Browsing by Subject "Hydroxymethylglutaryl CoA Reductases"
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Item Elucidation of Molecular Mechanisms Underlying Regulation Of Cholesterol Synthesis(2007-05-22) Lee, Peter Chang-whan; DeBoseBoyd, Russell A.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.Item Insig-Mediated Regulation of Mammalian HMG COA Reductase Ubiquitnation and Degradation(2004-12-15) Sever, Navdar; Brown, Michael S.3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase (HMGR) catalyzes the conversion of HMG CoA to mevalonate, which is the rate limiting step in the production of cholesterol and numerous nonsterol isoprenoid products. Mammalian HMGR is regulated by transcriptional and post-transcriptional feedback mechanisms. The transcriptional regulation is mediated by sterol regulatory element binding proteins (SREBPs), which are synthesized as inactive precursors in the endoplasmic reticulum (ER) membrane. In the absence of sterols, SREBP cleavage activating protein (SCAP) escorts SREBPs from ER to the Golgi apparatus, where SREBPs are cleaved by site 1 and site 2 proteases so as to release their amino terminal transcription factor domains to the nucleus. Sterols inhibit the exit of SCAP-SREBP complex from the ER by promoting the binding of two related polytopic ER membrane proteins, Insig-1 and Insig-2, to the membrane domain of SCAP. Insig-1, but not Insig-2, is an SREBP target gene, causing Insig-1 levels to drop in the presence of sterols, when it is expected to exert its action. The degradation of HMGR requires both sterols and a nonsterol product of the mevalonate pathway and the eight membrane spanning segments in its amino terminus. The membrane domains of HMGR and SCAP bear sequence similarity prompting the investigation of whether Insig proteins can also bind to HMGR. Indeed, Insig-1 and Insig-2 were found to interact with HMGR in a regulated manner and mediate its proteasomal degradation. This effect can be specifically inhibited by overexpressing the membrane domain of SCAP. Insigs were shown to promote the ubiquitination of HMGR on lysine 248 in the cytoplasmic loop between transmembrane segments 6 and 7. In an attempt to achieve a better understanding of the mechanism by which HMGR is degraded, a genetic approach was developed to select mutant somatic cells that cannot degrade HMGR in the presence of sterols. The isolation and characterization of Chinese hamster ovary cells deficient in Insig-1 confirmed the endogenous requirement of Insig-1 for HMGR degradation and revealed the role of differential regulation of Insig-1 and Insig-2 in terms of SREBP processing. These studies revealed a complex feedback regulatory system governing cholesterol homeostasis.Item Oxygen-Mediated Regulation of Cholesterol Synthesis through Accelerated Degradation of HMG COA Reductase(2009-09-04) Nguyen, Andrew Tuan Duc; DeBose-Boyd, RussellEndoplasmic reticulum-associated degradation of the enzyme 3-hydroxy-3-methylglutaryl CoA reductase represents one mechanism by which cholesterol synthesis is controlled in mammalian cells. The key reaction in this degradation is binding of reductase to Insig proteins in the endoplasmic reticulum, which is stimulated by the methylated cholesterol precursors lanosterol and 24,25-dihydrolanosterol. Conversion of these sterols to cholesterol requires the removal of three methyl groups, which consumes nine molecules of oxygen. Here, we report that oxygen deprivation (hypoxia) slows the rate of demethylation of lanosterol and its reduced metabolite 24,25-dihydrolanosterol, causing both sterols to accumulate in cells. These methylated sterols serve as one signal to stimulate rapid Insig-mediated degradation of reductase. In addition, hypoxia increases the expression of Insig-2 in a response mediated by hypoxia-inducible factor. Our analysis of the mouse Insig-2 gene revealed the presence of a functional hypoxia response element in the first intron. Importantly, hepatic Insig-2a expression is upregulated in three independent mouse models of hypoxia. These studies establish that Insig-2 is a target gene of hypoxia-inducible factor. The hypoxia-dependent increase in Insig levels confers cells with enhanced sensitivity to sterol-induced degradation of reductase. In this way, hypoxia-inducible factor-mediated induction of Insig-2 provides a second signal for stimulating reductase degradation. To address the specificity of methylated sterols in promoting reductase degradation, we reconstituted Insig-dependent, sterol-accelerated degradation of the membrane domain of mammalian reductase in Drosophila S2 cells. Studies in this system revealed that 24,25-dihydrolanosterol, and lanosterol, is active in accelerating degradation of reductase. These results were confirmed by examining ubiquitination of reductase in vitro using permeabilized mammalian cells. Collectively, these studies show that under hypoxic conditions reductase undergoes accelerated Insig-dependent degradation as the combined result of two events: 1) accumulation of 24,25-dihydrolanosterol and 2) hypoxia-inducible factor-mediated upregulation of Insig-2. Degradation of reductase ultimately slows a rate-determining step in cholesterol synthesis. These results highlight the importance of 24,25-dihydrolanosterol as a physiologic regulator of reductase degradation and define a novel oxygen-sensing mechanism in the mammalian cholesterol biosynthetic pathway.