Variations in Mevalonate Pathway Flux in Human Cells with Familial Hypercholesterolemia




Su, Shan

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BACKGROUND: 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.

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