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)

Limited proteolysis of grass carp alcohol dehydrogenase by alkaline protease or subtilisin BPN' at 30 degrees C for 6 hr generated a nicked species that was catalytically active. Electrophoresis on a denaturing SDS-PAGE showed that the 40 kDa subunit of the intact enzyme was cleaved to produce subunits of 27 and 13 kDa, which remained tightly associated with each other under native condition. Such a proteolytically nicked form was catalytically more active than the original intact form of the enzyme. The Vmax value toward the oxidation of ethanol at pH 10 increased by 7.8 fold whereas the K(m) value also exhibited a 140 fold increase. On the other hand, when the same protease treatment was applied to horse liver alcohol dehydrogenase, no activation nor any specific cleavage can be observed.
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PMID:Activation of grass carp liver alcohol dehydrogenase by limited proteolysis. 898 21

Dihydrotestosterone (DHT) decreases rat liver alcohol dehydrogenase (ADH) due principally to an increased rate of degradation of the enzyme. The pathway of degradation of ADH was investigated. Exposure of hepatocytes in culture to lactacystin or to MG132, which are inhibitors of the ubiquitin-proteasome pathway of protein degradation, resulted in higher ADH. Furthermore, both lactacystin and MG132 prevented the decrease in ADH caused by DHT. By contrast, the lysosomal proteolytic inhibitors 3-methyladenine and leupeptin as well as inhibitors of the calcium-activated neutral protease calpain system had no effect on ADH in the absence or presence of DHT. ADH isolated by immunoprecipitation from hepatocytes exposed to DHT reacted specifically with anti-ubiquitin antibody. Ubiquitinated ADH was also demonstrated in hepatocytes exposed to MG132. The combination of DHT and MG132 resulted in more ubiquitinated ADH than exposure to either compound alone. These results suggest that the ubiquitin-proteasome pathway plays a role in the degradation of ADH and in the enhanced degradation of this enzyme by DHT.
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PMID:Liver alcohol dehydrogenase is degraded by the ubiquitin-proteasome pathway. 1145 41

We tested the influence of IFNgamma on proteasome activity in parental Hep G2 cells that do not metabolize ethanol, as well as in recombinant Hep G2-derived cells that express either or both alcohol dehydrogenase (ADH) and cytochrome P4502E1 (CYP2E1). IFNgamma treatment increased proteasome activity in VL-17A (ADH(+), CYP2E1(+)) and E-47 (CYP2E1(+)) cells, but not in Hep G2, VI-R2 (parental cells with empty vectors) or in VA-13 (ADH(+)) cells. Proteasome activation by IFNgamma correlated positively with the level of CYP2E1 activity. Treatment of VL-17A cells with agents that inhibit CYP2E1 or the inducible nitric oxide synthase (iNOS) or that prevent the formation of peroxynitrite also blocked proteasome activation by IFNgamma, indicating that the proteasome may be directly activated by products of CYP2E1 and iNOS catalysis. While IFNgamma treatment increased proteasome activity, it also decreased CYP2E1 activity. Both effects were mediated via the Janus kinase-signal transducer and activator of transcription 1 (JAK-STAT1) pathway, as both were blocked by the JAK2 inhibitor, tyrphostin AG 490. Ethanol treatment of VL-17A cells also caused a similar blockage of these same IFNgamma-mediated effects, by inhibiting STAT1 phosphorylation. This inhibition was largely due to ethanol metabolism, as 4-methylpyrazole, an ethanol metabolism inhibitor, restored IFNgamma-mediated STAT1 phosphorylation in ethanol-treated cells. Our results lead us to propose that IFNgamma initiates signal transduction, which alters the activities of CYP2E1 and iNOS, thereby producing reactive oxygen species. One of these oxidants, possibly peroxynitrite, may be directly involved in proteasome activation. Ethanol metabolism by VL-17A cells suppresses IFNgamma-mediated induction of proteasome activity, in part, by preventing STAT1 phosphorylation.
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PMID:Interferon gamma enhances proteasome activity in recombinant Hep G2 cells that express cytochrome P4502E1: modulation by ethanol. 1294 50

Possible target proteins of cytosolic thioredoxin in higher plants have been investigated in the cell lysate of dark-grown Arabidopsis thaliana whole tissues. We immobilized a mutant of cytosolic thioredoxin, in which an internal cysteine at the active site was substituted with serine, on CNBr activated resin, and used the resin for the thioredoxin-affinity chromatography. By using this resin, the target proteins for thioredoxin in the higher plant cytosol were efficiently acquired. The obtained proteins were separated by two-dimensional gel electrophoresis and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Thus we have identified proteins of the anti-oxidative stress system proteins (ascorbate peroxidase, germin-like protein, and monomeric type II peroxiredoxin), proteins involved in protein biosynthesis (elongation factor-2 and eukaryotic translation initiation factor 4A), proteins involved in protein degradation (the regulatory subunit of 26S proteasome), and several metabolic enzymes (alcohol dehydrogenase, fructose 1,6-bis phosphate aldolase-like protein, cytosolic glyceraldehyde 3-phosphate dehydrogenase, cytosolic malate dehydrogenase, and vitamin B(12)-independent methionine synthase) together with some chloroplast proteins (chaperonin 60-alpha and 60-beta, heat shock protein 70, and glutamine synthase). The results in this study and recent proteomics studies on the target proteins of chloroplast thioredoxin indicate the versatility and the physiological significance of thioredoxin as reductant in plant cell.
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PMID:Target proteins of the cytosolic thioredoxins in Arabidopsis thaliana. 1474 82

The lipid peroxidation product 4-hydroxynonenal (4-HNE) has been shown to interfere with protein function. The goal of this study was to determine the effects of substrate modification by 4-HNE on protein degradation. Equine liver alcohol dehydrogenase (ADH, EC 1.1.1.1) treated with 2-fold molar excess 4-HNE was degraded by a rabbit reticulocyte lysate (RRL) system approximately 1.5-fold faster than control, while treatment with concentrations up to 100-fold molar excess aldehyde were inhibitory to degradation. Involvement of the 26S proteasome (EC 3.4.99.46) was demonstrated through the use of specific proteasome and ATPase inhibitors, and confirmed by measuring the extent of ADH polyubiquitination. Tryptic digestion and LC/MS analysis of 4-HNE-treated ADH identified modification of two zinc chelating Cys residues. Through molecular modeling experiments a conformational shift in both zinc-containing regions was predicted, with an approximate doubling of the distance between the structural zinc and its respective chelating residues. Modification of residues in the active site zinc binding motif resulted in less pronounced alteration in protein structure. The data presented here demonstrate accelerated ubiquitination and proteasomal degradation of ADH modified with 4-HNE, and suggest a conformational change after 4-HNE docking as a mechanism behind these observations.
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PMID:4-Hydroxynonenal regulates 26S proteasomal degradation of alcohol dehydrogenase. 1545 82

HepG2 cells were transfected with recombinant plasmids, one carrying the murine alcohol dehydrogenase (ADH) gene and the other containing the gene encoding human cytochrome P450 2E1 (CYP2E1). One of recombinant clones called VL-17A exhibited ADH and CYP2E1 specific activities comparable to those in isolated rat hepatocytes. VL-17A cells oxidized ethanol and generated acetaldehyde, the levels of which depended upon the initial ethanol concentration. Compared with unexposed VL-17A cells, ethanol exposure increased the cellular redox (lactate:pyruvate ratio) and caused cell toxicity, indicated by increased leakage of lactate dehydrogenase into the medium,. Exposure of VL-17A cells to 100mM ethanol significantly elevated caspase 3 activity, an indicator of apoptosis, but this ethanol concentration did not affect caspase 3 activity in parental HepG2 cells. Because ethanol consumption causes a decline in hepatic protein catabolism, we examined the influence of ethanol exposure on proteasome activity in HepG2, VL-17A, E-47 (CYP2E1(+)) and VA-13 (ADH(+)) cells. Exposure to 100mM ethanol caused a 25% decline in the chymotrypsin-like activity of the proteasome in VL-17A cells, but the enzyme was unaffected in the other cell types. This inhibitory effect on the proteasome was blocked when ethanol metabolism was blocked by 4-methyl pyrazole. We conclude that recombinant VL-17A cells, which express both ADH and CYP2E1 exhibit hepatocyte-like characteristics in response to ethanol. Furthermore, the metabolism of ethanol by these cells via ADH and CYP2E1 is sufficient to bring about an inhibition of proteasome activity that may lead to apoptotic cell death.
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PMID:Recombinant Hep G2 cells that express alcohol dehydrogenase and cytochrome P450 2E1 as a model of ethanol-elicited cytotoxicity. 1618

We previously showed that IFNgamma signal transduction was suppressed by ethanol in recombinant HepG2 cells (VL-17A cells), which express alcohol dehydrogenase (ADH) and CYP2E1. We examined the mechanisms by which STAT1 phosphorylation is blocked by ethanol treatment in VL-17A cells. Cells were exposed to 0 or 100 mmol/L ethanol for 72 hours. STAT1 phosphorylation was determined by Western blot after 1 hour IFNgamma exposure. Reduction of STAT1 phosphorylation by ethanol was prevented in the presence of 4MP, DAS, or uric acid, indicating that the oxidative products from ethanol metabolism were partly responsible for suppression of STAT1 phosphorylation. Ethanol exposure decreased STAT1 tyrosine phosphorylation, whereas serine phosphorylation on the protein was unchanged. These effects of ethanol were mimicked by the peroxynitrite (PN) donor, SIN-1, which also blocked tyrosine, but not serine phosphorylation, on STAT1. When cells expressing either ADH (VA-13 cells) or CYP2E1 (E-47 cells) were exposed to ethanol, both ADH- and CYP2E1-generated products reduced STAT1 phosphorylation. In addition, SOCS1, a negative regulator of IFNgamma signaling and which is degraded by the proteasome, was stabilized by ethanol treatment, presumably because of inhibited proteasome activity. Furthermore, SIN-1 treatment elevated SOCS1 levels in VL-17A cells, indicating that PN has a role in SOCS1 elevation. In conclusion, under conditions of ethanol-elicited oxidative stress, PN prevents STAT1 phosphorylation by stabilization of SOCS1, and possibly by nitration of tyrosine residues in STAT1 protein.
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PMID:Ethanol metabolism alters interferon gamma signaling in recombinant HepG2 cells. 1625 53

Volatile esters, primarily synthesized in peel tissues, are major aromatic components of apple fruits [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.]. The use of cold storage combined with 1-methylcyclopropene (1-MCP) treatment prolongs the life of apples but represses the regeneration of esters during poststorage ripening. In this study, the regeneration of total esters was significantly increased in apple fruits treated with salicylic acid (SA) and Ethephon (ETH) that had been treated once or twice with 1-MCP. However, methyl jasmonate (MeJA) treatment resulted in regeneration of total esters after a single 1-MCP treatment. To determine the mechanism by which SA, ETH, and MeJA regulate ester regeneration, the apple alcohol acyltransferase gene (MdAAT2) was investigated at the mRNA, protein, and enzyme activity levels. Genes associated with ethylene perception were also investigated by RT-PCR. The results suggest that MdAAT2 controls ester regeneration and that MdETR1 plays a key role in ethylene perception and regulation of downstream MdAAT2 gene expression during poststorage. Ester compounds and concentrations differed in peels treated with different signal molecules, indicating that regulation of the pathway upstream of straight-chain ester biosynthesis depended on the regulation of lipoxygenase (LOX) and alcohol dehydrogenase (ADH) activity by SA, ETH, and MeJA during poststorage ripening.
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PMID:Salicylic acid, ethephon, and methyl jasmonate enhance ester regeneration in 1-MCP-treated apple fruit after long-term cold storage. 1671 11

Efficiency of nutrient utilization is high in neonates with normal birth weights but is reduced in those with intrauterine growth restriction (IUGR). However, the underlying mechanisms are largely unknown. This study was conducted with the piglet model and proteomics technology to test the hypothesis that IUGR affects expression of key proteins that regulate growth and development of the small intestine, liver, and muscle, the major organs involved in the digestion, absorption, and metabolism of dietary nutrients. Jejunum, liver, and gastrocnemius muscle were obtained from IUGR and normal birth-weight piglets at birth for analysis of proteomes using the 2-dimensional-PAGE MS technology. The results indicate that IUGR decreased the levels of proteins that regulate immune function (immunoglobulins and annexin A1), oxidative defense (peroxiredoxin 1, transferrin, and zeta-crystallin), intermediary metabolism (creatine kinase, alcohol dehydrogenase, L-lactate dehydrogenase, prostaglandin F synthase, apolipoprotein AI, catecho O-methyltransferase, and phosphoglycerate kinase 1), protein synthesis (eukaryotic translation initiation factor-3), and tissue growth (beta-actin, desmin, and keratin 10) in a tissue-specific manner. In addition, IUGR increased the levels of proteins that are involved in proteolysis (proteasome alpha-5 and alpha-1 subunits), response to oxidative stress (scavenger-receptor protein and alpha-1 acid glycoprotein), and ATP hydrolysis (F1-ATPase). These novel findings suggest that cellular signaling defects, redox imbalance, reduced protein synthesis, and enhanced proteolysis may be the major mechanisms responsible for abnormal absorption and metabolism of nutrients, as well as reduced growth and impaired development of the small intestine, liver, and muscle in IUGR neonates.
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PMID:Intrauterine growth restriction affects the proteomes of the small intestine, liver, and skeletal muscle in newborn pigs. 1815 5

Acute and chronic ethanol administration increase autophagic vacuole (i.e., autophagosome; AV) content in liver cells. This enhancement depends on ethanol oxidation. Here, we used parental (nonmetabolizing) and recombinant (ethanol-metabolizing) Hep G2 cells to identify the ethanol metabolite that causes AV enhancement by quantifying AVs or their marker protein, microtubule-associated protein 1 light chain 3-II (LC3-II). The ethanol-elicited rise in LC3-II was dependent on ethanol dose, was seen only in cells that expressed alcohol dehydrogenase (ADH) and was augmented in cells that coexpressed cytochrome CYP2E1 (P450 2E1). Furthermore, the rise in LC3-II was inversely related to a decline in proteasome activity. AV flux measurements and colocalization of AVs with lysosomes or their marker protein Lysosomal-Associated Membrane Protein 1 (LAMP1) in ethanol-metabolizing VL-17A cells (ADH (+) /CYP2E1 (+) ) revealed that ethanol exposure not only enhanced LC3-II synthesis but also decreased its degradation. Ethanol-induced accumulation of LC3-II in these cells was similar to that induced by the microtubule inhibitor, nocodazole. After we treated cells with either 4-methylpyrazole to block ethanol oxidation or GSH-EE to scavenge reactive species, there was no enhancement of LC3-II by ethanol. Furthermore, regardless of their ethanol-metabolizing capacity, direct exposure of cells to acetaldehyde enhanced LC3-II content. We conclude that both ADH-generated acetaldehyde and CYP2E1-generated primary and secondary oxidants caused LC3-II accumulation, which rose not only from enhanced AV biogenesis, but also from decreased LC3 degradation by the proteasome and by lysosomes.
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PMID:Multilevel regulation of autophagosome content by ethanol oxidation in HepG2 cells. 2309 Jan 41


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