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)

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

Plasma fibrinogen is synthesized primarily in hepatocytes and assembly of the three component chains (A alpha, B beta, and gamma) into its final form as a six-chain dimer (A alpha, B beta, gamma)2 occurs rapidly in the lumen of the endoplasmic reticulum (ER). Assembly takes place in a stepwise manner with single chains interacting with each other to form A alpha-gamma and B beta-gamma complexes. The two-chain complexes then acquire another chain to form half-molecules (A alpha, B beta, gamma)1, which in a final step are linked to form the six-chain (A alpha, B beta, gamma)2 complex. As with other secreted glycoproteins, N-linked glycosylation of B beta and gamma chains commences in the ER and is completed in Golgi organelles. Sulfation and phosphorylation occur at post-ER stages of the secretory process. Since some ER chaperones coisolate with nascent fibrinogen chains they have been implicated in assisting chain assembly. Studies with recombinant systems, using deletion and substitution mutants, indicate that initial chain assembly depends on hydrophobic interactions present in the C-terminal half of the coil-coil domains and that inter- and intra-disulfide bonds that stabilize fibrinogen are needed to complete chain assembly. Not all the chains that are synthesized are assembled into fibrinogen and the unassembled chains are not secreted. HepG2 cells contain surplus A alpha and gamma chains that accumulate as free gamma chains and as an A alpha-gamma complex. A alpha-gamma is degraded by lysosomes whereas the gamma chain is degraded by the proteasome-ubiquitin system. Studies with expression of single chains by COS cells confirm that gamma and B beta are hydrolyzed by proteasomes and indicate that A alpha is degraded partially both by lysosomes and proteasomes. The role of surplus chains in regulating fibrinogen assembly is not understood but overexpression of any one chain, elicited by transfection of HepG2 cells, results in the upregulation of the other two genes, increased fibrinogen synthesis and secretion, and maintenance of surplus intracellular A alpha and gamma chains. HepG2 cells, programmed in this manner to increase basal fibrinogen expression, have higher HMG-CoA reductase mRNA levels, enhanced cholesterol and cholesterol ester synthesis, and increased secretion of apolipoprotein B (apoB). Overexpression of basal levels of fibrinogen does not affect synthesis of other acute phase proteins. Enhanced secretion of apoB is due to diminished degradation of nascent apoB by proteasomes and not to increased expression. Increased secretion of apoB is associated with increased basal expression of fibrinogen and is not affected when fibrinogen expression is stimulated by interleukin-6. In HepG2 cells, a feedback mechanism exists and extracellular sterols specifically downregulate expression of the three fibrinogen genes. These studies link, at the cellular level, basal fibrinogen expression with lipid metabolism.
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PMID:Fibrinogen biosynthesis. Assembly, intracellular degradation, and association with lipid synthesis and secretion. 1146 May 6

Sterols accelerate degradation of the ER enzyme 3-hydroxy-3-methylglutaryl CoA reductase (HMG CoA reductase), which catalyzes a rate-controlling step in cholesterol biosynthesis. This degradation contributes to feedback inhibition of synthesis of cholesterol and nonsterol isoprenoids. Here, we show that degradation of HMG CoA reductase is accelerated by the sterol-induced binding of its sterol-sensing domain to the ER protein insig-1. Accelerated degradation is inhibited by overexpression of the sterol-sensing domain of SREBP cleavage-activating protein (SCAP), suggesting that both proteins bind to the same site on insig-1. Whereas insig-1 binding to SCAP leads to ER retention, insig-1 binding to HMG CoA reductase leads to accelerated degradation that is blocked by proteasome inhibitors. Insig-1 appears to play an essential role in the sterol-mediated trafficking of two proteins with sterol-sensing domains, HMG CoA reductase and SCAP.
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PMID:Accelerated degradation of HMG CoA reductase mediated by binding of insig-1 to its sterol-sensing domain. 1253 18

Apomine, a novel 1,1-bisphosphonate ester, has been shown to lower plasma cholesterol concentration in several species. Here we show that Apomine reduced the levels of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the rate-limiting enzyme in the mevalonate pathway, both in rat liver and in cultured cells. Apomine resembles sterols such as 25-hydroxycholesterol in its ability to potently accelerate the rate of HMGR degradation by the ubiquitin-proteasome pathway, a process that depends on the transmembrane domain of the enzyme. The similarity between Apomine and sterols in promoting rapid HMGR degradation extends to its acute requirements for ongoing protein synthesis and mevalonate-derived non-sterol product(s) as a co-regulator. Yet, at suboptimal concentrations, sterols potentiated the effect of Apomine in stimulating HMGR degradation, indicating that these agents act via distinct modes. Furthermore, unlike sterols, Apomine inhibited the activity of acyl-CoA:cholesterol acyltransferase in intact cells but not in cell-free extracts. Apomine stimulated the cleavage of the precursor of sterol-regulatory element-binding protein-2 and increased the activity of low density lipoprotein receptor pathway. This Apomine-enhanced activation of sterol-regulatory element-binding protein-2 was prevented by sterols or mevalonate. Taken together, our results provide a molecular mechanism for the hypocholesterolemic activity of Apomine.
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PMID:Apomine, a novel hypocholesterolemic agent, accelerates degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and stimulates low density lipoprotein receptor activity. 1462 8

As the main risk factor for cardiovascular disease, hypercholesterolemia is one of the most studied age-related metabolic alterations. In the liver, cholesterol homeostasis is strictly regulated through the modulation of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), the key enzyme of cholesterol biosynthesis. With ageing, hepatic HMG-CoA reductase becomes completely activated and cholesterol content increases in the blood. The research reported in this paper uses the regulatory enzymes of reductase (i.e., the AMP-dependent kinase (AMPK) and the protein phosphatase 2A (PP2A)), the HMG-CoA reductase thermodependent activity and the "in vitro" enzyme degradation to elucidate the role played by the HMG-CoA reductase regulation and its membrane interaction. Related experiments were performed on 3 and 24 months "ad libitum" (AL) fed rats and 24 months caloric-restricted rats. The results show no changes in the PP2A level and the activation state of AMP dependent kinase in aged "ad libitum" fed rats. By contrast, the activation state of the kinase is enhanced in the aged caloric-restricted animals. With respect to the adult, the thermodependent activity of reductase remains unchanged, while the degradation rate of the HMG-CoA reductase is slower and independent on proteasome. These findings support the hypothesis that a different arrangement of the HMG-CoA reductase membrane domain in aged rats is a cause of reductase deregulation.
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PMID:Mechanisms underlying the impaired regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in aged rat liver. 1549 82

In contrast with the accelerated degradation observed in tumor cells in response to sterols, hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase turnover in whole animals was not increased by dietary cholesterol. Furthermore, treating rats with lovastatin to lower hepatic cholesterol levels did not decrease the rate of degradation. The half-life remained in the 6 h range. Co-immunoprecipitation studies revealed that the amount of ubiquitin associated with the reductase was entirely dependent upon the amount of microsomal protein subjected to immunoprecipitation. The results indicate that in liver, neither the rate of reductase protein degradation nor the ubiquitin-proteasome system appear to play roles in mediating changes in HMG-CoA reductase protein levels in response to dietary cholesterol.
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PMID:Degradation of HMG-CoA reductase in rat liver is cholesterol and ubiquitin independent. 1591 82

Inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, known as statins, effectively prevent cardiovascular events. In addition to their lipid lowering properties, a variety of pleiotropic effects on cardiovascular cells were demonstrated in vitro and in vivo. It has been hypothized that statins deploy a part of their effects by targeting the proteasome. Statin-induced effects remarkably overlap with effects obtained by inhibition of the proteasome in endothelial and vascular smooth muscle cells (e.g., endothelial nitric oxide synthase (eNOS)-upregulation, attenuation of nuclear factor kappa B (NF-kappaB) activation, inhibition of proliferation). We therefore examined, whether statins modulate the proteasomal activity of vascular cells. We studied the effect of simvastatin, atorvastatin, and pravastatin as well as of the proteasome inhibitor clasto-lactacystin on morphology, proliferation, viability, and proteasomal activity in two mammalian endothelial cell lines (CPAE and Ea.hy962), and in primary vascular smooth muscle cells (VSMCs). Both statins and lactacystin induced comparable morphological changes and attenuated proliferation of calf pulmonary artery cell line (CPAE). Whereas the statin-induced effects were reversed by mevalonic acid, however, the lactacystin-induced alterations were not influenced by mevalonic acid. As expected, lactacystin caused a significant loss of proteasomal activity measured in the extract of treated CPAE cells, whereas the extracts of statin-treated CPAEs exhibited unchanged activities. This result was also confirmed in Ea.hy926 cells and in primary rat VSMCs. We show here, that even high doses of statins do not modulate the activities of purified human 20S proteasomes. We conclude that the similar biological effects of statins and proteasome inhibitors in vascular cells are not due to a common inhibitory mechanism of action on the proteasome.
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PMID:Effect of statins on the proteasomal activity in mammalian endothelial and vascular smooth muscle cells. 1599 38

The membrane-anchored ubiquitin ligase gp78 promotes degradation of misfolded endoplasmic reticulum (ER) proteins and sterol-regulated degradation of HMG-CoA reductase. It was known previously that Ufd1 plays a critical role in ER-associated degradation (ERAD) together with Npl4 and VCP. The VCP-Ufd1-Npl4 complex recognizes polyubiquitin chains and transfers the ubiquitinated proteins to the proteasome. Here we show that Ufd1 directly interacts with gp78 and functions as a cofactor. Ufd1 enhances the E3 activity of gp78, accelerates the ubiquitination and degradation of reductase, and eventually promotes receptor-mediated uptake of low-density lipoprotein. Furthermore, we demonstrate that the monoubiquitin-binding site in Ufd1 is required for the enhancement of gp78 activity and that the polyubiquitin-binding site in Ufd1 is critical for a postubiquitination step in ERAD. In summary, our study identifies Ufd1 as a cofactor of gp78, reveals an unappreciated function of Ufd1 in the ubiquitination reaction during ERAD, and illustrates that Ufd1 plays a critical role in cholesterol metabolism.
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PMID:Ufd1 is a cofactor of gp78 and plays a key role in cholesterol metabolism by regulating the stability of HMG-CoA reductase. 1768 Nov 47

Simvastatin is a competitive inhibitor of HMG-CoA reductase, the rate-limiting enzyme of the mevalonate pathway required for the biosynthesis of cholesterol and higher isoprenoids such as geranylgeranyl pyrophosphate (GGPP). Apart from its capacity to lower cholesterol plasma levels and to protect against cardiovascular disease, simvastatin induces apoptosis in various cancer cells. We have generated human Namalwa Burkitt lymphoma cells that display general apoptosis resistance and hyperproliferation due to increased expression and proteolytic activity of 26S proteasomes in response to continuous treatment of the cells with the proteasome inhibitor bortezomib. In these cells, simvastatin does not inhibit proteasome activity, but induces apoptosis, G2/M cell cycle arrest and accumulation of p21(Waf1/Cip1), and effectively inhibits hyperproliferation. These effects are reversed by the addition of GGPP. GGPP-dependent plasma membrane localization of the small GTPase RhoA that is required for RhoA-mediated oncogenic signaling is completely inhibited by simvastatin. Finally, bortezomib but not simvastatin induces accumulation and stabilization of the anti-apoptotic protein Mcl-1, which is known to confer resistance to apoptosis in cancer cells. Thus, simvastatin overcomes bortezomib-induced apoptosis resistance by inhibiting synthesis of GGPP and disrupting a GGPP-dependent survival pathway.
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PMID:HMG-CoA reductase inhibitor simvastatin overcomes bortezomib-induced apoptosis resistance by disrupting a geranylgeranyl pyrophosphate-dependent survival pathway. 1862 2

The endoplasmic reticulum (ER) glycoprotein HMG-CoA reductase (HMGR) catalyzes the rate-limiting step in sterols biosynthesis. Mammalian HMGR is ubiquitinated and degraded by the proteasome when sterols accumulate in cells, representing the best example for metabolically controlled ER-associated degradation (ERAD). This regulated degradation involves the short-lived ER protein Insig-1. Here, we investigated the dislocation of these ERAD substrates to the cytosol en route to proteasomal degradation. We show that the tagged HMGR membrane region, HMG(350)-HA, the endogenous HMGR, and Insig-1-Myc, all polytopic membrane proteins, dislocate to the cytosol as intact full-length polypeptides. Dislocation of HMG(350)-HA and Insig-1-Myc requires metabolic energy and involves the AAA-ATPase p97/VCP. Sterols stimulate HMG(350)-HA and HMGR release to the cytosol concurrent with removal of their N-glycan by cytosolic peptide:N-glycanase. Sterols neither accelerate dislocation nor stimulate deglycosylation of ubiquitination-defective HMG(350)-HA((K89 + 248R)) mutant. Dislocation of HMG(350)-HA depends on Insig-1-Myc, whose dislocation and degradation are sterol independent. Coimmunoprecipitation experiments demonstrate sterol-stimulated association between HMG(350)-HA and Insig-1-Myc. Sterols do not enhance binding to Insig-1-Myc of HMG(350)-HA mutated in its sterol-sensing domain or of HMG(350)-HA((K89 + 248R)). Wild-type HMG(350)-HA and Insig-1-Myc coimmunoprecipitate from the soluble fraction only when both proteins were coexpressed in the same cell, indicating their encounter before or during dislocation, raising the possibility that they are dislocated as a tightly bound complex.
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PMID:Dislocation of HMG-CoA reductase and Insig-1, two polytopic endoplasmic reticulum proteins, en route to proteasomal degradation. 1945 99


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