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:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The I kappaB alpha protein is a key molecular target involved in the control of NF-kappaB/Rel transcription factors during viral infection or inflammatory reactions. This NF-kappaB-inhibitory factor is regulated by posttranslational phosphorylation and ubiquitination of its amino-terminal signal response domain that targets I kappaB alpha for rapid proteolysis by the 26S proteasome. In an attempt to identify regulators of the I kappaB alpha inhibitory activity, we undertook a yeast two-hybrid genetic screen, using the amino-terminal end of I kappaB alpha as bait, and identified 12 independent interacting clones. Sequence analysis identified some of these cDNA clones as Dlc-1, a sequence encoding a small, 9-kDa human homolog of the outer-arm dynein light-chain protein. In the two-hybrid assay, Dlc-1 also interacted with full-length I kappaB alpha protein but not with N-terminal-deletion-containing versions of I kappaB alpha. I kappaB alpha interacted in vitro with a glutathione S-transferase-Dlc-1 fusion protein, and RelA(p65) did not displace this association, demonstrating that p65 and Dlc-1 contact different protein motifs of I kappaB alpha. Importantly, in HeLa and 293 cells, endogenous and transfected I kappaB alpha coimmunoprecipitated with Myc-tagged or endogenous Dlc-1. Indirect immunofluorescence analyzed by confocal microscopy indicated that Dlc-1 and I kappaB alpha colocalized with both nuclear and cytoplasmic distribution. Furthermore, Dlc-1 and I kappaB alpha were found to associate with the microtubule organizing center, a perinuclear region from which microtubules radiate. Likewise, I kappaB alpha colocalized with alpha-tubulin filaments. Taken together, these results highlight an intriguing interaction between the I kappaB alpha protein and the human homolog of a member of the dynein family of motor proteins and provide a potential link between cytoskeleton dynamics and gene regulation.
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PMID:I kappaB alpha physically interacts with a cytoskeleton-associated protein through its signal response domain. 937 68

Activating transcription factor 2 (ATF2) is regulated by phosphorylation via the Jun N-terminal kinase, and its binding activity is markedly induced at late stages of T and B lymphocyte activation (Feuerstein, N., Firestein, R., Aiyer, N., Xiao, H., Murasko, D., and Cristofalo, V. (1996) J. Immunol. 156, 4582-4593). To identify proteins that interact specifically with ATF2 in lymphocytes, the yeast two-hybrid interaction system was employed using ATF2 cDNA as a "bait." In two separate screenings, a clone was identified that revealed a novel sequence with homology to several members of the ubiquitin-conjugating enzyme family. An identical sequence was recently reported as the human homolog of the yeast UBC9, hUBC9. Northern blot analysis revealed a 1.3-kilobase RNA transcript, which showed differential levels of expression in various human tissues and a moderate induction after a 48-h stimulation of peripheral blood T lymphocytes. An antibody that was generated against the bacterially expressed glutathione S-transferase-hUBC9 detected a approximately 19-kDa protein, which localizes predominantly in the nuclei of T cells. Further quantitative assays using the yeast two-hybrid system confirmed a high and specific level of interaction of hUBC9 with ATF2 and lack of interaction with lamin or control vectors. Two other cyclic AMP-responsive element-binding transcription factors, CREB and ATF1, also showed significant levels of interaction with hUBC9. However, this interaction was severalfold lower as compared with ATF2. Far Western blot analysis confirmed the specific binding of ATF2 and hUBC9 also in vitro. Evidence is presented that indicates a physiological significance for the interaction of hUBC9 with ATF2. (a) We show that ATF2 is ubiquitinated in vivo and in vitro, and (b) ATF2 ubiquitination in vitro is facilitated by addition of purified hUBC9. (c) ATF2 is shown to undergo a proteolytic process, which is rapidly regulated upon T cell activation concomitant with induction of ATF2 phosphorylation. (d) A proteasome inhibitor delays the down-regulation of ATF2 phophorylation after T cell activation. Taken collectively, these results implicate a role for hUBC9 and the ubiquitin/proteasome pathway in regulation of ATF2 in T cells.
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PMID:Association of activating transcription factor 2 (ATF2) with the ubiquitin-conjugating enzyme hUBC9. Implication of the ubiquitin/proteasome pathway in regulation of ATF2 in T cells. 948 27

Rad23 is an evolutionarily conserved protein that is important for nucleotide excision repair. A regulatory role has been proposed for Rad23 because rad23 mutants are sensitive to ultraviolet light but are still capable of incising damaged DNA. Here we show that Rad23 interacts with the 26S proteasome through an amino-terminal ubiquitin-like domain (UbL[R23]). The carboxy terminus of Rad23 binds to the Rad4 DNA repair protein and creates a link between the DNA repair and proteasome pathways. The ultraviolet sensitivity caused by deletion of the UbL(R23) domain may therefore arise from its inability to interact with the proteasome. The fusion proteins glutathione S-transferase (GST)-Rad23 and Rad4-haemagglutinin (HA), and the proteasome subunits Cim3 and Cim5, cofractionate through consecutive chromatography steps. The ubiquitin-like domain of human Rad23 (UbL[HRB]) also interacts with the human proteasome. These results demonstrate that ubiquitin-like domains (UbLs) represent a new class of proteasome-interacting motifs.
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PMID:Rad23 links DNA repair to the ubiquitin/proteasome pathway. 949 Apr 18

Bleomycin hydrolase (BH) is a highly conserved cysteine proteinase that deamidates and inactivates the anticancer drug bleomycin. Yeast BH self-assembles to form a homohexameric structure, which resembles a 20 S proteasome and may interact with other proteins. Therefore, we searched for potential human BH (hBH) partners using the yeast two-hybrid system with a HeLa cDNA library and identified the full-length human homologue of yeast ubiquitin-conjugating enzyme 9 (UBC9). Cotransformation assays using hBH deletion mutants revealed that the carboxyl terminus of hBH (amino acids 356-455), which contains two of the three essential catalytic amino acids, was not critical for protein binding in the yeast two-hybrid environment. In vitro translated human UBC9 was precipitated by glutathione S-transferase-hBH fusion protein but not by glutathione S-transferase. Efficient in vitro binding occurred in the absence of the first 24 amino acids of UBC9 and the catalytic Cys93 of UBC9. We confirmed that hBH and UBC9 interacted in vivo by affinity copurification of proteins overexpressed in mammalian cells. Using immunocytochemical analysis, hBH was colocalized with UBC9. Coexpression of hBH and UBC9 in mammalian cells did not markedly alter the bleomycin-hydrolyzing activity of hBH or apparent small ubiquitin-related modifier 1 addition. This is the first reported heteromeric interaction with hBH, and it suggests a role for hBH in intracellular protein processing and degradation.
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PMID:An evolutionarily conserved cysteine protease, human bleomycin hydrolase, binds to the human homologue of ubiquitin-conjugating enzyme 9. 985 22

The ubiquitin/proteasome pathway is a highly conserved mechanism of proteolysis in all eukaryotes. Ubiquitin (Ub) is conjugated to proteolytic substrates through the sequential action of ubiquitin-activating (E1/Uba) and ubiquitin-conjugating (E2/Ubc) enzymes. The mechanism of substrate recognition and ubiquitination is an area of active investigation, and we have begun a site-directed mutagenesis approach to define the biochemical and biophysical properties of ubiquitin-conjugating enzymes. We have characterized a specific mutation in Ubc4 (Ubc4(P62S)) which was previously shown to cause a temperature-sensitive growth defect in several other Ubc's. Ubc4(P62S) was rapidly degraded in vivo, contributing to the loss of function. However, reconstitution experiments revealed that the catalytic activity of Ubc4(P62S) was reversibly inactivated at 37 degrees C, demonstrating that the primary defect of Ubc4(P62S) is its inability to form a ubiquitin thioester bond at high temperature. The in vivo defect is compounded by increased susceptibility of Ubc4(P62S) to degradation by the ubiquitin/proteasome pathway. We have exploited the temperature-dependent degradation of the P62S mutant to destabilize an otherwise stable test protein (glutathione S-transferase). The use of this mutant may provide a useful cis-acting temperature-inducible degradation signal.
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PMID:Characterization of a temperature-sensitive mutant of a ubiquitin-conjugating enzyme and its use as a heat-inducible degradation signal. 1041 98

Oltipraz (OPZ) is a potent chemopreventive agent against chemically-induced carcinogenesis in several animal models. It affects the expression and/or activity of xenobiotic-metabolizing enzymes and its effects are altered in the course of inflammation in liver. The present study was undertaken to analyse the effect of OPZ alone or in combination with Escherichia coli lipopolysaccharide (LPS) on the expression and activities of glutathione S-transferases (GSTs) and cytochrome P450 (CYPs) in rat lung and kidney. Male Wistar rats were fed a diet containing OPZ for 1-5 days. LPS was injected 24 h before the end of OPZ treatment (from 48 to 72 h). Total GST activity, measured using 1-chloro-2,4-dinitrobenzene as a substrate, increased slightly in both lung and kidney during OPZ treatment. As previously demonstrated in the liver, OPZ induced rat GSTP1 in both kidney and lung and this effect was totally (kidney) or partially (lung) inhibited by co-treatment with LPS. CYP1A expression and activity were strongly increased in both tissues 24 h after starting OPZ treatment and maintained for 5 days. This increase was suppressed during the acute-phase response to endotoxin. OPZ has no effect on CYP2B1 mRNA expression in the lung, but it dramatically decreased the amount and activity of the corresponding apoprotein. The OPZ-dependent decrease in the CYP2B1 apoprotein was abolished and its corresponding activity partially reversed during LPS treatment. In reconstitution experiments using cytosol from OPZ-treated or control rat lungs and microsomal fractions, CYP2B1 apoprotein was rapidly degraded in the presence of cytosol from treated rats. This effect was partially reversed in the presence of MG132, a proteasome inhibitor. These observations support the conclusion that the decrease of CYP2B1 by OPZ involves proteasome-dependent degradation and represents a new mechanism of regulation by this compound.
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PMID:Differential effects of oltipraz on CYP1A and CYP2B in rat lung. 1115 40

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

Injury to motor neurons associated with mutant Cu,Zn-superoxide dismutase (SOD1)-related familial amyotrophic lateral sclerosis (FALS) results from a toxic gain-of-function of the enzyme. The mechanisms by which alterations to SOD1 elicit neuronal death remain uncertain despite intensive research effort. Analysis of the cellular proteins that are differentially expressed in the presence of mutant SOD1 represents a novel approach to investigate further this toxic gain-of-function. By using the motor neuron-like cell line NSC34 stably transfected with wild-type, G93A, or G37R mutant human SOD1, we investigated the effects of mutant human SOD1 on protein expression using proteomic approaches. Seven up-regulated proteins were identified as argininosuccinate synthase, argininosuccinate lyase, neuronal nitric-oxide synthase, RNA-binding motif protein 3, peroxiredoxin I, proteasome subunit beta 5 (X), and glutathione S-transferase (GST) Alpha 2. Seven down-regulated proteins were identified as GST Mu 1, GST Mu 2, GST Mu 5, a hypothetical GST Mu, GST Pi B, leukotriene B(4) 12-hydroxydehydrogenase, and proteasome subunit beta5i (LMP7). GST assays demonstrated a significant reduction in the total GST activity of cells expressing mutant human SOD1. Proteasome assays demonstrated significant reductions in chymotrypsin-like, trypsin-like, and post-glutamylhydrolase proteasome activities. Laser capture microdissection of spinal cord motor neurons from human FALS cases, in conjunction with reverse transcriptase-PCR, demonstrated decreased levels of mRNA encoding GST Mu 1, leukotriene B(4) 12-hydroxydehydrogenase, and LMP7. These combined approaches provide further evidence for involvement of alterations in antioxidant defenses, proteasome function, and nitric oxide metabolism in the pathophysiology of FALS.
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PMID:Analysis of the cytosolic proteome in a cell culture model of familial amyotrophic lateral sclerosis reveals alterations to the proteasome, antioxidant defenses, and nitric oxide synthetic pathways. 1247 80

The mechanism by which hypoxia induces gene transcription involves the inhibition of hypoxia-inducible factor (HIF)-1alpha prolyl hydroxylase activity, which prevents von Hippel-Lindau (vHL)-dependent targeting of HIF-1alpha to the ubiquitin-proteasome pathway. HIF-1alpha is stabilized, translocates to the nucleus, interacts with hypoxia-responsive elements, and promotes the activation of target genes. This report shows that cyclosporin A (CsA) interferes with the hypoxic signaling cascade in C6 glioma cells. CsA inhibits hypoxia-dependent gene transcription in a reporter gene assay and prevents the hypoxic accumulation of HIF-1alpha. Addition of the 530-603 C-terminal oxygen-dependent degradation (ODD) domain of HIF-1alpha to the green fluorescent protein (GFP) destabilized the protein in an oxygen-dependent manner. CsA prevented the hypoxic stabilization of an ODD.GFP fusion protein. An assay for 2-oxoglutarate-dependent dioxygenases was developed using a light mitochondrial kidney fraction as a source of enzyme. It uses the capacity of specific peptides to stimulate the degradation of [(14)C]2-oxoglutarate. CsA stimulated the enzymatic activity in the presence of a peptide that mimicked the 557-576 sequence of HIF-1alpha. The enzyme promoted [(35)S]vHL binding to glutathione S-transferase (GST).ODD fusion protein. This association increased in the presence of CsA. CsA effects were not observed when the proline residue corresponding to Pro-564 in the HIF-1alpha sequence was replaced by a hydroxyproline or an alanine residue. Finally, CsA increased vHL-ODD interaction during hypoxia. We conclude that CsA destabilizes HIF-1alpha by promoting hydroxylation of Pro-564 in the ODD domain. Such a mechanism may prevent hypoxic adaptation during CsA-induced nephrotoxicity and contribute to the adverse effects of this drug.
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PMID:Cyclosporin A prevents the hypoxic adaptation by activating hypoxia-inducible factor-1alpha Pro-564 hydroxylation. 1258 29

Polyubiquitination is required for retrotranslocation of proteins from the endoplasmic reticulum back into the cytosol, where they are degraded by the proteasome. We have tested whether the release of a polypeptide chain into the cytosol is caused by a ratcheting mechanism in which the attachment of polyubiquitin prevents the chain from moving back into the endoplasmic reticulum. Using a permeabilized cell system in which major histocompatibility complex class I heavy chains are retrotranslocated under the influence of the human cytomegalovirus protein US11, we demonstrate that polyubiquitination alone is insufficient to provide the driving force for retrotranslocation. Substrate release into the cytosol requires an additional ATP-dependent step. Release requires a lysine 48 linkage of ubiquitin chains. It does not occur when polyubiquitination of the substrate is carried out with glutathione S-transferase (GST)-ubiquitin, and this correlates with poly-GST-ubiquitin not being recognized by a ubiquitin-binding domain in the Ufd1-Npl4 cofactor of the ATPase p97. These data suggest that polyubiquitin does not serve as a ratcheting molecule. Rather, it may serve as a recognition signal for the p97-Ufd1-Npl4 complex, a component implicated in the movement of substrate into the cytosol.
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PMID:Polyubiquitin serves as a recognition signal, rather than a ratcheting molecule, during retrotranslocation of proteins across the endoplasmic reticulum membrane. 1281 30


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