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Sterol-induced dislocation of 3-hydroxy-3-methylglutaryl coenzyme A reductase from membranes of permeabilized cells.

ABSTRACT: 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.

SUBMITTER: Elsabrouty R

PROVIDER: S-EPMC3814148 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Sterol-induced dislocation of 3-hydroxy-3-methylglutaryl coenzyme A reductase from membranes of permeabilized cells.

Elsabrouty Rania R Jo Youngah Y Dinh Tammy T TT DeBose-Boyd Russell A RA

Molecular biology of the cell 20130911 21

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 reductas ...[more]

PMID: 24025715

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Project description:ObjectiveIn 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.MethodsThe 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.ResultsStatin 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.ConclusionStatins 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.

Dataset Information

Sterol-induced dislocation of 3-hydroxy-3-methylglutaryl coenzyme A reductase from membranes of permeabilized cells.

Publications

Sterol-induced dislocation of 3-hydroxy-3-methylglutaryl coenzyme A reductase from membranes of permeabilized cells.

Similar Datasets

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