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)

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

Ubiquitin-conjugating enzyme variants share significant sequence similarity with typical E2 (ubiquitin-conjugating) enzymes of the protein ubiquitination pathway but lack their characteristic active site cysteine residue. The MMS2 gene of Saccharomyces cerevisiae encodes one such ubiquitin-conjugating enzyme variant that is involved in the error-free DNA postreplicative repair pathway through its association with Ubc13, an E2. The Mms2-Ubc13 heterodimer is capable of linking ubiquitin molecules to one another through an isopeptide bond between the C terminus and Lys-63. Using highly purified components, we show here that the human forms of Mms2 and Ubc13 associate into a heterodimer that is stable over a range of conditions. The ubiquitin-thiol ester form of the heterodimer can be produced by the direct activation of its Ubc13 subunit with E1 (ubiquitin-activating enzyme) or by the association of Mms2 with the Ubc13-ubiquitin thiol ester. The activated heterodimer is capable of transferring its covalently bound ubiquitin to Lys-63 of an untethered ubiquitin molecule, resulting in diubiquitin as the predominant species. In (1)H (15)N HSQC ((1)H (15)N heteronuclear single quantum coherence) NMR experiments, we have mapped the surface determinants of tethered and untethered ubiquitin that interact with Mms2 and Ubc13 in both their monomeric and dimeric forms. These results have identified a surface of untethered ubiquitin that interacts with Mms2 in the monomeric and heterodimeric form. Furthermore, the C-terminal tail of ubiquitin does not participate in this interaction. These results suggest that the role of Mms2 is to correctly orient either a target-bound or untethered ubiquitin molecule such that its Lys-63 is placed proximally to the C terminus of the ubiquitin molecule that is linked to the active site of Ubc13.
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PMID:Noncovalent interaction between ubiquitin and the human DNA repair protein Mms2 is required for Ubc13-mediated polyubiquitination. 1150 15

A small molecule inhibitor of NF-kappaB-dependent cytokine expression was discovered that blocked tumor necrosis factor (TNF) alpha-induced IkappaB(alpha) degradation in MM6 cells but not the degradation of beta-catenin in Jurkat cells. Ro106-9920 blocked lipopolysaccharide (LPS)-dependent expression of TNFalpha, interleukin-1beta, and interleukin-6 in fresh human peripheral blood mononuclear cells with IC(50) values below 1 microm. Ro106-9920 also blocked TNFalpha production in a dose-dependent manner following oral administration in two acute models of inflammation (air pouch and LPS challenge). Ro106-9920 was observed to inhibit an ubiquitination activity that does not require betaTRCP but associates with IkappaB(alpha) and will ubiquitinate IkappaB(alpha) S32E,S36E (IkappaB(alpha)(ee)) specifically at lysine 21 or 22. Ro106-9920 was identified in a cell-free system as a time-dependent inhibitor of IkappaB(alpha)(ee) ubiquitination with an IC(50) value of 2.3 +/- 0.09 microm. The ubiquitin E3 ligase activity is inhibited by cysteine-alkylating reagents, supported by E2UBCH7, and requires cIAP2 or a cIAP2-associated protein for activity. These activities are inconsistent with what has been reported for SCF(betaTRCP), the putative E3 for IkappaB(alpha) ubiquitination. Ro106-9920 was observed to be selective for IkappaB(alpha)(ee) ubiquitination over the ubiquitin-activating enzyme (E1), E2UBCH7, nonspecific ubiquitination of cellular proteins, and 97 other molecular targets. We propose that Ro106-9920 selectively inhibits an uncharacterized but essential ubiquitination activity associated with LPS- and TNFalpha-induced IkappaB(alpha) degradation and NF-kappaB activation.
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PMID:A small molecule ubiquitination inhibitor blocks NF-kappa B-dependent cytokine expression in cells and rats. 1195 Aug 39

The fission yeast Schizosaccharomyces pombe has three putative ubiquitin-protein ligases of the Nedd4/Rsp5 family, named Pub1p, Pub2p and Pub3p. Pub1p has been reported to be involved in cell cycle regulation and proliferation under acidic pH conditions. Here we characterize Pub2p, which contains a conserved HECT domain and a WW domain but lacks a C2 domain. Transcription of the pub2(+) gene was constitutive and further enhanced by nitrogen starvation. A pub2-null mutation gave no remarkable phenotypes, but intensified temperature sensitivity in a pub1Delta background. Moderately overexpressed pub2(+) suppressed the temperature sensitivity of pub1Delta cells, which suggests that the function of Pub2p overlaps with that of Pub1p. Overexpression of pub2(+) by a strong nmt1 promoter in wild-type strains caused growth arrest and cell elongation, probably owing to defects in G2 progression or the G2/M transition. Unlike Pub1p, however, overexpression of Pub2p did not reduce the levels of Cdc25p. Pub2-GFP was found throughout the cell, especially at the cell surface in the polar regions. Pub2p contains a conserved cysteine residue (Cys639) in its putative catalytic HECT domain that can be thiol-ubiquitinated. Substitution of Cys639 by alanine (Pub2CA) caused a functional defect, because growth arrest and cell elongation were not induced by overexpression of Pub2CA. A chimeric Pub1 protein, in which the HECT domain was replaced by the Pub2 HECT domain, completely suppressed the temperature sensitivity of pub1Delta cells, suggesting that the HECT domain of Pub2p has the catalytic activity of a ubiquitin ligase. We conclude that Pub2p is a HECT-type ubiquitin-protein ligase that shares partially overlapping function with Pub1p.
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PMID:The novel HECT-type ubiquitin-protein ligase Pub2p shares partially overlapping function with Pub1p in Schizosaccharomyces pombe. 1195 16

The androgen receptor (AR) N-terminal domain plays a critical role in androgen-responsive gene regulation. A novel AR N-terminal-interacting protein (ARNIP) was isolated using the yeast two-hybrid system and its interaction with amino acids 11-172 of the normal or corresponding region of the polyglutamine-expanded human AR confirmed by glutathione S-transferase pulldown assays. ARNIP cDNAs cloned from NSC-34 (mouse neuroblastoma/spinal cord) or PC-3 (human prostate adenocarcinoma) mRNA encoded highly homologous 30 kDa (261 amino acids) cysteine-rich proteins with a RING-H2 (C3H2C3 zinc finger) domain; this motif is highly conserved in predicted ARNIP-homologous proteins from several other species. Expression of the approximately 1.7 kb ARNIP mRNA was detected in various tissues by Northern blotting, but was highest in mouse testes, kidney and several neuronal cell lines. In addition, the human ARNIP protein was found to be encoded by nine exons spanning 32 kb on chromosome 4q21. In COS-1 cells, coexpression of ARNIP and AR did not affect AR ligand-binding kinetics, nor did ARNIP act as a coactivator or corepressor in transactivation assays. However, AR N-terminal:C-terminal interaction was reduced in the presence of ARNIP. Intriguingly, ARNIP, and in particular its RING-H2 domain, functioned as a ubiquitin-protein ligase in vitro in the presence of a specific ubiquitin-conjugating enzyme, Ubc4-1. Mutation of a single cysteine residue in the ARNIP RING-H2 domain (Cys145Ala) abolished this E3 ubiquitin ligase activity. Fluorescent protein tagging studies revealed that AR-ARNIP interaction was hormone-independent in COS-1 cells, and suggest that colocalization of both AR and ARNIP to the nucleus upon androgen addition may allow ARNIP to play a role in nuclear processes. Thus, identification of a novel AR-interacting protein with ubiquitin ligase activity will stimulate further investigation into the role of ubiquitination and the ubiquitin-proteasome system in AR-mediated cellular functions.
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PMID:Cloning and characterization of an androgen receptor N-terminal-interacting protein with ubiquitin-protein ligase activity. 1220 Feb 28

The anaphase-promoting complex (APC) is a ubiquitin-protein ligase (E3) that targets cell cycle regulators such as cyclin B and securin for degradation. The APC11 subunit functions as the catalytic core of this complex and mediates the transfer of ubiquitin from a ubiquitin-conjugating enzyme (E2) to the substrate. APC11 contains a RING-H2-finger domain, which includes one histidine and seven cysteine residues that coordinate two Zn(2+) ions. We now show that exposure of purified APC11 to H(2)O(2) (0.1 to 1 mM) induced the release of bound zinc as a result of the oxidation of cysteine residues. It also impaired the physical interaction between APC11 and the E2 enzyme Ubc4 as well as inhibited the ubiquitination of cyclin B1 by APC11. The release of HeLa cells from metaphase arrest in the presence of exogenous H(2)O(2) inhibited the ubiquitination of cyclin B1 as well as the degradation of cyclin B1 and securin that were apparent in the absence of H(2)O(2). The presence of H(2)O(2) also blocked the co-immunoprecipitation of Ubc4 with APC11 and delayed the exit of cells from mitosis. Inhibition of APC11 function by H(2)O(2) thus likely contributes to the delay in cell cycle progression through mitosis that is characteristic of cells subjected to oxidative stress.
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PMID:The RING-H2-finger protein APC11 as a target of hydrogen peroxide. 1525 23

Protein ubiquitination requires the sequential activity of three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-ligase (E3). The ubiquitin-transfer machinery is hierarchically organized; for every ubiquitin-activating enzyme, there are several ubiquitin-conjugating enzymes, and most ubiquitin-conjugating enzymes can in turn interact with multiple ubiquitin ligases. Despite the central role of ubiquitin-activating enzyme in this cascade, a crystal structure of a ubiquitin-activating enzyme is not available. The enzyme is thought to consist of an adenylation domain, a catalytic cysteine domain, a four-helix bundle, and possibly, a ubiquitin-like domain. Its adenylation domain can be modeled because it is clearly homologous to the structurally known adenylation domains of the activating enzymes for the small ubiquitin-like modifier (SUMO) and for the protein encoded by the neuronal precursor cell-expressed, developmentally down-regulated gene 8 (NEDD8). Low sequence similarity and vastly different domain lengths make modeling difficult for the catalytic cysteine domain that results from the juxtaposition of two catalytic cysteine half-domains. Here, we present a biochemical and crystallographic characterization of the two half-domains and the crystal structure of the larger, second catalytic cysteine half-domain of mouse ubiquitin-activating enzyme. We show that the domain is organized around a conserved folding motif that is also present in the NEDD8- and SUMO-activating enzymes, and we propose a tentative model for full-length ubiquitin-activating enzyme.
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PMID:Crystal structure of a fragment of mouse ubiquitin-activating enzyme. 1577 60

5-Azacytidine- and 5-aza-deoxycytidine (5-aza-CdR)-mediated reactivation of tumor suppressor genes silenced by promoter methylation has provided an alternate approach in cancer therapy. Despite the importance of epigenetic therapy, the mechanism of action of DNA-hypomethylating agents in vivo has not been completely elucidated. Here we report that among three functional DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), the maintenance methyltransferase, DNMT1, was rapidly degraded by the proteasomal pathway upon treatment of cells with these drugs. The 5-aza-CdR-induced degradation, which occurs in the nucleus, could be blocked by proteasomal inhibitors and required a functional ubiquitin-activating enzyme. The drug-induced degradation occurred even in the absence of DNA replication. Treatment of cells with other nucleoside analogs modified at C-5, 5-fluorodeoxyuridine and 5-fluorocytidine, did not induce the degradation of DNMT1. Mutation of cysteine at the catalytic site of Dnmt1 (involved in the formation of a covalent intermediate with cytidine in DNA) to serine (CS) did not impede 5-aza-CdR-induced degradation. Neither the wild type nor the catalytic site mutant of Dnmt3a or Dnmt3b was sensitive to 5-aza-CdR-mediated degradation. These results indicate that covalent bond formation between the enzyme and 5-aza-CdR-incorporated DNA is not essential for enzyme degradation. Mutation of the conserved KEN box, a targeting signal for proteasomal degradation, to AAA increased the basal level of Dnmt1 and blocked its degradation by 5-aza-CdR. Deletion of the catalytic domain increased the expression of Dnmt1 but did not confer resistance to 5-aza-CdR-induced degradation. Both the nuclear localization signal and the bromo-adjacent homology domain were essential for nuclear localization and for the 5-aza-CdR-mediated degradation of Dnmt1. Polyubiquitination of Dnmt1 in vivo and its stabilization upon treatment of cells with a proteasomal inhibitor indicate that the level of Dnmt1 is controlled by ubiquitin-dependent proteasomal degradation. Overexpression of the substrate recognition component, Cdh1 but not Cdc20, of APC (anaphase-promoting complex)/cyclosome ubiquitin ligase reduced the level of Dnmt1 in both untreated and 5-aza-CdR-treated cells. In contrast, the depletion of Cdh1 with small interfering RNA increased the basal level of DNMT1 that blocked 5-aza-CdR-induced degradation. Dnmt1 interacted with Cdh1 and colocalized in the nucleus at discrete foci. Both Dnmt1 and Cdh1 were phosphorylated in vivo, but only Cdh1 was significantly dephosphorylated upon 5-aza-CdR treatment, suggesting its involvement in initiating the proteasomal degradation of DNMT1. These results demonstrate a unique mechanism for the selective degradation of DNMT1, the maintenance DNA methyltransferase, by well-known DNA-hypomethylating agents.
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PMID:5-Aza-deoxycytidine induces selective degradation of DNA methyltransferase 1 by a proteasomal pathway that requires the KEN box, bromo-adjacent homology domain, and nuclear localization signal. 2971 69

Mutations in the parkin gene, encoding an E3 ubiquitin-protein ligase, are a frequent cause of autosomal recessive parkinsonism and are also involved in sporadic Parkinson's disease. Loss of Parkin function is thought to compromise the polyubiquitylation and proteasomal degradation of specific substrates, leading to their deleterious accumulation. Several studies have analyzed the effects of parkin gene mutations on the biochemical properties of the protein. However, the absence of a cell-free system for studying intrinsic Parkin activity has limited the interpretation of these studies. Here we describe the biochemical characterization of Parkin and 10 pathogenic variants carrying amino-acid substitutions throughout the sequence. Mutations in the RING fingers or the ubiquitin-like domain decreased the solubility of the protein in detergent and increased its tendency to form visible aggregates. None of the mutations studied compromised the binding of Parkin to a series of known protein partners/substrates. Moreover, only two variants with substitutions of conserved cysteine residues of the second RING finger were inactive in a purely in vitro ubiquitylation assay, demonstrating that loss of ligase activity is a minor pathogenic mechanism. Interestingly, in this in vitro assay, Parkin catalyzed the linkage of single ubiquitin molecules only, whereas the ubiquitin-protein ligases CHIP and Mdm2 promoted the formation of polyubiquitin chains. Similarly, in mammalian cells Parkin promoted the multimonoubiquitylation of its substrate p38, rather than its polyubiquitylation. Thus, Parkin may mediate polyubiquitylation or proteasome-independent monoubiquitylation depending on the protein context. The discovery of monoubiquitylated Parkin species in cells hints at a novel post-translational modification potentially involved in the regulation of Parkin function.
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PMID:Biochemical analysis of Parkinson's disease-causing variants of Parkin, an E3 ubiquitin-protein ligase with monoubiquitylation capacity. 1671

UBE1 is known as the human ubiquitin-activating enzyme (E1), which activates ubiquitin in an ATP-dependent manner. Here, we identified a novel human ubiquitin-activating enzyme referred to as UBE1L2, which also shows specificity for ubiquitin. The UBE1L2 sequence displays a 40% identity to UBE1 and also contains an ATP-binding domain and an active site cysteine conserved among E1 family proteins. UBE1L2 forms a covalent link with ubiquitin in vitro and in vivo, which is sensitive to reducing conditions. In an in vitro polyubiquitylation assay, recombinant UBE1L2 could activate ubiquitin and transfer it onto the ubiquitin-conjugating enzyme UbcH5b. Ubiquitin activated by UBE1L2 could be used for ubiquitylation of p53 by MDM2 and supported the autoubiquitylation of the E3 ubiquitin ligases HectH9 and E6-AP. The UBE1L2 mRNA is most abundantly expressed in the testis, suggesting an organ-specific regulation of ubiquitin activation.
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PMID:UBE1L2, a novel E1 enzyme specific for ubiquitin. 1758 Mar 10


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