Characteristics of rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase.
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ABSTRACT:
SUBMITTER: Ness GC
PROVIDER: S-EPMC1152999 | biostudies-other | 1986 Jan
REPOSITORIES: biostudies-other
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Project description:Dietary cholesterol lowers the activity of rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase without affecting various other liver microsomal enzymes. This is consistent with a specific regulatory mechanism and distinguishes the action of cholesterol on 3-hydroxy-3-methylglutaryl-CoA reductase from that of at least one other stimulus known to affect this enzyme.
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Project description:The objective of this study was to detect the prevalence of anti-3-hydroxyl-3- methylglutaryl coenzyme A reductase (anti-HMGCR) antibodies in Chinese patients with idiopathic inflammatory myopathies (IIMs), and to analyze the clinical features of the antibody-positive IIM patients.The presence of anti-HMGCR antibodies was detected in 405 patients with IIMs, 90 healthy controls, and 221 patients with other rheumatic diseases by using an ELISA kit. Clinical data from anti-HMGCR antibody-positive and -negative patients were compared. Long-term follow-up of the anti-HMGCR antibody-positive patients was conducted to evaluate the role of anti-HMGCR antibody in IIM disease prognosis.Of the 405 IIM patients, 22 (5.4%) were found to carry the anti-HMGCR antibody. These IIM patients were predominantly female (73%), and only 3 anti-HMGCR antibody-positive patients with IIM were exposure to statins. Most patients experienced progressive onset, and presented with muscular weakness. Dysphagia was observed in half of the patients (p < 0.01), and 15% of these patients experienced the complication of interstitial lung disease (ILD) (p > 0.05). Mean creatine kinase (CK) levels were higher in antibody-positive patients than in antibody-negative patients (p < 0.05). Muscle biopsies were available from 12 anti-HMGCR antibody-positive patients, eight who experienced myofiber necrosis and showed very little or no evidence of inflammatory cell infiltrates in their muscle biopsies. Of these eleven patients who were followed-up 2.5- to 29-month, 73% experienced improvement after treatment. A cross-sectional study showed that anti-HMGCR antibody levels were significantly associated with CK levels (r = 0.486, p = 0.026) as well as with Myositis Disease Activity Assessment (MYOACT) scores (r = -0.67, p = 0.003) during the initial visit. However, changes in serum anti-HMGCR antibody levels did not correlate with changes in CK levels, Manual Muscle Testing 8 (MMT-8) scores or MYOACT scores in long-term follow-up.The major clinical features of anti-HMGCR antibody-positive Chinese IIM patients were muscle weakness and dysphagia, which were seen in patients with and without statin exposure. This subtype of patients were responsive to immunosuppressive treatment and received good prognoses after treatment, but serum levels of the anti-HMGCR antibody do not correlate with disease activity.
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Project description:Objective: The intestine occupies the critical interface between cholesterol absorption and excretion. Despite this, surprisingly little is known about the role of de novo cholesterol synthesis in this organ, and its relationship to whole body cholesterol homeostasis. In addition to cholesterol, the mevalonate pathway is responsible for the synthesis of numerous non-sterol isoprenoids. Here we investigate the physiological importance of the mevalonate pathway within the intestine, through genetic deletion of the rate-limiting enzyme. Approach and Results: Mice lacking 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr) in intestinal villus and crypt epithelial cells were generated using a Villin-Cre transgene. In contrast to intestine-specific Srebp-2 and Scap knockouts, mice with intestinal-specific loss of Hmgcr are viable through adulthood and fertile. Hmgcr was efficiently deleted based on mRNA levels, as well as quantitative analysis of Hmgcr alleles by droplet digital PCR. Lipidomics revealed substantial reductions in the abundance of numerous non-sterol isoprenoids and sterol intermediates within the epithelial layer, while cholesterol levels were preserved. Although the intestinal knockout mice are born smaller, there is no net defect in feed efficiency or triglyceride absorption due to compensatory changes in bile acid composition and intestinal growth. At the cellular level, loss of Hmgcr is compensated for quickly after weaning through a dramatic expansion of the stem cell compartment within the crypts. Conclusions: Genetic loss of Hmgcr in the intestine is compatible with life, through mechanisms involving compensatory changes in bile acid composition, increased absorptive surface area, and expansion of the resident stem cell compartment.
2021-01-12 | GSE164589 | GEO
Project description:In this study, we take advantage of the ability of HMG-CoA reductase (HMGR) from Pseudomonas mevalonii to remain active while in its crystallized form to study the changing interactions between the ligands and protein as the first reaction intermediate is created. HMG-CoA reductase catalyzes one of the few double oxidation-reduction reactions in intermediary metabolism that take place in a single active site. Our laboratory has undertaken an exploration of this reaction space using structures of HMG-CoA reductase complexed with various substrate, nucleotide, product, and inhibitor combinations. With a focus in this publication on the first hydride transfer, our structures follow this reduction reaction as the enzyme converts the HMG-CoA thioester from a flat sp(2)-like geometry to a pyramidal thiohemiacetal configuration consistent with a transition to an sp(3) orbital. This change in the geometry propagates through the coenzyme A (CoA) ligand whose first amide bond is rotated 180° where it anchors a web of hydrogen bonds that weave together the nucleotide, the reaction intermediate, the enzyme, and the catalytic residues. This creates a stable intermediate structure prepared for nucleotide exchange and the second reduction reaction within the HMG-CoA reductase active site. Identification of this reaction intermediate provides a template for the development of an inhibitor that would act as an antibiotic effective against the HMG-CoA reductase of methicillin-resistant Staphylococcus aureus.
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Project description:Treatment of rats with pharmacological doses of oestrogen resulted in a 3-fold decrease in the activity of hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) and a 4-fold increase in saturable binding of 125I-labelled chylomicron remnants to liver membranes in vitro. Intragastric administration of mevalonolactone to rats did not affect the capacity of the liver membranes to bind to labelled chylomicron remnants even though there was a substantial decrease in the activity of HMG-CoA reductase. Similar results were obtained after cholesterol feeding. Simultaneous treatment of rats with cholestyramine and compactin increased hepatic HMG-CoA reductase activity 6-fold. However, liver membranes derived from these animals showed no change in their capacity to bind to labelled chylomicron remnants in vitro. Administration of mevalonolactone to the cholestyramine/compactin-treated animals also failed to produce a change in remnant-binding capacity. Although administration of mevalonolactone alone produced a significant 3-fold decrease in the activity of hepatic HMG-CoA reductase it was unable to suppress significantly the increase in enzyme activity caused by treatment with cholestyramine and compactin.
| S-EPMC1153325 | biostudies-other
Project description:In addition to inducing a self-limited myopathy, statin use is associated with an immune-mediated necrotizing myopathy (IMNM), with autoantibodies that recognize ?200-kd and ?100-kd autoantigens. The purpose of this study was to identify these molecules to help clarify the disease mechanism and facilitate diagnosis.The effect of statin treatment on autoantigen expression was addressed by immunoprecipitation using sera from patients. The identity of the ?100-kd autoantigen was confirmed by immunoprecipitation of in vitro-translated 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) protein. HMGCR expression in muscle was analyzed by immunofluorescence. A cohort of myopathy patients was screened for anti-HMGCR autoantibodies by enzyme-linked immunosorbent assay and genotyped for the rs4149056 C allele, a predictor of self-limited statin myopathy.Statin exposure induced expression of the ?200-kd/?100-kd autoantigens in cultured cells. HMGCR was identified as the ?100-kd autoantigen. Competition experiments demonstrated no distinct autoantibodies recognizing the ?200-kd protein. In muscle biopsy tissues from anti-HMGCR-positive patients, HMGCR expression was up-regulated in cells expressing neural cell adhesion molecule, a marker of muscle regeneration. Anti-HMGCR autoantibodies were found in 45 of 750 patients presenting to the Johns Hopkins Myositis Center (6%). Among patients ages 50 years and older, 92.3% had taken statins. The prevalence of the rs4149056 C allele was not increased in patients with anti-HMGCR.Statins up-regulate the expression of HMGCR, the major target of autoantibodies in statin-associated IMNM. Regenerating muscle cells express high levels of HMGCR, which may sustain the immune response even after statins are discontinued. These studies demonstrate a mechanistic link between an environmental trigger and the development of sustained autoimmunity. Detection of anti-HMGCR autoantibodies may facilitate diagnosis and direct therapy.
| S-EPMC3335400 | biostudies-literature
Project description:The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR) catalyzes the first committed step of the mevalonate pathway, which is used across biology in the biosynthesis of countless metabolites. HMGR consumes 2 equiv of the cofactor NAD(P)H to perform the four-electron reduction of HMG-CoA to mevalonate toward the production of steroids and isoprenoids, the largest class of natural products. Recent structural data have shown that HMGR contains a highly mobile C-terminal domain (CTD) that is believed to adopt many different conformations to permit binding and dissociation of the substrate, cofactors, and products at specific points during the reaction cycle. Here, we have characterized the HMGR from Delftia acidovorans as an NADH-specific enzyme and determined crystal structures of the enzyme in unbound, mevalonate-bound, and NADH- and citrate-bound states. Together, these structures depict ligand binding in both the active site and the cofactor-binding site while illustrating how a conserved helical motif confers NAD(P)H cofactor specificity. Unexpectedly, the NADH-bound structure also reveals a new conformation of the CTD, in which the domain has "flipped" upside-down, while directly binding the cofactor. By capturing these structural snapshots, this work not only expands the known range of HMGR domain movement but also provides valuable insight into the catalytic mechanism of this biologically important enzyme.
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Project description:The polytopic endoplasmic reticulum (ER)-localized enzyme 3-hydroxy-3-methylglutaryl CoA reductase catalyzes a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids. Excess sterols cause the reductase to bind to ER membrane proteins called Insig-1 and Insig-2, which are carriers for the ubiquitin ligases gp78 and Trc8. The resulting gp78/Trc8-mediated ubiquitination of reductase marks it for recognition by VCP/p97, an ATPase that mediates subsequent dislocation of reductase from ER membranes into the cytosol for proteasomal degradation. Here we report that in vitro additions of the oxysterol 25-hydroxycholesterol (25-HC), exogenous cytosol, and ATP trigger dislocation of ubiquitinated and full-length forms of reductase from membranes of permeabilized cells. In addition, the sterol-regulated reaction requires the action of Insigs, is stimulated by reagents that replace 25-HC in accelerating reductase degradation in intact cells, and is augmented by the nonsterol isoprenoid geranylgeraniol. Finally, pharmacologic inhibition of deubiquitinating enzymes markedly enhances sterol-dependent ubiquitination of reductase in membranes of permeabilized cells, leading to enhanced dislocation of the enzyme. Considered together, these results establish permeabilized cells as a viable system in which to elucidate mechanisms for postubiquitination steps in sterol-accelerated degradation of reductase.
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Project description:Characteristics of 3-hydroxy-3-methylglutaryl-CoA reductase from normal liver, Morris hepatomas 5123C, 5123t.c. and 9618A, and host liver were studied. Animals were fed on control and 5%-cholesterol diets. Microsomal membranes from all tissues were found to accumulate cholesterol after 3 days on the 5%-cholesterol diet. The enzyme of the tumours showed no feedback inhibition by dietary cholesterol, and that of host liver gave a variable response, whereas that of control liver was constantly inhibited by 90% or more. Arrhenius-plot analysis was conducted on the microsomal enzyme isolated from the various tissues. Control animals showed that the phase transition present at 27 degrees C was removed when animals were fed on 5%-cholesterol diet for 12 h. The hepatomas failed to show this change even after 3 days of 5%-cholesterol diet and a significant increase in microsomal cholesterol. This failure to remove the break in Arrhenius plots also occurred in host liver, even though enzyme inhibition occurred. The reason why hepatomas fail to regulate 3-hydroxy-3-methylglutaryl-CoA reductase activity in response to dietary cholesterol may be a decreased membrane-enzyme interaction.
| S-EPMC1158372 | biostudies-other
Project description:Fungal strain 14919 was originally isolated from a soil sample collected at Mt. Kiyosumi, Chiba Prefecture, Japan. It produces FR901512, a potent and strong 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor. The genome sequence of fungal strain 14919 was determined and annotated to improve the productivity of FR901512.
| S-EPMC5383895 | biostudies-literature