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 endoplasmic reticulum (ER) membrane protein 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is subject to regulated degradation when cells are presented with an excess of sterols or mevalonate. In this report, we demonstrate the degradation of HMG-CoA reductase in ER membranes prepared from cells which have been pretreated with mevalonate or sterols prior to membrane purification. Degradation of HMG-CoA reductase in membranes prepared from pretreated cells is more rapid than in membranes prepared from cells which have received no regulatory molecules. In vitro degradation is blocked by protease inhibitors previously shown to inhibit reductase degradation in vivo and is specific for intact HMG-CoA reductase. The lumenal contents of the ER membranes are dispensible for the regulated proteolysis and the proteases responsible for reductase degradation are stably associated with the ER membrane. Regulated proteolysis of HMG-CoA reductase is inhibited by lactacystin, a newly defined inhibitor of the multicatalytic protease, the proteasome, and in vitro degradation of reductase correlates with the presence of proteasome subunits in purified ER membranes. The ubiquitin system for protein degradation, which has recently been shown to be required for the degradation of several ER membrane proteins, is not required for the degradation of HMG-CoA reductase. Finally, we conclude that the regulated proteolysis of HMG-CoA reductase in response to regulatory molecules such as mevalonate or sterols is mediated by increased susceptibility of the reductase to ER proteases, rather than the induction of a new proteolytic activity.
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PMID:Degradation of 3-hydroxy-3-methylglutaryl-CoA reductase in endoplasmic reticulum membranes is accelerated as a result of increased susceptibility to proteolysis. 881 Mar 39

The cytochrome P-450 family of enzymes performs an incredibly diverse range of detoxification and oxidation reactions within the cell and constitutes between 5 and 10% of protein in hepatic endoplasmic reticulum. In this report it is demonstrated that constitutively expressed membranous P-450s are targeted for destruction by the proteasome, in a process which is ubiquitin-independent and is demonstrated in vitro to require prior labilization of the enzyme. This process was specific for P-450s CYP1A2, CYP2E1, CYP3A, and CYP4A and was not demonstrated to be involved in the turnover of CYP1A1, CYP2B1/2, or NADPH reductase. In reconstitution experiments using purified proteasomes and microsomal fractions, labilized P-450 conformations are protected from 20 S proteasome degradation by substrate addition, with proteolysis occurring while P-450s are still attached to the endoplasmic reticulum.
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PMID:Evidence of proteasome-mediated cytochrome P-450 degradation. 909 10

The title compound Ta6Br(2+)12 is of interest for the analysis of biological structures as a heavy-metal derivative with great potential for the structure determination of large protein systems. In macromolecular crystallography the phases of the measured structure factor amplitudes have to be determined. The most widely used method for novel structures is isomorphous replacement by introducing electron-rich compounds into the protein crystals. These compounds produce measurable changes of the diffraction intensities, which allow phase determination. We synthetized the Ta6Br(2+)12 cluster in high yields, crystallized it, and determined its crystal structure by X-ray diffraction analysis at atomic resolution. The cluster is a regular octahedron consisting of six metal atoms with 12 bridging bromine atoms along the 12 edges of the octahedron. The cluster is compact, of approximately spherical shape with about 4.3 A radius and highly symmetrical. One Ta6Br(2+)12 ion adds 856 electrons to a protein, a considerable contribution to the scattering power even of large proteins or multimeric systems. At low resolution all atoms of the cluster scatter in phase and act as a super heavy-atom, which is easy to locate in the difference Patterson map. We investigated its binding sites in the biologically significant high-resolution structures of an antibody V(L) domain, dimethyl sulfoxide reductase, GTP-cyclohydrolase I, and the proteasome. With the randomly oriented cluster, treated as a single site scatterer, phases could be used only up to 6 A resolution. In contrast, when the cluster is correctly oriented, phases calculated from its 18 atom sites can be used to high resolution. We present the atomic structure of the Ta6Br(2+)12, describe a method to determine its localization and orientation in the unit cell of protein crystals of two different proteins, and analyse its phasing power. We show that phases can be calculated to high resolution. The phase error is lower by more than 30 degrees compared to the single site approximation, using a resolution of 2.2 A. Furthermore, Ta6Br(2+)12 has two different strong anomalous scatterers tantalum and bromine to be used for phase determination.
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PMID:Ta6Br(2+)12, a tool for phase determination of large biological assemblies by X-ray crystallography. 923 95

Hydroxymethylglutaryl-coenzyme A reductase degradation occurs in the endoplasmic reticulum, and is regulated by the mevalonate pathway. In order to discover the molecules that mediate the degradation process and its control, we conducted a genetic analysis of the degradation of the yeast Hmg2p isozyme of hydroxymethylglutaryl-coenzyme A reductase. Hmg2p degradation occurs by the action of HRD genes that direct Hmg2p to the ubiquitin-proteasome pathway. Regulation of HRD-dependent Hmg2p degradation appears to occur by the action of a separate set of CRD genes.
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PMID:Genetic analysis of hydroxymethylglutaryl-coenzyme A reductase regulated degradation. 955 64

We have recently shown that the endoplasmic reticulum (ER) membrane protein, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is cleaved in isolated membrane fractions enriched for endoplasmic reticulum. Importantly, the cleavage rate is accelerated when the membranes are prepared from cells that have been pretreated with mevalonate or sterols, physiological regulators of the degradation process in vivo (McGee, T. P., Cheng, H. H., Kumagai, H., Omura, S., and Simoni, R. D. (1996) J. Biol. Chem. 271, 25630-25638). In the current study, we further characterize this in vitro cleavage of HMG-CoA reductase. E64, a specific inhibitor of cysteine-proteases, inhibits HMG-CoA reductase cleavage in vitro. In contrast, lactacystin, an inhibitor of the proteasome, inhibits HMG-CoA reductase degradation in vivo but does not inhibit the in vitro cleavage. Purified ER fractions contain lactacystin-sensitive and E64-insensitive proteasome activity as measured by succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin hydrolysis. We removed the proteasome from purified ER fractions by solubilization with heptylthioglucoside and observed that the detergent extracted, proteasome-depleted membrane fractions retain regulated cleavage of HMG-CoA reductase. This indicates that ER-associated proteasome is not involved in degradation of HMG-CoA reductase in vitro. In order to determine the site(s) of proteolysis of HMG-CoA reductase in vitro, four antisera were prepared against peptide sequences representing various domains of HMG-CoA reductase and used for detection of proteolytic intermediates. The sizes and antibody reactivity of the intermediates suggest that HMG-CoA reductase is cleaved in the in vitro degradation system near the span 8 membrane region, which links the N-terminal membrane domain to the C-terminal catalytic domain of the protein. We conclude that HMG-CoA reductase can be cleaved in the membrane-span 8 region by a cysteine protease(s) tightly associated with ER membranes.
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PMID:Degradation of HMG-CoA reductase in vitro. Cleavage in the membrane domain by a membrane-bound cysteine protease. 970 46

In this paper we present the finding that lovastatin arrests cells by inhibiting the proteasome, which results in the accumulation of p21 and p27, leading to G1 arrest. Lovastatin is an inhibitor of hydroxymethyl glutaryl (HMG)-CoA reductase, the rate-limiting enzyme in cholesterol synthesis. Previously, we reported that lovastatin can be used to arrest cultured cells in the G1 phase of the cell cycle, resulting in the stabilization of the cyclin-dependent kinase inhibitors (CKIs) p21 and p27. In this report we show that this stabilization of p21 and p27 may be the result of a previously unknown function of the pro-drug, beta-lactone ring form of lovastatin to inhibit the proteasome degradation of these CKIs. The lovastatin mixture used in this study is 80% open-ring form and 20% pro-drug, beta-lactone form. We show that while the lovastatin open-ring form and pravastatin (a lovastatin analogue, 100% open ring) inhibit the HMG-CoA reductase enzyme, lovastatin pro-drug inhibits the proteasome but does not inhibit HMG-CoA reductase. In addition, many of the properties of proteasome inhibition by the pro-drug are the same as the specific proteasome inhibitor lactacystin. Lastly, mevalonate (used to rescue cells from lovastatin arrest) unexpectedly abrogates the lactacystin and lovastatin pro-drug inhibition of the proteasome. Mevalonate increases the activity of the proteasome, which results in degradation of the CKIs, allowing lovastatin- and lactacystin-arrested cells to resume cell division. The lovastatin-mediated inhibition of the proteasome suggests a unique mechanism for the chemopreventative effects of this agent seen in human cancer.
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PMID:Lovastatin-mediated G1 arrest is through inhibition of the proteasome, independent of hydroxymethyl glutaryl-CoA reductase. 1039 1

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the key regulatory enzyme in the mevalonate (MVA) pathway, is rapidly degraded in mammalian cells supplemented with sterols or MVA. This accelerated turnover was blocked by N-acetyl-leucyl-leucyl-norleucinal (ALLN), MG-132, and lactacystin, and to a lesser extent by N-acetyl-leucyl-leucyl-methional (ALLM), indicating the involvement of the 26 S proteasome. Proteasome inhibition led to enhanced accumulation of high molecular weight polyubiquitin conjugates of HMGR and of HMGal, a chimera between the membrane domain of HMGR and beta-galactosidase. Importantly, increased amounts of polyubiquitinated HMGR and HMGal were observed upon treating cells with sterols or MVA. Cycloheximide inhibited the sterol-stimulated degradation of HMGR concomitantly with a marked reduction in polyubiquitination of the enzyme. Inhibition of squalene synthase with zaragozic acid blocked the MVA- but not sterol-stimulated ubiquitination and degradation of HMGR. Thus, similar to yeast, the ubiquitin-proteasome pathway is involved in the metabolically regulated turnover of mammalian HMGR. Yet, the data indicate divergence between yeast and mammals and suggest distinct roles for sterol and nonsterol metabolic signals in the regulated ubiquitination and degradation of mammalian HMGR.
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PMID:The ubiquitin-proteasome pathway mediates the regulated degradation of mammalian 3-hydroxy-3-methylglutaryl-coenzyme A reductase. 1096 18

We have recently shown that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, an endoplasmic reticulum (ER) membrane protein, is degraded in ER membranes prepared from sterol pretreated cells and that such degradation is catalyzed by a cysteine protease within the reductase membrane domain. The use of various protease inhibitors suggested that degradation of HMG-CoA reductase in vitro is catalyzed by a cathepsin L-type cysteine protease. Purified ER contains E-64-sensitive cathepsin L activity whose inhibitor sensitivity was well matched to that of HMG-CoA reductase degradation in vitro. CLIK-148 (cathepsin L inhibitor) inhibited degradation of HMG-CoA reductase in vitro. Purified cathepsin L also efficiently cleaved HMG-CoA reductase in isolated ER preparations. To determine whether a cathepsin L-type cysteine protease is involved in sterol-regulated degradation of HMG-CoA reductase in vivo, we examined the effect of E-64d, a membrane-permeable cysteine protease inhibitor, in living cells. While lactacystin, a proteasome-specific inhibitor, inhibited sterol-dependent degradation of HMG-CoA reductase, E-64d failed to do so. In contrast, degradation of HMG-CoA reductase in sonicated cells was inhibited by E-64d, CLIK-148, and leupeptin but not by lactacystin. Our results indicate that HMG-CoA reductase is degraded by the proteasome under normal conditions in living cells and that it is cleaved by cathepsin L leaked from lysosomes during preparation of the ER, thus clarifying the apparently paradoxical in vivo and in vitro results. Cathepsin L-dependent proteolysis was observed to occur preferentially in sterol-pretreated cells, suggesting that sterol treatment results in conformational changes in HMG-CoA reductase that make it more susceptible to such cleavage.
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PMID:3-hydroxy-3-methylglutaryl coenzyme A reductase is sterol-dependently cleaved by cathepsin L-type cysteine protease in the isolated endoplasmic reticulum. 1136 43

Adaptation to hypoxia is a topic of considerable clinical relevance, as it influences the pathophysiology of anaemia, polycythaemia, tissue ischaemia and cancer. A growing number of physiologically relevant genes are regulated in response to changes in intracellular oxygen tension. These include genes encoding erythropoietin, vascular endothelial growth factor and tyrosine hydroxylase. Studies on the regulation of the erythropoietin gene have provided insights into the common mechanism of oxygen sensing and signal transduction, leading to activation of the hypoxia-inducible transcription factor 1 (HIF-1). Activation of HIF-1 by hypoxia depends on rescue of its alpha-subunit from oxygen-dependent degradation in the proteasome, allowing it to form a heterodimer with HIF-1 beta. This then translocates to the nucleus. There, HIF-1 assembles with a highly conserved orphan nuclear receptor, HNF-4, and a critical transcriptional adaptor, p300. This complex binds to a 3' enhancer on the erythropoietin gene, enabling transcription of erythropoietin. HIF-1 also activates other genes, the cis-acting elements of which contain cognate hypoxia response elements. There is growing evidence that the oxygen sensor is a flavohaem protein and that the signal transduction pathway involves changes in the level of intracellular reactive oxygen intermediates. We have recently cloned a novel fusion protein called cytochrome b5/b5 reductase, which is a cyanide-insensitive NADPH oxidase and, therefore, a candidate to be the oxygen sensor. This flavohaem protein is widely expressed in cell lines and tissues, with localization in the perinuclear space. In the presence of oxygen and iron, it may induce oxidative modifications that target HIF-1 alpha for ubiquitination and degradation.
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PMID:Detecting and responding to hypoxia. 1181 5

A number of clinical studies suggest that the use of the lipid-lowering agents collectively referred to as statins (hydroxymethyl glutaryl coenzyme A [HMG-CoA] reductase inhibitors) is associated with increased bone density, reduced fracture risk, and net bone anabolism. Statins (< or =5 micromol/L) stimulate rodent bone formation, but the mechanistic basis remains unclear. Since statins and the proteasome inhibitor lactacystin are structurally similar, and high doses (> or =40 micromol/L) of statins can inhibit the chymotryptic activity of the proteasome, it has been hypothesized that statins exert their anabolic effects on bone, in part, by inhibiting the proteasome, the major eukaryotic intracellular regulatory protease. This hypothesis conflicts with reports that statins stimulate proteasome activity and that proteasome-catalyzed degradation of specific substrates is required for cell proliferation, differentiation, and survival. Our chief objective was to determine the effects of statins (< or =10 micromol/L) on the chymotryptic activity of the proteasome in the 20 S proteasome and intact murine MC3T3-E1 cells cultured to low density (preosteoblasts) or high density (differentiated osteoblasts). Lovastatin (0.001 micromol/L to 5.0 micromol/L) stimulated the chymotryptic activity of the highly purified 20 S proteasome. Preosteoblasts and differentiated osteoblasts treated with 1, 5, or 10 micromol/L lovastatin for 1 hour exhibited morphologic abnormalities that were ameliorated by preincubation and treatment with 20 micromol/L mevalonate. The chymotryptic activity of the preosteoblast proteasome increased after 2 days of 1.0 micromol/L or 5.0 micromol/L lovastatin treatment. In addition, the DNA and protein contents of 1.0 micromol/L or 5.0 micromol/L lovastatin-treated preosteoblast cultures were lower those that observed in vehicle-, 0.01 micromol/L lovastatin-, or 0.10 micromol/L lovastatin-treated cultures. The chymotryptic activity of the proteasome was much lower in differentiated osteoblasts than in preosteoblasts. Two days of treatment with 1 micromol/L lovastatin modestly stimulated the chymotryptic activity of the proteasome in differentiated osteoblasts, but had no effects on total protein or DNA, compared to cultures treated with vehicle or lower doses of lovastatin. Thus, the data support the hypothesis that statins stimulate proteasome activities in highly purified proteasome preparations and preosteoblastic cells. Treating preosteoblastic or differentiated MC3T3-E1 cells with lovastatin concentrations > or = 1 micromol/L resulted in abnormal morphology and reduced the DNA and protein levels in preosteoblastic cultures, confirming the adverse effects of statins previously reported for other cells. In conclusion, the hypothesis that lovastatin exerts its anabolic effects on bone by inhibiting the proteasome activity of the osteoblast was refuted, and the effects of lovastatin on MC3T3-E1 cells were found to be highly dose- and development-dependent.
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PMID:The effects of lovastatin on proteasome activities in highly purified rabbit 20 S proteasome preparations and mouse MC3T3-E1 osteoblastic cells. 1220 Jul 60


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