Browsing by Subject "Sterols"
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Item The Roles of ATP-Binding Cassette Transporters G5 and G8 in Liver X Receptor-Mediated Sterol Trafficking(2007-12-03) York, Jennifer Lynn; Hobbs, Helen H.The liver X receptor (LXR) is a nuclear receptor that plays a critical role in orchestrating the trafficking of sterols between tissues. Treatment of wild type mice with a potent and specific nonsteroidal LXR agonist, T0901317, is associated with increased biliary cholesterol secretion, decreased fractional cholesterol absorption, and increased fecal neutral sterol excretion. The following studies show that expression of two target genes of LXRalpha , the ATP-binding cassette (ABC) transporters Abcg5 and Abcg8, is required for the increase in sterol excretion, the decrease in fractional cholesterol absorption, and the increase in fecal neutral sterol excretion associated with LXR agonist treatment. Mice lacking ABCG5 and ABCG8 (G5G8-/- mice) and wild type littermate controls were treated for 7 days with T0901317. In control animals, the LXR agonist produced a 3-fold increase in biliary cholesterol concentration, a 25% reduction in fractional cholesterol absorption, and a 4-fold elevation in fecal neutral sterol excretion. In contrast, treatment of G5G8-/- mice with the LXR agonist did not significantly affect any of these parameters. These results demonstrate that ABCG5 and ABCG8 are required for LXR agonist-associated changes in dietary and biliary sterol trafficking and that increased expression of these proteins promotes cholesterol excretion in vivo.Item Sequential Actions of VCP/p97 and the Proteasome 19S Regulatory Particle in Sterol-Accelerated, ER-Associated Degradation of HMG CoA Reductase(2014-05-28) Morris, Lindsey LaChelle; Goodman, Joel M.; DeBose-Boyd, Russell A.; Lehrman, Mark A.; De Martino, GeorgeAccelerated endoplasmic reticulum (ER)-associated degradation (ERAD) of the cholesterol biosynthetic enzyme HMG CoA reductase results from its sterol-induced binding to ER membrane proteins called Insig-1 and Insig-2. This binding allows for subsequent ubiquitination of reductase by Insig-associated ubiquitin ligases. Once ubiquitinated, reductase becomes dislocated from ER membranes into the cytosol for degradation by 26S proteasomes through poorly defined reactions mediated by the AAA-ATPase VCP/p97 and augmented by the nonsterol isoprenoid geranylgeraniol. Here, we report that the oxysterol 25-hydroxycholesterol and geranylgeraniol combine to trigger extraction of reductase across ER membranes prior to its cytosolic release. This conclusion was drawn from studies utilizing a novel assay that measures membrane extraction of reductase by determining susceptibility of a lumenal epitope in the enzyme to in vitro protease digestion. Susceptibility of the lumenal epitope to protease digestion, and thus membrane extraction of reductase, was tightly regulated by 25-hydroxycholesterol and geranylgeraniol. The reaction was inhibited by RNA interference mediated knockdown of either Insigs or VCP/p97. In contrast, reductase continued to become membrane extracted, but not cytosolically dislocated, in cells deficient for AAA-ATPases of the proteasome 19S regulatory particle. These findings establish sequential roles for VCP/p97 and the 19S regulatory particle in the sterol-accelerated ERAD of reductase that may be applicable to the ERAD of other substrates.Item Variations in Mevalonate Pathway Flux in Human Cells with Familial Hypercholesterolemia(2020-05-01T05:00:00.000Z) Su, Shan; DeBose-Boyd, Russell A.; Liang, Guosheng; Radhakrishnan, ArunBACKGROUND: HMG-CoA reductase (HMGCR) is a membrane protein of the endoplasmic reticulum (ER) that catalyzes the reduction of HMG-CoA to mevalonate, a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids. Sterol and nonsterol isoprenoids exert stringent feedback control on HMGCR through multiple mechanisms. This ensures constant synthesis of essential nonsterol isoprenoids, while avoiding toxic overaccumulation of cholesterol. One regulator of HMGCR is UBIAD-1, a vitamin K2 biosynthetic enzyme. Individuals with familial hypercholesterolemia (FH) suffer from cholesterol excess due to the inability of cells to take up cholesterol from the environment, leading to a cholesterol depleted cellular state and an increase in cholesterol production. OBJECTIVE: In this study, we examine the effect of sterol and nonsterol isoprenoid depletion via statins followed by mevalonate treatment on the expression of genes and proteins in the mevalonate pathway and localization of UBIAD-1 in human fibroblasts. METHODS: Cells expressing FH mutations and control cells were grown on culture plates or coverslips and fed media containing FCS, or FCS plus compactin and 0.05 mM, 0.2 mM, 1 mM, 3 mM, or 10 mM mevalonate. After overnight feeding, cells were harvested for immunofluorescence visualization, and qRT-PCR and immunoblot analysis of genes and proteins related to cholesterol and nonsterol isoprenoid synthesis. RESULTS: Immunoblot analysis indicates that FH cells generally express higher amounts of sterol biosynthetic enzymes but lower amounts of CoQ10 biosynthetic enzymes than control cells. qRT-PCR showed that genes of the CoQ10 pathway in FH cells are expressed to a significantly less extent than in control cells, and that sterol synthetic genes are relatively unaffected in FH cells but upregulated in control cells fed compactin and mevalonate. Immunofluorescence and quantitation of UBIAD-1 Golgi localization indicate that compactin causes UBIAD-1 to migrate to the ER in both cells, and that FH cells require a greater concentration of mevalonate following the addition of compactin to restore Golgi localization. CONCLUSION: The FH phenotype causes a cellular deficiency of sterols, leading cells to upregulate mechanisms toward sterol synthesis at the expense of CoQ10 synthesis, which results in a relative CoQ10 deficiency in FH cells.