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 ubiquitin-protein ligase (E3), hRPF1/Nedd4, is a component of the ubiquitin-proteasome pathway responsible for substrate recognition and specificity. Although previously characterized as a regulator of the stability of cytoplasmic proteins, hRPF1/Nedd4 has also been suggested to have a role in the nucleus. However, in light of the cytoplasmic localization of hRPF1/Nedd4, it is unclear whether bona fide nuclear substrates of hRPF1/Nedd4 exist, and if so, what mechanism may allow a cytoplasmic ubiquitin ligase to manifest nuclear activity. Our search for nuclear substrates led to the identification of the human proline-rich transcript, brain-expressed (hPRTB) protein, the ubiquitination and degradation of which is regulated by hRPF1/Nedd4. Interestingly, hPRTB colocalizes with the splicing factor SC35 in nuclear speckles. Finally, we demonstrate that hRPF1/Nedd4 is indeed capable of entering the nucleus; however, the presence of a functional Rev-like nuclear export sequence in hRPF1/Nedd4 ensures a predominant cytoplasmic localization. Cumulatively, these findings highlight a nuclear role for the ubiquitin ligase hRPF1/Nedd4 and underscore cytoplasmic/nuclear localization as an important regulatory component of hRPF1/Nedd4-substrate recognition.
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PMID:Nuclear import/export of hRPF1/Nedd4 regulates the ubiquitin-dependent degradation of its nuclear substrates. 1134 38

The von Hippel-Lindau tumor-suppressor protein (pVHL) forms a protein complex (VCB-Cul2) with elongin C, elongin B, Cul-2, and Rbx1, which functions as a ubiquitin-protein ligase (E3). The alpha-subunits of the hypoxia-inducible factors have been identified as targets for the VCB-Cul2 ubiquitin ligase. However, a variety of cellular defects caused by the depletion of pVHL cannot be explained solely by the ubiquitin-mediated degradation of hypoxia-inducible factor-alpha. We show here that a member of the atypical protein kinase C (PKC) group, PKClambda, is ubiquitinated by the pVHL-containing E3 enzyme. An active PKClambda mutant is ubiquitinated more extensively than wild-type PKClambda in HEK293 cells, and the ubiquitination is further enhanced by the overexpression of pVHL. The activation of wild-type PKClambda by serum stimulation of cells enhances the ubiquitination of the protein, supporting the notion that active PKClambda is preferentially ubiquitinated by VCB-Cul2 ubiquitin ligase. Furthermore, we show that PKClambda can be ubiquitinated in vitro in a cell-free ubiquitination assay using purified recombinant components including VCB-Cul2. Given the known function of aPKC in the regulation of cell polarity and cell growth, PKClambda may be a target of pVHL in its function as a tumor suppressor.
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PMID:The von Hippel-Lindau tumor suppressor protein mediates ubiquitination of activated atypical protein kinase C. 1157 46

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

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

The anaphase-promoting complex (APC/C) is a large ubiquitin-protein ligase which controls progression through anaphase by triggering the degradation of cell cycle regulators such as securin and B-type cyclins. The APC/C is an unusually complex ligase containing at least 10 different, evolutionarily conserved components. In contrast to APC/C's role in cell cycle regulation little is known about the functions of individual subunits and how they might interact with each other. Here, we have analyzed Swm1/Apc13, a small subunit recently identified in the budding yeast complex. Database searches revealed proteins related to Swm1/Apc13 in various organisms including humans. Both the human and the fission yeast homologues are associated with APC/C subunits, and they complement the phenotype of an SWM1 deletion mutant of budding yeast. Swm1/Apc13 promotes the stable association with the APC/C of the essential subunits Cdc16 and Cdc27. Accordingly, Swm1/Apc13 is required for ubiquitin ligase activity in vitro and for the timely execution of APC/C-dependent cell cycle events in vivo.
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PMID:Swm1/Apc13 is an evolutionarily conserved subunit of the anaphase-promoting complex stabilizing the association of Cdc16 and Cdc27. 1506 Jan 74

Mutations in the parkin gene cause autosomal-recessive juvenile parkinsonism. Parkin encodes a ubiquitin-protein ligase characterized by having the RBR domain, composed of two RING fingers plus an IBR/DRIL domain. The RBR family is defined as the group of genes whose products contain an RBR domain. RBR family members exist in all eukaryotic species for which significant sequence data is available, including animals, plants, fungi, and several protists. The integration of comparative genomics with structural and functional data allows us to conclude that RBR proteins have multiple roles, not only in protein quality control mechanisms, but also as indirect regulators of transcription. A recently formulated hypothesis, based on a case of gene fusion, suggested that RBR proteins may be often part of cullin-containing ubiquitin ligase complexes. Recent data on Parkin protein agrees with that hypothesis. We discuss the involvement of RBR proteins in several neurodegenerative diseases and cancer.
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PMID:Parkin and relatives: the RBR family of ubiquitin ligases. 1515 79

We have analyzed the chromosome 6q21 breakpoint of a non-constitutional t(6;15)(q21;q21) rearrangement in sporadic Wilms' tumor. This identified a novel gene encoding a protein with six N-terminal ankyrin repeats linked to a C-terminal HECT ubiquitin-protein ligase domain. We therefore designated this gene HACE1 (HECT domain and Ankyrin repeat Containing E3 ubiquitin-protein ligase 1). HACE1 is widely expressed in human tissues, including mature and fetal kidney. We show that Hace1 protein possesses intrinsic ubiquitin ligase activity, utilizes UbcH7 as a candidate partner E2 enzyme and localizes predominantly to the endoplasmic reticulum. Although the HACE1 locus was not directly interrupted by the translocation in the index Wilms' case, its expression was markedly lower in tumor tissue compared with adjacent normal kidney. Moreover, HACE1 expression was virtually undetectable in the SK-NEP-1 Wilms' tumor cell line and in four of five additional primary Wilms' tumor cases compared with patient-matched normal kidney. We found no evidence of HACE1 mutations or deletions, but hypermethylation of two upstream CpG islands correlates with low HACE1 expression in tumor samples. Our findings implicate Hace1 as a novel ubiquitin-protein ligase and demonstrate that its expression is very low in primary Wilms' tumors.
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PMID:Differential expression of a novel ankyrin containing E3 ubiquitin-protein ligase, Hace1, in sporadic Wilms' tumor versus normal kidney. 1525 18

Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease. It is urgently needed to elucidate the cause of the disease and to establish neuroprotective treatment. We have been working on the etiology and pathogenesis of PD for many years and we found selective loss of mitochondrial complex I and the alpha-ketoglutarate dehydrogenase complex in the nigral neurons of patients with PD. Our observation firmly established mitochondrial defects in PD. Mitochondrial respiratory failure induces oxidative damage in neurons, and we found increase in hydroxynonenal and 8-oxo-deoxyguanine, indices of oxidative damage, in the nigral neurons of PD. These abnormalities can trigger apoptotic cell death. The primary events which induce mitochondrial failure and oxidative damage are not known, however, it has been postulated that the interaction of genetic risk factors and environmental factors would initiate the degenerative process. Based on this assumption, we conducted genetic association studies by the candidate gene methods. We found that polymorphic mutations of superoxide dismutase-2 and 24-kDa subunit of mitochondrial complex I were associated increased risk of developing Parkinson's disease. While we were doing this genetic association study, we found a family, in which parkinsonian phenotype completely segregated with a polymorphic mutation of the superoxide dismutase-2 gene. In this family, 4 out of 6 siblings were affected with early onset parkinsonism and the parents were apparently normal. Thus the mode of inheritance appeared to be autosomal recessive and this type is now called as AR-JP or Park2. We confirmed the linkage of this type of familial Parkinson's disease to the superoxide dismutase loci that is located in the telomeric region of chromosome 6 by the linkage analysis using microsatellite markers in this region. Then we found another family, in which an affected patient showed lack of one of the microsatellite markers (D6S315), which we were using in the linkage analysis. This observation prompted us to initiate the molecular cloning of the disease gene utilizing D6S315 as the initial probe. The molecular cloning was done with the collaboration with Professor Nobuyoshi Shimizu of Keio University. We identified a novel gene and confirmed that mutations of this novel gene were found only in the patients with autosomal recessive Parkinson's disease. The novel gene was named parkin. We conducted mutational analysis on more than 700 families with Parkinson's disease. We also established a method to detect compound heterozygotes of parkin mutations. Mutinous of the parkin gene were found in approximately 50% of autosomal recessive families. Many kinds of exonic deletions and point mutations were found. This type of familial Parkinson's disease had been considered to be unique among Japanese, but since we started mutational analysis of the parkin gene, we confirmed the world wide distribution of parkin gene mutations. Then we analyzed functions of parkin protein with the collaboration with Dr. Keiji Tanaka of Tokyo Metropolitan Institute of Medical Sciences. We found that parkin protein was a ubiquitin-protein ligase of the ubiquitin system. Now we are working on the candidate substrates of parkin protein as a ubiquitin ligase. We found that CDCrel-1, a synaptic vesicle protein, was a candidate substrate of parkin protein. In addition, we found two additional candidate proteins, i.e., alpha-synuclein 22 and PAEL receptor, with the collaboration of Professor Denis Selkoe of Harvard Medical School and Dr. Ryosuke Takahashi of RIKEN, respectively. Accumulation of PAEL receptor in the endoplasmic reticulum causes endoplasmic reticulum stress and apoptotic cell death. We found evidence to indicate accumulation of PAEL receptor and the presence of endoplasmic reticulum stress in a patient with AR-JP (Park2). Thus our studies firmly established that a genetic defect of an enzyme in the ubiquitin-proteasome system induces selective nigral neuronal death. We indicated the important role of the ubiquitin-proteasome system in neurodegeneration in general. In many other neurodegenerative disorders, such as Alzheimer's disease, Huntington's disease, Machado-Joseph disease, dentatorubral-pallidoluysian atrophy, and ALS, ubiquitinated proteins are accumulated in neurons. Thus protein handling in the ubiquitin-proteasome system appears to be affected in these neurodegenerative disorders despite the difference in the primary defects. Our studies also suggest many potential approaches for the discovery of neuroprotective treatment for not only Parkinson's disease but also other neurodegenerative disorders.
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PMID:[Etiology and pathogenesis of Parkinson's disease: from mitochondrial dysfunctions to familial Parkinson's disease]. 1528 6

It is established that neuronal nitric-oxide synthase (nNOS) is ubiquitylated and proteasomally degraded. The proteasomal degradation of nNOS is enhanced by suicide inactivation of nNOS or by the inhibition of hsp90, which is a chaperone found in a native complex with nNOS. In the current study, we have examined whether CHIP, a chaperone-dependent E3 ubiquitin-protein isopeptide ligase that is known to ubiquitylate other hsp90-chaperoned proteins, could act as an ubiquitin ligase for nNOS. We found with the use of HEK293T or COS-7 cells and transient transfection methods that CHIP overexpression causes a decrease in immunodetectable levels of nNOS. The extent of the loss of nNOS is dependent on the amount of CHIP cDNA used for transfection. Lactacystin (10 microM), a selective proteasome inhibitor, attenuates the loss of nNOS in part by causing the nNOS to be found in a detergent-insoluble form. Immunoprecipitation of the nNOS and subsequent Western blotting with an anti-ubiquitin IgG shows an increase in nNOS-ubiquitin conjugates because of CHIP. Moreover, incubation of nNOS with a purified system containing an E1 ubiquitin-activating enzyme, an E2 ubiquitin carrier protein conjugating enzyme (UbcH5a), CHIP, glutathione S-transferase-tagged ubiquitin, and an ATP-generating system leads to the ubiquitylation of nNOS. The addition of purified hsp70 and hsp40 to this in vitro system greatly enhances the amount of nNOS-ubiquitin conjugates, suggesting that CHIP is an E3 ligase for nNOS whose action is facilitated by (and possibly requires) its interaction with nNOS-bound hsp70.
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PMID:Ubiquitylation of neuronal nitric-oxide synthase by CHIP, a chaperone-dependent E3 ligase. 1546 72


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