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 level of cellular ceramide, an apoptotic rheostat, is increased by sphingomyelinase or de novo synthesis. The expression of the glutathione S-transferase (GST) gene, whose induction accounts for cell viability, is regulated by activation of CCAAT/enhancer binding protein-beta (C/EBPbeta) and NF-E2-related factor-2 (Nrf2). Hepatic nuclear factor-1 (HNF1) is a transcription factor necessary for cell survival. This study investigated the role of HNF1 in GSTA2 gene transactivation, the ubiquitin proteasomal degradation of HNF1, and the inhibition of activating HNF1 by ceramide for GSTA2 repression. C2-ceramide (C2), a cell-permeable analog, repressed the GSTA2 expression in H4IIE cells, whereas dihydro-C2, an inactive analog, had no effect. Immunoblot, immunocytochemical, and gel shift analyses revealed that C2 decreased the level of nuclear HNF1 and protein binding to the HNF response element (HRE). Deletion of the HRE or the GSTA2 gene promoter region containing the HRE reduced luciferase reporter expression. Immunoprecipitation and immunoblot analyses showed that C2 decreased the level of ubiquitinated HNF1, which was reversed by treatment with MG132, a proteasome inhibitor. C2 suppressed GSTA2 induction by oltipraz via inhibition of inducible HNF1 DNA binding. The functional role of HRE for C2 repression of GSTA2 gene transactivation by oltipraz was verified by both the luciferase reporter gene expression and the transfection experiment with DeltaHNF-pGL-1651 lacking the HRE. C2 similarly repressed the induction of GSTA2 promoter-luciferase by tert-butylhydroquinone via HNF1 suppression, suggesting that constitutive HNF1 activation is required for GSTA2 induction. C2 also inhibited GSTA3/5 expression. In conclusion, the HRE in the GSTA2 promoter region is functionally active for the constitutive and inducible gene expression, and ceramide inhibits GST gene transactivation through decrease in nuclear HNF1, which is degraded by the ubiquitin proteasome system.
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PMID:Ceramide negatively regulates glutathione S-transferase gene transactivation via repression of hepatic nuclear factor-1 that is degraded by the ubiquitin proteasome system. 1515 40

Ceramide is a sphingolipid that acts as a second messenger in signaling systems. Sphingomyelinase generates ceramide in response to cytotoxic stimuli. CCAAT/enhancer binding protein-beta (C/EBPbeta) and NF-E2-related factor-2 (Nrf2) are both involved in the regulation of the genes encoding phase II detoxification enzymes including glutathione S-transferase (GST). In the present study, we examined the effects of ceramide on C/EBPbeta or Nrf2 activation and on the inducible GSTA2 gene transactivation. C2-ceramide (C2), a cell-permeable analog, inhibited GSTA2 induction by oltipraz or tert-butylhydroquinone (t-BHQ) in H4IIE cells, whereas dihydro-C2-ceramide (dihydro-C2), an inactive analog, had no effect. Immunoblot analysis revealed that C2 prevented increase in the level of nuclear C/EBPbeta by oltipraz, whereas the level of C/EBPbeta in total cell lysates was not changed. Increase in nuclear Nrf2 by t-BHQ was also prevented by C2 treatment. Decreases in nuclear C/EBPbeta and Nrf2 by C2 were reversed by treatment of cells with N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132), a proteasome inhibitor, verifying the previous observations that the transcription factors were degraded by the proteasome system. In another study, we found that ceramide decreased nuclear hepatic nuclear factor-1 (HNF1), whose binding to the HNF1-response element in the GSTA2 gene was responsible for the constitutive and inducible gene expression. To define the role of C/EBPbeta or Nrf2 repression in GST expression under the condition excluding the negative regulation by C2-mediated HNF1 suppression, luciferase activity was determined in the cells transfected with DeltaHNF-pGL-1651 plasmid lacking the HNF1-response element. In the cells transfected with DeltaHNF-pGL-1651, C2 decreased the luciferase induction by oltipraz or t-BHQ. Thus, ceramide inhibits C/EBPbeta or Nrf2 activation, which contributes to repression of GSTA2 gene transactivation.
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PMID:Ceramide, an apoptotic rheostat, inhibits CCAAT/enhancer binding protein-beta and NF-E2-related factor-2 activation: the role in glutathione S-transferase A2 gene repression. 1531 26

Peptide:N-glycanase (PNGase) has been proposed to participate in the proteasome-dependent glycoprotein degradation pathway. The finding that yeast PNGase interacts with the 19S proteasome subunit through the protein Rad23 supports this hypothesis. In this report, we have used immunofluorescence, subcellular fractionation, coimmunoprecipitation, and in vitro GST pull-down techniques for detecting intracellular localization and interactions of PNGase, HR23B, and S4 by using human (h) and mouse (m) homologs. Immunofluorescence studies revealed that hPNGase, hHR23B, and hS4 are present in close proximity to the endoplasmic reticulum (ER) when calnexin was used as an ER marker in HeLa cells. Subcellular fractionation suggests not only cytoplasmic but also ER association of hPNGase in HeLa cells. Immunoprecipitation analysis revealed the interaction of h/mPNGase with the 19S proteasome subunit, hS4, through hHR23B. Using an in vitro GST pull-down assay, we also have shown that recombinant mPNGase requires its N terminus and middle domain for interaction with mHR23B. Finally, using misfolded yeast carboxypeptidase Y and chicken ovalbumin as glycoprotein substrates, we have established that mHR23B acts as a receptor for deglycosylated proteins. Based on this finding, we propose that after deglycosylation of misfolded glycoproteins by PNGase, the aglyco forms of these proteins are recognized by HR23B and targeted for degradation.
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PMID:A complex between peptide:N-glycanase and two proteasome-linked proteins suggests a mechanism for the degradation of misfolded glycoproteins. 1535 61

A widely expressed protein containing UBA (ubiquitin-associated) and UBX (ubiquitin-like) domains was identified as a substrate of SAPKs (stress-activated protein kinases). Termed SAKS1 (SAPK substrate-1), it was phosphorylated efficiently at Ser200 in vitro by SAPK3/p38gamma, SAPK4/p38delta and JNK (c-Jun N-terminal kinase), but weakly by SAPK2a/p38alpha, SAPK2b/p38beta2 or ERK (extracellular-signal-regulated kinase) 2. Ser200, situated immediately N-terminal to the UBX domain, became phosphorylated in HEK-293 (human embryonic kidney) cells in response to stressors. Phosphorylation was not prevented by SB 203580 (an inhibitor of SAPK2a/p38alpha and SAPK2b/p38beta2) and/or PD 184352 (which inhibits the activation of ERK1 and ERK2), and was similar in fibroblasts lacking both SAPK3/p38gamma and SAPK4/p38delta or JNK1 and JNK2. SAKS1 bound ubiquitin tetramers and VCP (valosin-containing protein) in vitro via the UBA and UBX domains respectively. The amount of VCP in cell extracts that bound to immobilized GST (glutathione S-transferase)-SAKS1 was enhanced by elevating the level of polyubiquitinated proteins, while SAKS1 and VCP in extracts were coimmunoprecipitated with an antibody raised against S5a, a component of the 19 S proteasomal subunit that binds polyubiquitinated proteins. PNGase (peptide N-glycanase) formed a 1:1 complex with VCP and, for this reason, also bound to immobilized GST-SAKS1. We suggest that SAKS1 may be an adaptor that directs VCP to polyubiquitinated proteins, and PNGase to misfolded glycoproteins, facilitating their destruction by the proteasome.
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PMID:A novel UBA and UBX domain protein that binds polyubiquitin and VCP and is a substrate for SAPKs. 1536 74

The ubiquitin-proteasome system is an essential mechanism for protein degradation in eukaryotes. Protein ubiquitination is composed of a series of enzymatic reactions. The ubiquitin-conjugating enzyme (E2) is one of the important enzymes involved in the process. A cDNA encoding an E2 enzyme was cloned from a Clonorchis sinensis cDNA library by large-scale sequencing. This new cDNA contains 862 bp with a putative open reading frame of 156 amino acids. The deduced amino acid sequence is 77% identical to the human E2, HHR6A and HHR6B. The coding region of this cDNA was expressed in E. coli as a GST-tagged protein, and was purified to electrophoretic homogeneity. Enzymatic assays showed that this E2 had the capacity to form a thiolester linkage, and could conjugate ubiquitin to histone H2A in an E3-independent manner in vitro, which indicated that the expressed protein was functionally active. The nucleotide sequence reported in this paper has been submitted to the Genbank Database with accession number AY632078.
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PMID:Molecular cloning and characterization of cDNA encoding a ubiquitin-conjugating enzyme from Clonorchis sinensis. 1548 Jul 85

Constitutive albumin uptake by the proximal tubule is achieved by a receptor-mediated process in which the Cl(-) channel, ClC-5, plays an obligate role. Here we investigated the functional interaction between ClC-5 and ubiquitin ligases Nedd4 and Nedd4-2 and their role in albumin uptake in opossum kidney proximal tubule (OK) cells. In vivo immunoprecipitation using an anti-HECT antibody demonstrated that ClC-5 bound to ubiquitin ligases, whereas glutathione S-transferase pull-downs confirmed that the C terminus of ClC-5 bound both Nedd4 and Nedd4-2. Nedd4-2 alone was able to alter ClC-5 currents in Xenopus oocytes by decreasing cell surface expression of ClC-5. In OK cells, a physiological concentration of albumin (10 mug/ml) rapidly increased cell surface expression of ClC-5, which was also accompanied by the ubiquitination of ClC-5. Albumin uptake was reduced by inhibiting either the lysosome or proteasome. Total levels of Nedd4-2 and proteasome activity also increased rapidly in response to albumin. Overexpression of ligase defective Nedd4-2 or knockdown of endogenous Nedd4-2 with small interfering RNA resulted in significant decreases in albumin uptake. In contrast, pathophysiological concentrations of albumin (100 and 1000 mug/ml) reduced the levels of ClC-5 and Nedd4-2 and the activity of the proteasome to the levels seen in the absence of albumin. These data demonstrate that normal constitutive uptake of albumin by the proximal tubule requires Nedd4-2, which may act via ubiquitination to shunt ClC-5 into the endocytic pathway.
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PMID:Nedd4-2 functionally interacts with ClC-5: involvement in constitutive albumin endocytosis in proximal tubule cells. 1548 23

Mitogen-activated protein (MAP) kinase phosphatases (MKPs) are dual-specificity phosphatases that dephosphorylate phosphothreonine and phosphotyrosine residues within MAP kinases. Here, we describe a novel posttranslational mechanism for regulating MKP-3/Pyst1/DUSP6, a member of the MKP family that is highly specific for extracellular signal-regulated kinase 1 and 2 (ERK1/2) inactivation. Using a fibroblast model in which the expression of either MKP-3 or a more stable MKP-3-green fluorescent protein (GFP) chimera was induced by tetracycline, we found that serum induces the phosphorylation of MKP-3 and its subsequent degradation by the proteasome in a MEK1 and MEK2 (MEK1/2)-ERK1/2-dependent manner. In vitro phosphorylation assays using glutathione S-transferase (GST)-MKP-3 fusion proteins indicated that ERK2 could phosphorylate MKP-3 on serines 159 and 197. Tetracycline-inducible cell clones expressing either single or double serine mutants of MKP-3 or MKP-3-GFP confirmed that these two sites are targeted by the MEK1/2-ERK1/2 module in vivo. Double serine mutants of MKP-3 or MKP-3-GFP were more efficiently protected from degradation than single mutants or wild-type MKP-3, indicating that phosphorylation of either serine by ERK1/2 enhances proteasomal degradation of MKP-3. Hence, double mutation caused a threefold increase in the half-life of MKP-3. Finally, we show that the phosphorylation of MKP-3 has no effect on its catalytic activity. Thus, ERK1/2 exert a positive feedback loop on their own activity by promoting the degradation of MKP-3, one of their major inactivators in the cytosol, a situation opposite to that described for the nuclear phosphatase MKP-1.
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PMID:Extracellular signal-regulated kinases phosphorylate mitogen-activated protein kinase phosphatase 3/DUSP6 at serines 159 and 197, two sites critical for its proteasomal degradation. 1563 84

Arsenic present in drinking water and mining environments in some areas has been associated with an increased rate of skin and internal cancers. Contrary to the epidemiological evidence in humans, arsenic does not induce cancer in animal models, but is able to enhance the mutagenicity of other agents. In order to achieve a better understanding of the interaction between arsenic and ionising radiation, an investigation was conducted to detect differences at the proteome level of human TK6 lymphoblastoid cells exposed to these agents. Cells were exposed to either a single dose of 1-Gy 137Cs-gamma-rays or to 1 microM arsenite (As(III)) or to both agents in combination. Two-dimensional (2D) electrophoresis and matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) were employed for the screening and identification of proteins, respectively. It proved possible to identify seven proteins with significantly affected abundance, three of which showed increased levels and the remaining four showed decreased levels under at least one of the exposure conditions. Following arsenite treatment or irradiation, a significant increase compared with that of the control was observed for glutathione (GSH) transferase omega 1 and proteasome subunit beta type 4 precursor. The combined exposure did not result in an induction of the enzymes. The expression of electron-transfer flavoprotein subunit alpha was found to be enhanced under all three-exposure conditions. Ubiquinol-cytochrome C reductase complex core protein I, adenine phosphoribosyl transferase and endoplasmic reticulum protein hERp29 showed decreased levels after irradiation or arsenite treatment, but not after the combined exposure. The level of serine/threonine protein phosphatase 1 alpha decreased with all treatments. The main conclusions are that both arsenite and gamma-radiation influence the levels of several proteins involved in major metabolic and regulatory pathways, either directly or by triggering the defence mechanisms of the cell. The combined effect of both exposures on the level of some essential proteins such as glutathione transferase, proteasome or serine/threonine phosphatase may contribute to the co-carcinogenic effect of arsenic.
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PMID:Combined effects of gamma radiation and arsenite on the proteome of human TK6 lymphoblastoid cells. 1572 13

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


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