-induced ubiquitination of p53 (refs 6-10), is an intermediate step in E6-AP-dependent ubiquitination. The order of ubiquitin transfer is from E1 to E2, from E2 to E6-AP, and finally from E6-AP to a substrate. This cascade of ubiquitin thioester complexes suggests that E3s have a defined enzymatic activity and do not function simply as docking proteins. The cysteine residue of E6-AP responsible for ubiquitin thioester formation was mapped to a region that is highly conserved among several proteins of unknown function, suggesting that these proteins share the ability to form thioesters with ubiquitin.
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PMID:Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade. 780 44

Little is known regarding the mechanism by which MHC class I-associated peptides are generated. Proteins can be targeted for degradation by the covalent attachment of ubiquitin. The first step in ubiquitin conjugation to proteins is its binding to E1 ubiquitin-activating enzyme. To study the role of ubiquitin-targeted protein degradation in Ag processing, we used two mutant cell lines with temperature-sensitive E1 proteins, and a recombinant vaccinia virus expressing wild-type human E1. One of the cell lines examined (hamster ts20 cells) was previously reported to have a minimal capacity after a 1-h incubation at 41 degrees C to present osmotically loaded OVA to a T cell hybridoma, as assessed by IL-2 release. Even after incubating the same cells for 1 h at 43 degrees C, we failed to detect an E1-related decrease in the presentation of biosynthesized or osmotically loaded OVA to splenic T cells, as measured by target cell lysis. We introduce the use of mouse tsA1S9 cells to Ag-processing studies and provide the initial biochemical characterization of their defect in protein ubiquitination. Relative to parental L929 cells, after thermal inactivation of E1, these cells actually demonstrate enhanced presentation of endogenous or exogenous viral Ags to T cells. Our findings do not support a role for protein ubiquitination in Ag processing, and indicate that either the temperature-sensitive cell lines examined do not exhibit a sufficient reduction in ubiquitin-conjugating activity to affect the generation of antigenic peptides, or that ubiquitin-targeted proteolysis is not essential for processing the two exogenous and six endogenous Ags examined.
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PMID:Presentation of endogenous and exogenous antigens is not affected by inactivation of E1 ubiquitin-activating enzyme in temperature-sensitive cell lines. 781 64

The ubiquitin conjugation system is a multi-step pathway in which ubiquitin is activated and conjugated to acceptor proteins, one function of which is to target acceptor proteins for rapid degradation within the cell. The conjugation system is involved in many aspects of cellular functions, including the cell cycle. Several cell-cycle arrest mutant cell lines have been characterized and appear to harbour a mutant ubiquitin-activating enzyme, E1, as their primary defect. One such cell line is ts20, which is derived from Chinese hamster ovary E36 cells. This cell line has been used to characterize some of the potential functions of the ubiquitin conjugation system in vivo, such as its involvement in the maturation of autophagic vacuoles. The present study describes the complete rescue of the complex ts20 phenotype following the expression of the cDNA for human E1. Stable transfectants expressing the human E1 cDNA in the CMVneo expression vector were measured for ubiquitin-conjugation activity, protein degradation and growth in culture at the nonpermissive temperature. This rescue confirms that the phenotype observed in the ts20 cells is due to a defect in the E1 enzyme. Thus, the ts20 cell line will serve as a useful tool to delineate the functions of the ubiquitin system in vivo.
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PMID:Rescue of the complex temperature-sensitive phenotype of Chinese hamster ovary E36ts20 cells by expression of the human ubiquitin-activating enzyme cDNA. 781 64

The strict evolutionary conservation of ubiquitin suggests an essential role for each residue in the folding, stability, or function of the protein but precludes identification of such contributions through interspecies comparison of ubiquitin sequences. However, site-directed mutagenesis potentially allows assignment of specific function(s) for each residue. The four arginines present on ubiquitin at positions 42, 54, 72, and 74 were independently mutated to leucine and their effects on the interaction of the resulting polypeptides with ubiquitin-activating enzyme (E1) were characterized. All of the mutants except UbR54L exhibited altered kinetics for E1-catalyzed ATP:PPi exchange compared to wild-type ubiquitin. In addition, the UbR72L mutant altered the mechanism of E1 from strictly order addition of substrates to random addition with respect to ATP and ubiquitin. Values for the intrinsic Kd of ubiquitin binding were determined by coupling the net forward reaction of E1 to the E232K-catalyzed conjugation of histone H2B. Only R54 and R72 residues participate in the initial binding of free ubiquitin, resulting in a 6- or 58-fold increase in Kd for UbR54L or UbR72L, respectively, compared to wild type. More significant effects of the UbR42L and UbR72L mutants were observed for binding of their respective ubiquitin adenylate intermediates within the E1 active site. Wild-type ubiquitin adenylate binds to E1 with an estimated Kd < or = 8 x 10(-12) M while intermediates formed with UbR42L or UbR72L each bind with ca. 10(3)-fold lower affinity, representing a destabilization of > or = 7 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Site-directed mutagenesis of ubiquitin. Differential roles for arginine in the interaction with ubiquitin-activating enzyme. 800 94


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