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Insig regulates HMG-CoA reductase by controlling enzyme phosphorylation in fission yeast.

ABSTRACT: Insig functions as a central regulator of cellular cholesterol homeostasis by controlling activity of HMG-CoA reductase (HMGR) in cholesterol synthesis. Insig both accelerates the degradation of HMGR and suppresses HMGR transcription through the SREBP-Scap pathway. The fission yeast Schizosaccharomyces pombe encodes homologs of Insig, HMGR, SREBP, and Scap, called ins1(+), hmg1(+), sre1(+), and scp1(+). Here, we characterize fission yeast Insig and demonstrate that Ins1 is dedicated to regulation of Hmg1, but not the Sre1-Scp1 pathway. Using a sterol-sensing domain mutant of Hmg1, we demonstrate that Ins1 binding to Hmg1 inhibits enzyme activity by promoting phosphorylation of the Hmg1 active site, which increases the K(M) for NADPH. Ins1-dependent phosphorylation of Hmg1 requires the MAP kinase Sty1/Spc1, and Hmg1 phosphorylation is physiologically regulated by nutrient stress. Thus, in fission yeast, Insig regulates sterol synthesis by a different mechanism than in mammalian cells, controlling HMGR phosphorylation in response to nutrient supply.

SUBMITTER: Burg JS

PROVIDER: S-EPMC2646361 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Insig regulates HMG-CoA reductase by controlling enzyme phosphorylation in fission yeast.

Burg John S JS Powell David W DW Chai Raymond R Hughes Adam L AL Link Andrew J AJ Espenshade Peter J PJ

Cell metabolism 20081201 6

Insig functions as a central regulator of cellular cholesterol homeostasis by controlling activity of HMG-CoA reductase (HMGR) in cholesterol synthesis. Insig both accelerates the degradation of HMGR and suppresses HMGR transcription through the SREBP-Scap pathway. The fission yeast Schizosaccharomyces pombe encodes homologs of Insig, HMGR, SREBP, and Scap, called ins1(+), hmg1(+), sre1(+), and scp1(+). Here, we characterize fission yeast Insig and demonstrate that Ins1 is dedicated to regulatio ...[more]

PMID: 19041767

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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

Insig regulates HMG-CoA reductase by controlling enzyme phosphorylation in fission yeast.

Publications

Insig regulates HMG-CoA reductase by controlling enzyme phosphorylation in fission yeast.

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