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Differential regulation of HMG-CoA reductase and Insig-1 by enzymes of the ubiquitin-proteasome system.

ABSTRACT: The endoplasmic reticulum (ER)-resident enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyzes the rate-limiting step in sterol production and is the therapeutic target of statins. Understanding HMG-CoA reductase regulation has tremendous implications for atherosclerosis. HMG-CoA reductase levels are regulated in response to sterols both transcriptionally, through a complex regulatory loop involving the ER Insig proteins, and posttranslationally, by Insig-dependent protein degradation by the ubiquitin-proteasome system. The ubiquitin ligase (E3) gp78 has been implicated in the sterol-regulated degradation of HMG-CoA reductase and Insig-1 through ER-associated degradation (ERAD). More recently, a second ERAD E3, TRC8, has also been reported to play a role in the sterol-accelerated degradation of HMG-CoA reductase. We interrogated this network in gp78(-/-) mouse embryonic fibroblasts and also assessed two fibroblast cell lines using RNA interference. Although we consistently observe involvement of gp78 in Insig-1 degradation, we find no substantive evidence to support roles for either gp78 or TRC8 in the robust sterol-accelerated degradation of HMG-CoA reductase. We discuss factors that might lead to such discrepant findings. Our results suggest a need for additional studies before definitive mechanistic conclusions are drawn that might set the stage for development of drugs to manipulate gp78 function in metabolic disorders.

SUBMITTER: Tsai YC

PROVIDER: S-EPMC3510011 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Differential regulation of HMG-CoA reductase and Insig-1 by enzymes of the ubiquitin-proteasome system.

Tsai Yien Che YC Leichner Gil S GS Pearce Margaret M P MM Wilson Gaye Lynn GL Wojcikiewicz Richard J H RJ Roitelman Joseph J Weissman Allan M AM

Molecular biology of the cell 20121019 23

The endoplasmic reticulum (ER)-resident enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyzes the rate-limiting step in sterol production and is the therapeutic target of statins. Understanding HMG-CoA reductase regulation has tremendous implications for atherosclerosis. HMG-CoA reductase levels are regulated in response to sterols both transcriptionally, through a complex regulatory loop involving the ER Insig proteins, and posttranslationally, by Insig-dependent protein degradati ...[more]

PMID: 23087214

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Project description:HMG-CoA reductase (HMGR) is the target of statins, cholesterol-lowering drugs prescribed to millions of patients worldwide. More recent research indicates that HMGR could be a useful target in the development of antimicrobial agents. Over the last seven decades, researchers have proposed a series of increasingly complex reaction mechanisms for this biomedically important enzyme. The maturation of the mechanistic proposals for HMGR have paralleled advances in a diverse set of research areas, such as molecular biology and computational chemistry. Thus, the development of the HMGR mechanism provides a useful case study for following the advances in state-of-the-art methods in enzyme mechanism research. Similarly, the questions raised by these mechanism proposals reflect the limitations of the methods used to develop them. The mechanism of HMGR, a four-electron oxidoreductase, is unique and far more complex than originally thought. The reaction contains multiple chemical steps, coupled to large-scale domain motions of the homodimeric enzyme. The first proposals for the HMGR mechanism were based on kinetic and labeling experiments, drawing analogies to the mechanism of known dehydrogenases. Advances in molecular biology and bioinformatics enabled researchers to use site-directed mutagenesis experiments and protein sequencing to identify catalytically important glutamate, aspartate, and histidine residues. These studies, in turn, have generated new and more complicated mechanistic proposals. With the development of protein crystallography, researchers solved HMGR crystal structures to reveal an unexpected lysine residue at the center of the active site. The many crystal structures of HMGR led to increasingly complex mechanistic proposals, but the inherent limitations of the protein crystallography left a number of questions unresolved. For example, the protonation state of the glutamate residue within the active site cannot be clearly determined from the crystal structure. The differing protonation state of this residue leads to different proposed mechanisms for the enzyme. As computational analysis of large biomolecules has become more feasible, the application of methods such as hybrid quantum mechanics/molecular mechanics (QM/MM) calculations to the HMGR mechanism have led to the most detailed mechanistic proposal yet. As these methodologies continue to improve, they prove to be very powerful for the study of enzyme mechanisms in conjunction with protein crystallography. Nevertheless, even the most current mechanistic proposal for HMGR remains incomplete due to limitations of the current computational methodologies. Thus, HMGR serves as a model for how the combination of increasingly sophisticated experimental and computational methods can elucidate very complex enzyme mechanisms.

Dataset Information

Differential regulation of HMG-CoA reductase and Insig-1 by enzymes of the ubiquitin-proteasome system.

Publications

Differential regulation of HMG-CoA reductase and Insig-1 by enzymes of the ubiquitin-proteasome system.

Similar Datasets

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