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 anaphase-promoting complex (APC) is a cell cycle-regulated ubiquitin-protein ligase that targets cyclin B, securin and other destruction box containing proteins for proteolysis. Nine APC subunits have been identified in vertebrates and eleven in yeast, but for none of them it is known how they contribute to the catalysis of ubiquitination reactions. Here we report the mass spectrometric identification of CDC26 and of the RING-H2 finger protein APC11 in the human APC. We have expressed these proteins and several other APC subunits in Escherichia coli and have tested their activities in vitro. We find that APC11 alone is sufficient to allow the synthesis of multiubiquitin chains in the presence of E1 and UBC4. These multiubiquitin chains are partly unanchored and partly bound to APC11 itself. APC11 and UBC4 are also able to ubiquitinate securin and cyclin B, but these reactions show a decreased dependency on the destruction box. The integrity of the putative zinc binding RING-H2 finger is required for the ability of APC11 to support ubiquitination reactions. These results suggest that APC11 and UBC4 catalyze the formation of isopeptide bonds in APC-mediated ubiquitination reactions.
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PMID:The RING-H2 finger protein APC11 and the E2 enzyme UBC4 are sufficient to ubiquitinate substrates of the anaphase-promoting complex. 1092 56

The endoplasmic reticulum contains a protein quality control system that discovers malfolded or unassembled secretory proteins and subjects them to degradation in the cytosol. This requires retrograde transport of the respective proteins from the endoplasmic reticulum back to the cytosol via the Sec61 translocon. In addition, a fully competent ubiquitination machinery and the 26 S proteasome are necessary for retrotranslocation and degradation. Ubiquitination of mutated and malfolded proteins of the endoplasmic reticulum is dependent mainly on the ubiquitin-conjugating enzyme Ubc7p. In addition, several new membrane components of the endoplasmic reticulum are required for degradation. Here we present the topology of the previously discovered RING-H2 finger protein Der3/Hrd1p, one of the new components of the endoplasmic reticulum membrane. The protein spans the membrane six times. The amino terminus and the carboxyl terminus containing the RING finger domain face the cytoplasm. Altogether, RING finger-dependent ubiquitination of malfolded carboxypeptidase yscY in vivo, as well as of Der3/Hrd1p itself in vitro and RING finger-dependent binding of Ubc7p, uncovers Der3/Hrd1p as the ubiquitin-protein ligase (E3) of the endoplasmic reticulum-associated protein degradation process.
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PMID:Membrane topology and function of Der3/Hrd1p as a ubiquitin-protein ligase (E3) involved in endoplasmic reticulum degradation. 1113 75

We recently reported the identification of a RING finger-containing protein, HHARI (human homologue of Drosophila ariadne), which binds to the human ubiquitin-conjugating enzyme UbcH7 in vitro. We now demonstrate that HHARI interacts and co-localizes with UbcH7 in mammalian cells, particularly in the perinuclear region. We have further defined a minimal interaction region of HHARI comprising residues 186-254, identified individual amino acid residues essential for the interaction, and determined that the distance between the RING1 finger and IBR (in between RING fingers) domains is critical to maintaining binding. We have also established that the RING1 finger of HHARI cannot be substituted for by the highly homologous RING finger domains of either of the ubiquitin-protein ligase components c-CBL or Parkin, despite their similarity in structure and their independent capabilities to bind UbcH7. Furthermore, mutation of the RING1 finger domain of HHARI from a RING-HC to a RING-H2 type abolishes interaction with UbcH7. These studies demonstrate that very subtle changes to the domains that regulate recognition between highly conserved components of the ubiquitin pathway can dramatically affect their ability to interact.
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PMID:Features of the parkin/ariadne-like ubiquitin ligase, HHARI, that regulate its interaction with the ubiquitin-conjugating enzyme, Ubch7. 1127 16

EL5, a rice gene responsive to N-acetylchitooligosaccharide elicitor, encodes a RING-H2 finger protein with structural features common to the plant-specific ATL family. We show that the fusion protein of EL5 with maltose binding protein (MBP) was polyubiquitinated by incubation with ubiquitin, ubiquitin-activating enzyme (E1), and the Ubc4/5 subfamily of the ubiquitin-conjugating enzyme (E2). EL5 possesses the activity to catalyse the transfer of ubiquitin to the MBP moiety, and the RING-H2 finger motif of EL5 is necessary for this activity. Thus, we concluded that EL5 represents a ubiquitin ligase (E3). We also show that two rice E2s (OsUBC5a, OsUBC5b) of the Ubc4/5 subfamily function as E2 which catalyses EL5-mediated ubiquitination, and OsUBC5b was induced by elicitor, as well as EL5. These results strongly suggest that EL5 and OsUBC5b have roles in plant defense response through the turnover of protein(s) via the ubiquitin/proteasome system.
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PMID:EL5, a rice N-acetylchitooligosaccharide elicitor-responsive RING-H2 finger protein, is a ubiquitin ligase which functions in vitro in co-operation with an elicitor-responsive ubiquitin-conjugating enzyme, OsUBC5b. 1202 74

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

Cellular as well as viral RNAs are usually found complexed with proteins. In an attempt to identify proteins that interact with transcripts of hepatitis B virus (HBV), a DNA virus that replicates through reverse transcription, a partial cDNA was isolated from a human cDNA expression library whose gene product bound to an HBV-derived RNA. Using an overlapping clone from a molecular hybridization screen a full-length cDNA was assembled. It contained a large open reading frame for a 1208 amino-acid protein of 138 kDa identical to the hypothetical product of the KIAA0675 clone. Closely related sequences are present in mouse cDNA libraries but not in the genomes of lower organisms. The protein sequence contained no known RNA-binding domain and, apart from a probable coiled-coil domain, the only significant homology involved a complete RING-H2 motif. This suggested that the protein might be a novel RNA-binding RING-dependent ubiquitin-protein ligase or E3 enzyme. A motif critical for RNA binding was experimentally mapped to a central Lys-rich region. Binding specificity is either broad or the protein has as yet unknown physiological targets; hence, at present, a potential importance for HBV biology remains open. The RING-H2 domain was functional in and essential for self- and trans-ubiquitylation in vitro and for proteasome-mediated turnover of the protein in vivo. We therefore termed it hRUL138 for human RNA-binding ubiquitin ligase of 138 kDa. hRUL138 mRNAs are expressed at low levels in most tissues. GFP-tagged hRUL138 derivatives were found associated with cytoplasmic structures, possibly the ER, but excluded from the nucleus. The combined presence of RNA binding and E3 activity in hRUL138 raises the possibility that both are mechanistically linked.
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PMID:hRUL138, a novel human RNA-binding RING-H2 ubiquitin-protein ligase. 1253 61

EL5, a RING-H2 finger protein, is rapidly induced by N-acetylchitooligosaccharides in rice cell. We expressed the EL5 RING-H2 finger domain in Escherichia coli and determined its structure in solution by NMR spectroscopy. The EL5 RING-H2 finger domain consists of two-stranded beta-sheets (beta1, Ala(147)-Phe(149); beta2, Gly(156)-His(158)), one alpha-helix (Cys(161)-Leu(166)), and two large N- and C-terminal loops. It is stabilized by two tetrahedrally coordinated zinc ions. This structure is similar to that of other RING finger domains of proteins of known function. From structural analogies, we inferred that the EL5 RING-H2 finger is a binding domain for ubiquitin-conjugating enzyme (E2). The binding site is probably formed by solvent-exposed hydrophobic residues of the N- and C-terminal loops and the alpha-helix. We demonstrated that the fusion protein with EL5-(96-181) and maltose-binding protein (MBP) was polyubiquitinated by incubation with ubiquitin, ubiquitin-activating enzyme (E1), and a rice E2 protein, OsUBC5b. This supported the idea that the EL5 RING finger domain is essential for ubiquitin-ligase activity of EL5. By NMR titration experiments, we identified residues that are critical for the interaction between the EL5 RING-H2 finger and OsUBC5b. We conclude that the RING-H2 finger domain of EL5 is the E2 binding site of EL5.
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PMID:High precision NMR structure and function of the RING-H2 finger domain of EL5, a rice protein whose expression is increased upon exposure to pathogen-derived oligosaccharides. 1258 69

The p53 tumor suppressor exerts anti-proliferative effects in response to various types of stress including DNA damage and abnormal proliferative signals. Tight regulation of p53 is essential for maintaining normal cell growth and this occurs primarily through posttranslational modifications of p53. Here, we describe Pirh2, a gene regulated by p53 that encodes a RING-H2 domain-containing protein with intrinsic ubiquitin-protein ligase activity. Pirh2 physically interacts with p53 and promotes ubiquitination of p53 independently of Mdm2. Expression of Pirh2 decreases the level of p53 protein and abrogation of endogenous Pirh2 expression increases the level of p53. Furthermore, Pirh2 represses p53 functions including p53-dependent transactivation and growth inhibition. We propose that Pirh2 is involved in the negative regulation of p53 function through physical interaction and ubiquitin-mediated proteolysis. Hence, Pirh2, like Mdm2, participates in an autoregulatory feedback loop that controls p53 function.
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PMID:Pirh2, a p53-induced ubiquitin-protein ligase, promotes p53 degradation. 1265 45

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

p53-Induced ring-H2 protein (Pirh2), a recently identified ubiquitin-protein ligase, interacts with p27(Kip1) to promote ubiquitination of p27(Kip1) independently of p53. High Pirh2 and low p27(Kip1) immunoreactivity are associated with a poor prognosis in several cancers, including resistant phenotypes. In the present study, we investigated the role of Pirh2 and p27(Kip1) in human hepatocellular carcinoma (HCC) progression. Immunohistochemical analysis was performed on formalin-fixed paraffin sections of 87 specimens. Statistical analysis showed that expression of Pirh2 was negatively related to p27(Kip1) expression (r = 0.787; P < .05), and Pirh2 expression correlated significantly with histologic grade (P < .001), venous invasion (P = .004), tumor size (P = .024), and the presence of multiple tumor-bearing lymph nodes (P = .017), whereas p27(Kip1) expression correlated significantly with histologic grade (P < .001), venous invasion (P = .048), and cirrhosis (P = .028). By Kaplan-Meier analysis, the survival curves of low versus high expressers of Pirh2 and p27(Kip1) showed significant separation (P < .01). Molecular interaction could be demonstrated between Pirh2 and p27(Kip1) in three HCC cell lines. In vitro, following release of two HCC cell lines from serum starvation, the expression of Pirh2 was upregulated, whereas p27(Kip1) was downregulated. Our results suggest that Pirh2 mediates the degradation of p27(Kip1) and participates in cell proliferation in human HCC. These findings provide a rational framework for further development of Pirh2 inhibitors as a novel class of anti-tumor agents.
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PMID:Expression of Pirh2, a p27(Kip1) ubiquitin ligase, in hepatocellular carcinoma: correlation with p27(Kip1) and cell proliferation. 2123 67


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