Elucidation of Molecular Mechanisms Underlying Regulation of Cholesterol Synthesis




Lee, Peter Chang-whan

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Insig-1 and Insig-2, a pair of ER membrane proteins, mediate feedback control of cholesterol synthesis through their sterol-dependent binding to two polytopic ER membrane proteins: SCAP and HMG CoA reductase. Sterol-induced binding of Insigs to SCAP prevents the proteolytic processing of SREBPs, membrane-bound transcription factors that enhance the synthesis of cholesterol, by retaining complexes between SCAP and SREBP in the ER. Sterol-induced binding of Insigs to reductase leads to the ubiquitination and ER-associated degradation of the enzyme, thereby slowing a rate-controlling step in cholesterol synthesis. The successful application of somatic cell genetics in unraveling the SREBP pathway, merits its use in the dissection of mechanisms for Insig-mediated, sterol-accelerated degradation of reductase or ER retention of SCAP. I have designed a genetic screen to isolate mutants of CHO cells that cannot degrade reductase when presented with sterols. CHO cells were mutagenized and selected for growth in cholesterol-free medium containing the SR-12813. SR-12813 blocks cholesterol synthesis by mimicking the action of sterols in accelerating reductase degradation. Using this screen I have isolated the following mutant cell lines. 1) SRD-14 cells, which do not produce Insig-1 mRNA and protein due to a partial deletion of the Insig-1 gene. Sterols fail to promote reductase ubiquitination/degradation and the rate at which sterols suppress SREBP processing is significantly slower in SRD-14 than wild type cells; 2) SRD-15 cells which are deficient in both Insig-1 and Insig-2. Sterols neither inhibit SREBP processing nor promote reductase ubiquitination/degradation in SRD-15 even upon prolonged treatment; 3) SRD-16, -17, and -18 cells contain a point mutation in one reductase allele. Sterols failed to promote ubiquitination and degradation of these reductase mutants, owing to their decreased affinity for Insigs; 4) SRD-19 cells have amplified the number of copies of the gene encoding SCAP, leading to the overproduction of SCAP mRNA and protein. Sterols fail to suppress processing of SREBPs, even though the cells express normal levels of Insig-2. These studies demonstrate 1) absolute requirement for Insig proteins in the regulatory system that mediates lipid homeostasis in animal cells; 2) the importance of interactions between Insigs and the membrane domain of reductase in feedback control of a rate-determining step in cholesterol synthesis; 3) the importance of Insig-SCAP ratios in the normal regulation of SREBP processing.

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