title: The Regulatory Domain of Squalene Monooxygenase Contains a Re-entrant Loop and Senses Cholesterol via a Conformational Change.
abstract: Squalene monooxygenase (SM) is an important control point in cholesterol synthesis beyond 3-hydroxy-3-methylglutaryl-CoA reductase. Although it is known to associate with the endoplasmic reticulum, its topology has not been determined. We have elucidated the membrane topology of the sterol-responsive domain of SM comprising the first 100 amino acids fused to GFP (SM N100-GFP) by determining the accessibility of 16 introduced cysteines to the cysteine-reactive, membrane-impermeable reagent PEG-maleimide. We have identified a region integrally associated with the endoplasmic reticulum membrane that is likely to interact with cholesterol or respond to cholesterol-induced membrane effects. By comparing cysteine accessibility with and without cholesterol treatment, we further present evidence to suggest that cholesterol induces a conformational change in SM N100-GFP. This change is likely to lead to its targeted degradation by the ubiquitin-proteasome system because degradation is blunted by treatment with the chemical chaperone glycerol, which retains SM N100-GFP in its native conformation. Furthermore, degradation can be disrupted by insertion of two N-terminal myc tags, implicating the N terminus in this process. Together, this information provides new molecular insights into the regulation of this critical control point in cholesterol synthesis.
authors.affiliation: From the School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney 2052, Australia and.
authors.forename: Vicky
authors.initials: V
authors.lastname: Howe
authors.affiliation: From the School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney 2052, Australia and.
authors.forename: Ngee Kiat
authors.initials: NK
authors.lastname: Chua
authors.affiliation: Department of Nutritional Sciences and Toxicology, University of California, Berkeley, California 94720.
authors.forename: Julian
authors.initials: J
authors.lastname: Stevenson
authors.affiliation: From the School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney 2052, Australia and aj.brown@unsw.edu.au.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Brown
mesh_terms: D000818:Animals; D016466:CHO Cells; D002784:Cholesterol / Q000096:biosynthesis / Q000378:metabolism*; D003412:Cricetulus; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050603:Squalene Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002784:Cholesterol; D050603:Squalene Monooxygenase; D003545:Cysteine
keywords: cholesterol; cholesterol regulation; conformational change; membrane; membrane topology; protein turnover; squalene monooxygenase