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)

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

The E6 protein of the oncogenic human papillomavirus types 16 and 18 facilitates the rapid degradation of the tumor-suppressor protein p53 via the ubiquitin-dependent proteolytic pathway. The E6 protein binds to a cellular protein of 100 kDa termed E6-AP. The complex of E6 and E6-AP specifically interacts with p53 and induces the ubiquitination of p53 in a reaction which requires the ubiquitin-activating enzyme (E1) and a cellular fraction thought to contain a mammalian ubiquitin-conjugating enzyme (E2). This mammalian E2 activity could be replaced with bacterially expressed UBC8 from Arabidopsis thaliana, which belongs to a subfamily of E2s including yeast UBC4 and UBC5 which are highly conserved at the amino acid level. In this paper we describe the cloning of a human cDNA encoding a human E2 that we have designated UbcH5 and that is related to Arabidopsis UBC8 and the other members of this subfamily. We demonstrate that UbcH5 can function in the E6/E6-AP-induced ubiquitination of p53.
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PMID:Identification of a human ubiquitin-conjugating enzyme that mediates the E6-AP-dependent ubiquitination of p53. 809 Jul 26

E6-AP, a 100-kDa cellular protein, was originally identified through its interaction with the E6 protein of the oncogenic human papillomavirus types 16 and 18. The complex of E6-AP and E6 specifically interacts with p53 and mediates ubiquitination of p53 in concert with the E1 ubiquitin-activating enzyme and the E2 ubiquitin-conjugating enzyme UbcH5. Recent results suggest that E6-AP is representative of a family of putative ubiquitin-protein ligases. Members of this family are characterized by a conserved C-terminal region, termed hect domain. In this paper, we describe the isolation of two human E2s, designated as UbcH6 and UbcH7, that in addition to UbcH5 can interact with E6-AP. UbcH6 is a novel member of an evolutionally conserved subfamily of E2s that includes UbcH5 and Saccharomyces cerevisiae UBC4. Although UbcH7 does not appear to be a member of this subfamily, UbcH7 efficiently substitutes for UbcH5 in E6-AP-dependent ubiquitination. Surprisingly, UbcH6 was only weakly active in this particular assay. In addition, UbcH5 but not UbcH6 or UbcH7 efficiently interacts with the heet protein RSP5. These results indicate that E6-AP can interact with at least two species of E2 and that different hect proteins may interact with different E2s.
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PMID:Cloning of human ubiquitin-conjugating enzymes UbcH6 and UbcH7 (E2-F1) and characterization of their interaction with E6-AP and RSP5. 857 57

In this study, we found that the E6-associated protein (E6-AP/UBE3A) directly interacts with and coactivates the transcriptional activity of the human progesterone receptor (PR) in a hormone-dependent manner. E6-AP also coactivates the hormone-dependent transcriptional activities of the other members of the nuclear hormone receptor superfamily. Previously, it was shown that E6-AP serves the role of a ubiquitin-protein ligase (E3) in the presence of the E6 protein from human papillomavirus types 16 and 18. Our data show that the ubiquitin-protein ligase function of E6-AP is dispensable for its ability to coactivate nuclear hormone receptors, showing that E6-AP possesses two separable independent functions, as both a coactivator and a ubiquitin-protein ligase. Disruption of the maternal copy of E6-AP is correlated with Angelman syndrome (AS), a genetic neurological disorder characterized by severe mental retardation, seizures, speech impairment, and other symptoms. However, the exact mechanism by which the defective E6-AP gene causes AS remains unknown. To correlate the E6-AP coactivator function and ubiquitin-protein ligase functions with the AS phenotype, we expressed mutant forms of E6-AP isolated from AS patients and assessed the ability of each of these mutant proteins to coactivate PR or provide ubiquitin-protein ligase activity. This analysis revealed that in the majority of the AS patients examined, the ubiquitin-protein ligase function of E6-AP was defective whereas the coactivator function was intact. This finding suggests that the AS phenotype results from a defect in the ubiquitin-proteosome protein degradation pathway.
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PMID:The Angelman syndrome-associated protein, E6-AP, is a coactivator for the nuclear hormone receptor superfamily. 989 Oct 52

The E6 protein of the high-risk human papillomaviruses (HPVs) and the cellular ubiquitin-protein ligase E6AP form a complex which causes the ubiquitination and degradation of p53. We show here that HPV16 E6 promotes the ubiquitination and degradation of E6AP itself. The half-life of E6AP is shorter in HPV-positive cervical cancer cells than in HPV-negative cervical cancer cells, and E6AP is stabilized in HPV-positive cancer cells when expression of the viral oncoproteins is repressed. Expression of HPV16 E6 in cells results in a threefold decrease in the half-life of transfected E6AP. E6-mediated degradation of E6AP requires (i) the binding of E6 to E6AP, (ii) the catalytic activity of E6AP, and (iii) activity of the 26S proteasome, suggesting that E6-E6AP interaction results in E6AP self-ubiquitination and degradation. In addition, both in vitro and in vivo experiments indicate that E6AP self-ubiquitination results primarily from an intramolecular transfer of ubiquitin from the active-site cysteine to one or more lysine residues; however, intermolecular transfer can also occur in the context of an E6-mediated E6AP multimer. Finally, we demonstrate that an E6 mutant that is able to immortalize human mammary epithelial cells but is unable to degrade p53 retains its ability to bind and degrade E6AP, raising the possibility that E6-mediated degradation of E6AP contributes to its ability to transform mammalian cells.
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PMID:Human papillomavirus type 16 E6 induces self-ubiquitination of the E6AP ubiquitin-protein ligase. 1086 52

In normal cells, p53 is maintained at a low level by ubiquitin-mediated proteolysis, but after genotoxic insult this process is inhibited and p53 levels rise dramatically. Ubiquitination of p53 requires the ubiquitin-activating enzyme Ubc5 as a ubiquitin conjugation enzyme and Mdm2, which acts as a ubiquitin protein ligase. In addition to the N-terminal region, which is required for interaction with Mdm2, the C-terminal domain of p53 modulates the susceptibility of p53 to Mdm2-mediated degradation. To analyze the role of the C-terminal domain in p53 ubiquitination, we have generated p53 molecules containing single and multiple lysine-to-arginine changes between residues 370 and 386. Although wild-type (WT) and mutant molecules show similar subcellular distributions, the mutants display a higher transcriptional activity than WT p53. Simultaneous mutation of lysine residues 370, 372, 373, 381, 382, and 386 to arginine residues (6KR p53 mutant) generates a p53 molecule with potent transcriptional activity that is resistant to Mdm2-induced degradation and is refractory to Mdm2-mediated ubiquitination. In contrast to WT p53, transcriptional activity directed by the 6KR p53 mutant fails to be negatively regulated by Mdm2. Those differences are also manifest in HeLa cells which express the human papillomavirus E6 protein, suggesting that p53 C-terminal lysine residues are also implicated in E6-AP-mediated ubiquitination. These data suggest that p53 C-terminal lysine residues are the main sites of ubiquitin ligation, which target p53 for proteasome-mediated degradation.
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PMID:Multiple C-terminal lysine residues target p53 for ubiquitin-proteasome-mediated degradation. 1104 42

Angelman syndrome (AS), characterized by motor dysfunction, mental retardation, and seizures, is caused by several genetic etiologies involving chromosome 15q11-q13, including mutations of the UBE3A gene. UBE3A encodes UBE3A/E6-AP, a ubiquitin-protein ligase, and shows brain-specific imprinting, with brain expression predominantly from the maternal allele. Lack of a functional maternal allele of UBE3A causes AS. In order to understand the causal relationship between maternal UBE3A mutations and AS, we have constructed a mouse model with targeted inactivation of Ube3a. The inactive allele contains a lacZ reporter gene for analysis of brain-specific imprinting. Maternal, but not paternal, transmission of the targeted allele leads to beta-galactosidase activity in hippocampal and cerebellar neurons. Maternal inheritance of the Ube3a mutant allele also causes impaired performance in tests of motor function and spatial learning, as well as abnormal hippocampal EEG recordings. As predicted from the dependence of UBE3A-mediated ubiquitination of p53 on HPV E6 protein, our maternal-deficient mice show normal brain p53 levels.
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PMID:Neurobehavioral and electroencephalographic abnormalities in Ube3a maternal-deficient mice. 1189 68

The tumor suppressor p53 is a short-lived protein that under normal conditions is reduced to a barely detectable level. The stability of p53 protein is primarily regulated in normal non-transformed cells by two interplayers: Mdm2 and p14(ARF). Relocation of p53, Mdm2, and p14(ARF) to the nucleolus seems to regulate, at least partially, the steady-state of p53. Moreover, there are alternative pathways of the regulation of p53 stability in unstressed cells. Jun-N(amino)-terminal kinase (JNK) and poly(ADP-ribose) polymerase-1 (PARP-1) are involved in the regulation of the steady-state of wild-type (wt) p53 protein. However, in most human cervical carcinomas, which express the high-risk human papilloma viruses (HPVs) E6 protein, a complete switch from Mdm2 to HPV E6-mediated degradation of p53 occurs. Virally encoded E6 protein utilizes the cellular ubiquitin-protein ligase termed E6-associated protein (E6-AP) to target p53 protein for proteolytic degradation. We recently addressed the question of whether p53 protein can be generally reactivated by chemotherapy in HeLa cells despite the E6 activity. We observed an increase of cellular p53 after cisplatin (CP) treatment. p53 protein accumulated preferentially in the nucleoli. We checked the cellular level of E6 during CP therapy. Six hours after application of CP the expression of E6 protein was markedly reduced. This coincided with the increase of cellular p53 level and preceded the nucleolar accumulation of p53 protein, thereby indicating that repression of virally coded E6 protein by CP contributes to the restoration of p53 expression.
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PMID:How the nucleolar sequestration of p53 protein or its interplayers contributes to its (re)-activation. 1503 32

Numerous reports have raised the level of national concern that chemicals found in the environment may have adverse effects on the health of humans and wildlife. Environmental exposure to pollutants, such as dioxin, has been implicated in gonadal tumor formation in Maine softshell clams (Mya arenaria). Prevalence of these tumors is as high as 40% in some populations. Although their etiology is still unknown, investigations into the mechanisms of tumor formation have revolved around a hypothesis of dioxin-induced toxicity. The aryl hydrocarbon receptor (AHR) was initially investigated, but was later determined to not bind the prototypical ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), suggesting that dioxin toxicity is mediated through an AHR-independent pathway. An alternative mechanism of tumor formation has been investigated, involving a protein with significant sequence similarity to mammalian E6AP, a HECT (homologous to E6AP carboxy terminus) E3 ubiquitin-protein ligase. E6AP, in association with the high-risk human papillomavirus (HPV) E6 protein, is involved in the abnormal degradation of the p53 tumor suppressor protein in human cervical cancer. Tumorigenic clam reproductive tissue revealed higher M. arenaria E3 (MaE3) protein levels concomitant with lower M. arenaria p53 (Map53) levels. While the function of MaE3 as a HECT E3 was verified, results from three methods agree that MaE3 does not associate with Map53. However, alteration in Map53 levels may still play a role in clam gonadal tumorigenesis. Due to upregulation of MaE3 in neoplastic reproductive tissue, further investigations will focus on determining the proteolytic targets of MaE3. In conjunction with our previous findings that dioxin toxicity in the softshell clam is not mediated by AHR, the results from our current investigation suggest a complex etiology for the clam germinomas.
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PMID:A HECT E3 ubiquitin-protein ligase with sequence similarity to E6AP does not target p53 for degradation in the softshell clam (Mya arenaria). 1528 42


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