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)

By having demonstrated previously that p27(Kip1), a potent inhibitor of G(1) cyclin-cyclin-dependent kinases complexes, increases markedly during intestinal epithelial cell differentiation, we examined the effect of p27(Kip1) on the activity of the transcription factor CDX2. The present results revealed the following. 1) p27(Kip1) interacts with the CDX2 transcription factor. 2) In contrast to CDX2 mRNA levels, CDX2 protein expression levels significantly increased as soon as Caco-2/15 cells reached confluence, slowed their proliferation, and began their differentiation. The mechanism of CDX2 regulation is primarily related to protein stability, because inhibition of proteasome activity increased CDX2 levels. The half-life of CDX2 protein was significantly enhanced in differentiated versus undifferentiated proliferative intestinal epithelial cells. 3) Cdk2 interacted with CDX2 and phosphorylated CDX2, as determined by pull-down glutathione S-transferase and immunoprecipitation experiments with proliferating undifferentiated Caco-2/15 cell extracts. 4) Treatment of Caco-2/15 cells with MG132 (a proteasome inhibitor) and (R)-roscovitine (a specific Cdk2 inhibitor) induced an increase in CDX2 protein levels. 5) Conversely, ectopic expression of Cdk2 resulted in decreased expression of CDX2 protein. 6) Of note, treatment of proliferative Caco-2/15 cells with (R)-roscovitine or leptomycin (an inhibitor of nuclear export through CRM1) led to an accumulation of CDX2 into the nucleus. These data suggest that CDX2 undergoes CRM1-dependent nuclear export and cytoplasmic degradation in cells in which Cdk2 is activated, such as in proliferative intestinal epithelial cells. The targeted degradation of CDX2 following its phosphorylation by Cdk2 identifies a new mechanism through which CDX2 activity can be regulated in coordination with the cell cycle machinery.
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PMID:Cdk2-dependent phosphorylation of homeobox transcription factor CDX2 regulates its nuclear translocation and proteasome-mediated degradation in human intestinal epithelial cells. 1574 Nov 63

A yeast two-hybrid screen using EBNA3C as bait revealed an interaction between this Epstein-Barr virus (EBV)-encoded nuclear protein and the C8 (alpha7) subunit of the human 20S proteasome. The interaction was confirmed by glutathione S-transferase (GST) pull-down experiments and these also revealed that the related proteins EBNA3A and EBNA3B can bind similarly to C8/alpha7. The interaction between these viral proteins and GST-C8/alpha7 was shown to be significantly more robust than the previously reported interaction between C8/alpha7 and the cyclin-dependent kinase inhibitor p21(WAF1/CIP1). Co-immunoprecipitation of the EBNA3 proteins with C8/alpha7 was also demonstrated after transfection of expression vectors into B cells. Consistent with this ability to bind directly to an alpha-subunit of the 20S proteasome, EBNAs 3A, 3B and 3C were all degraded in vitro by purified 20S proteasomes. However, surprisingly, no sign of proteasome-mediated turnover of these latent viral proteins in EBV-immortalized B cells could be detected, even in the presence of gamma interferon. In actively proliferating lymphoblastoid cell lines, EBNAs 3A, 3B and 3C appear to be remarkably stable, with no evidence of either de novo synthesis or proteasome-mediated degradation.
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PMID:Epstein-Barr virus EBNA3 proteins bind to the C8/alpha7 subunit of the 20S proteasome and are degraded by 20S proteasomes in vitro, but are very stable in latently infected B cells. 1583 37

Exposure of cells to a wide variety of chemoprotective compounds confers resistance to a broad set of carcinogens. For a subset of the chemoprotective compounds, protection is generated by an increase in the abundance of phase 2 detoxification enzymes such as glutathione S-transferases (GSTs). Transcription factor Nrf2, which is sequestered in the cytoplasm by Keap1 (Kelch-like ECH-associated protein-1) under unstimulated conditions, regulates the induction of phase 2 enzymes. In this study, to explore the role of the proteasome in the detoxification response, we tested the effect of proteasome inhibitors such as MG132, clasto-lactacystin beta-lactone, and lactacystin on the induction of GST isozymes and found that these inhibitors selectively induced the class Pi GST isozyme (GST P1). Down-regulation of the proteasome by antisense oligonucleotides or RNA interference indeed resulted in significant up-regulation of GST P1, suggesting that a decline in the proteasome activity could be directly or indirectly linked to the induction of GST P1. From the functional analysis of various deletion constructs of the upstream regulatory region of the GST P1 promoter, GST P1 enhancer I was identified as the response element for proteasome inhibition. Overexpression of the wild-type and dominant-negative forms of Nrf2 and Keap1 had little effect on the induction of GST P1 not only by the proteasome inhibitor, but also by phase 2-inducing isothiocyanate, suggesting that there may be a process of GST P1 induction distinct from other phase 2 gene induction mechanisms. Because GST P1 is highly and specifically induced during early hepatocarcinogenesis as well as in hepatocellular carcinoma cells, these data may provide a potential critical role for the proteasome in the induction of a cellular defense program associated with carcinogenesis.
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PMID:Selective induction of the tumor marker glutathione S-transferase P1 by proteasome inhibitors. 1586 7

The mechanism by which hypoxia induces gene transcription involves the inhibition of HIF-1alpha (hypoxia-inducible factor-1 alpha subunit) PHD (prolyl hydroxylase) activity, which prevents the VHL (von Hippel-Lindau)-dependent targeting of HIF-1alpha to the ubiquitin/proteasome pathway. HIF-1alpha thus accumulates and promotes gene transcription. In the present study, first we provide direct biochemical evidence for the presence of a conserved hypoxic signalling pathway in Drosophila melanogaster. An assay for 2-oxoglutarate-dependent dioxygenases was developed using Drosophila embryonic and larval homogenates as a source of enzyme. Drosophila PHD has a low substrate specificity and hydroxylates key proline residues in the ODD (oxygen-dependent degradation) domains of human HIF-1alpha and Similar, the Drosophila homologue of HIF-1alpha. The enzyme promotes human and Drosophila [(35)S]VHL binding to GST (glutathione S-transferase)-ODD-domain fusion protein. Hydroxylation is enhanced by proteasomal inhibitors and was ascertained using an anti-hydroxyproline antibody. Secondly, by using transgenic flies expressing a fusion protein that combined an ODD domain and the green fluorescent protein (ODD-GFP), we analysed the hypoxic cascade in different embryonic and larval tissues. Hypoxic accumulation of the reporter protein was observed in the whole tracheal tree, but not in the ectoderm. Hypoxic stabilization of ODD-GFP in the ectoderm was restored by inducing VHL expression in these cells. These results show that Drosophila tissues exhibit different sensitivities to hypoxia.
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PMID:Analysis of the hypoxia-sensing pathway in Drosophila melanogaster. 1617 82

Using a proteomic approach, we characterized different protein expression profiles in anterior gills of the Chinese mitten crab, Eriocheir sinensis, after cadmium (Cd) exposure. Two experimental conditions were tested: (i) an acute exposure (i.e. 500 microg Cd l(-1) for 3 days) for which physiological, biochemical and ultrastructural damage have been observed previously; (ii) a chronic exposure (i.e. 50 microg Cd l(-1) for 30 days) resulting in physiological acclimation, i.e. increased resistance to a subsequent acute exposure. Two-dimensional gel electrophoresis (2-DE) revealed six protein spots differentially expressed after acute, and 31 after chronic Cd exposure. From these spots, 15 protein species were identified using MS/MS micro-sequencing and MS BLAST database searches. Alpha tubulin, glutathione S-transferase and crustacean calcium-binding protein 23 were down-regulated after an acute exposure, whereas another glutathione S-transferase isoform was up-regulated. Furthermore, analyses revealed the over-expression of protein disulfide isomerase, thioredoxin peroxidase, glutathione S-transferase, a proteasome subunit and cathepsin D after chronic exposure. Under the same condition, ATP synthase beta, alpha tubulin, arginine kinase, glyceraldehyde-3-phosphate dehydrogenase and malate dehydrogenase were down-regulated. These results demonstrate that acute and chronic exposure to waterborne Cd induced different responses at the protein expression level. Protein identification supports the idea that Cd mainly exerts its toxicity through oxidative stress induction and sulfhydryl-group binding. As a result, analyses showed the up-regulation of several antioxidant enzymes and chaperonins during acclimation process. The gill proteolytic capacity seems also to be increased. On the other hand, the clearly decreased abundance of several enzymes involved in energy transfer suggests that chronic metal exposure induced an important metabolic reshuffling.
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PMID:Differential protein expression profiles in anterior gills of Eriocheir sinensis during acclimation to cadmium. 1624 38

Although vitamin B6 has been supposed to have anti-inflammatory effects, the molecular mechanism is not fully understood. To explore the mechanism of anti-inflammatory effects of vitamin B6, we have examined the effect of vitamin B6 on lipopolysaccharide (LPS)-stimulated inflammatory response in RAW 264.7 macrophages. This study demonstrated that vitamin B6 (pyridoxal) pretreatment of RAW cells inhibited LPS-induced expression of iNOS and COX-2 at the mRNA and protein levels. Vitamin B6 inhibited LPS-induced nuclear translocation of the NF-kappaB, the proinflammatory transcription factor, with reduction of the extent of LPS-induced IkappaBalpha degradation in RAW cells. Although vitamin B6 did not affect cellular proteasome activity, in vitro phosphorylation analysis with glutathione S-transferase-fused IkappaBalpha has shown that vitamin B6 suppressed LPS-induced IkappaB kinase activation. Furthermore, we demonstrated that elevating dietary vitamin B6 suppressed NO production in vivo in response to LPS administration. These observations suggest that the anti-inflammatory effect of vitamin B6 is mediated by suppression of NF-kappaB activation.
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PMID:Vitamin B6 suppresses NF-kappaB activation in LPS-stimulated mouse macrophages. 1627 88

The antiestrogen fulvestrant (ICI 182,780) causes immobilization of estrogen receptor-alpha (ERalpha) in the nuclear matrix accompanied by rapid degradation by the ubiquitin-proteasome pathway. In this study we tested the hypothesis that fulvestrant induces specific nuclear matrix protein-ERalpha interactions that mediate receptor immobilization and turnover. A glutathione S-transferase (GST)-ERalpha-activating function-2 (AF2) fusion protein was used to isolate and purify receptor-interacting proteins in cell lysates prepared from human MCF-7 breast cancer cells. After SDS-PAGE and gel excision, mass spectrometry was used to identify two major ERalpha-interacting proteins, cytokeratins 8 and 18 (CK8.CK18). We determined, using ERalpha-activating function-2 mutants, that helix 12 (H12) of ERalpha, but not its F domain, is essential for fulvestrant-induced ERalpha-CK8 and CK18 interactions. To investigate the in vivo role of H12 in fulvestrant-induced ERalpha immobilization/degradation, transient transfection assays were performed using wild type ERalpha,ERalpha with a mutated H12, and ERalpha with a deleted F domain. Of those, only the ERalpha H12 mutant was resistant to fulvestrant-induced immobilization to the nuclear matrix and protein degradation. Fulvestrant treatment caused ERalpha degradation in CK8.CK18-positive human breast cancer cells, and CK8 and CK18 depletion by small interference RNAs partially blocked fulvestrant-induced receptor degradation. Furthermore, fulvestrant-induced ERalpha degradation was not observed in CK8 or CK18-negative cancer cells, suggesting that these two intermediate filament proteins are necessary for fulvestrant-induced receptor turnover. Using an ERalpha-green fluorescent protein construct in fluorescence microscopy revealed that fulvestrant-induced cytoplasmic localization of newly synthesized receptor is mediated by its interaction with CK8 and CK18. In summary, this study provides the first direct evidence linking ERalpha immobilization and degradation to the nuclear matrix. We suggest that fulvestrant induces ERalpha to interact with CK8 and CK18, drawing the receptor into close proximity to nuclear matrix-associated proteasomes that facilitate ERalpha turnover.
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PMID:Fulvestrant (ICI 182,780)-dependent interacting proteins mediate immobilization and degradation of estrogen receptor-alpha. 1645 37

The ubiquitin-proteasome pathway (UPP) is involved in regulation of multiple cellular processes. Hypoxia-inducible factor 1 alpha (HIF-1 alpha) is a prototypic target of the UPP and, as such, is stabilized under conditions of proteasomal inhibition. Using carbonic anhydrase IX (CAIX) and vascular endothelial growth factor (VEGF) expression as paradigmatic markers of HIF-1 activity, we found that proteasomal inhibitors (PI) abrogated hypoxia-induced CAIX expression in all cell lines tested and VEGF expression in two out of three. Mapping of the inhibitory effect identified the C-terminal activation domain (CAD) of HIF-1 alpha as the primary target of PI. PI specifically inhibited the HIF-1 alpha CAD despite activating the HIF-1 alpha coactivator p300 and another p300 cysteine/histidine-rich domain 1-dependent transcription factor, STAT-2. Coimmunoprecipitation and glutathione S-transferase pull downs indicated that PI does not disrupt interactions between HIF-1 alpha and p300. Mutational analysis failed to confirm involvement of sites of known or putative posttranslational modifications in regulation of HIF-1 alpha CAD function by PI. Our data provide evidence for the counterintuitive hypothesis that inhibition of HIF-1 function could be responsible for at least some of the antitumor effects of proteasomal inhibition. Further studies of the mechanism of the PI-induced attenuation of HIF-1alpha will provide important, potentially novel insight into regulation of HIF-1 activity and possibly identify new targets for HIF-directed therapy.
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PMID:Proteasomal inhibition attenuates transcriptional activity of hypoxia-inducible factor 1 (HIF-1) via specific effect on the HIF-1alpha C-terminal activation domain. 1684 40

The clinical manifestations of West Nile virus (WNV), a member of the Flavivirus family, include febrile illness, sporadic encephalitis, and paralysis. The capsid (Cp) of WNV is thought to participate in these processes by inducing apoptosis through mitochondrial dysfunction and activation of caspase-9 and caspase-3. To further identify the molecular mechanism of the WNV capsid protein (WNVCp), yeast two-hybrid assays were employed using WNV-Cp as bait. Jab1, the fifth subunit of the COP9 signalosome, was subsequently identified as a molecule that interacts with WNVCp. Immunoprecipitation and glutathione S-transferase pulldown assays confirmed that direct interaction could occur between WNVCp and Jab1. Immunofluorescence microscopy demonstrated that the overexpressed WNVCp, which localized to the nucleolus, was translocated to the cytoplasm upon its co-expression with Jab1. When treated with leptomycin B, Jab1-facilitated nuclear exclusion of WNVCp was prevented, which indicated that the CRM1 complex is required for Jab1-facilitated nuclear export of WNVCp. Moreover, Jab1 promoted the degradation of WNVCp in a proteasome-dependent way. Consistent with this, WNVCp-mediated cell cycle arrest at the G(2) phase in H1299 was prevented by exogenous Jab1. Finally, an analysis of WNVCp deletion mutants indicated that the first 15 amino acids were required for interaction with Jab1. Furthermore, the double-point mutant of the WNVCp, P5A/P8A, was incapable of binding to Jab1. These results indicate that Jab1 has a potential protective effect against pathogenic WNVCp and might provide a novel target site for the treatment of disease caused by WNV.
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PMID:Jab1 mediates cytoplasmic localization and degradation of West Nile virus capsid protein. 1688 64

Itch is a ubiquitin ligase that has been implicated in the regulation of a number of cellular processes. We previously have identified Itch as a binding partner for the endocytic protein Endophilin and found it to be localized to endosomes. Using affinity purification coupled to mass spectrometry, we have now identified the ubiquitin-protease FAM/USP9X as a binding partner of Itch. The association between Itch and FAM/USP9X was confirmed in vitro by glutathione S-transferase pulldown and in vivo through coimmunoprecipation. Itch and FAM partially colocalize in COS-7 cells at the trans-Golgi network and in peripheral vesicles. We mapped the FAM-binding domain on Itch to the WW domains, a region known to be involved in substrate recognition. However, transient overexpression of FAM/USP9X resulted in the deubiquitylation of Itch. Moreover, we show that Itch auto-ubiquitylation leads to its degradation in the proteasome. By examining the amounts of Itch and FAM in various cell lines and rat tissues, a positive correlation was found in the expression of both proteins. This observation suggests that the levels of FAM expression could have an influence on Itch in cells. Experimental decrease in FAM levels by RNA interference leads to a significant reduction in intracellular levels of endogenous Itch, which can be prevented by treatment with the proteasome inhibitor lactacystin. Accordingly, overexpression of FAM/USP9X resulted in a marked increase in endogenous Itch levels. These results demonstrate an intriguing interplay between a ubiquitin ligase and a ubiquitin protease, based on direct interaction between the two proteins.
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PMID:The ubiquitin ligase itch is auto-ubiquitylated in vivo and in vitro but is protected from degradation by interacting with the deubiquitylating enzyme FAM/USP9X. 1703 27


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