Abstract
Mevalonic aciduria (MA) and hyper-IgD and periodic fever syndrome (HIDS) are two inherited disorders both caused by depressed mevalonate kinase (MK) activity. MK is the first enzyme to follow the highly regulated 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (HMGR), which catalyzes the rate-limiting step in the isoprenoid/cholesterol biosynthesis pathway. In fibroblasts of MA patients, but not of HIDS patients, HMGR activity is elevated under normal growth conditions. This activity is down-regulated when cells are supplemented with the isoprenoid precursors geraniol, farnesol, and geranylgeraniol, and a mixture of 25-hydroxycholesterol and cholesterol. This indicates that the regulation of the pathway in these cells is not disturbed. The elevated HMGR activity is probably due to a shortage of non-sterol isoprenoid end products, as indicated by normal HMGR mRNA levels in MA fibroblasts. Furthermore, the HMGR activity in MA cells was more sensitive to geranylgeraniol suppression and less sensitive to sterol suppression than the HMGR activity in low density lipoprotein receptor-deficient cells. HMGR activity in MA cells was down-regulated also by addition of its product mevalonate to the culture medium. Thus, it appears that the elevation of mevalonate levels, which are high in MA patients and moderate in HIDS patients, allows the cells to compensate for the depressed MK activity. Indeed, the isoprenylation of Ras and RhoA protein appeared normal in HIDS and MA fibroblasts under normal conditions but showed increased sensitivity toward inhibition of HMGR by simvastatin. Our results indicate that MK-deficient cells maintain the flux through the isoprenoid/cholesterol biosynthesis pathway by elevating intracellular mevalonate levels.
Highlights
Mal recessive disorders both caused by a deficient activity of the enzyme mevalonate kinase (MK, ATP:mevalonate-5-phosphotransferase, EC 2.7.1.36) due to functional significant mutations in the encoding gene (MVK) [1,2,3,4,5]
Mevalonic aciduria (MA) and hyper-IgD and periodic fever syndrome (HIDS) are syndromes both caused by a deficiency of MK enzyme activity but to variable degrees
HMGR that catalyzes the conversion of HMG-CoA into mevalonate, which is the enzyme step preceding the one catalyzed by MK, is believed to perform the main rate-limiting step in isoprenoid biosynthesis and is among the most highly regulated enzymes in nature [8]
Summary
Materials—HMG-CoA, geraniol (GOH), FOH, geranylgeraniol (GGOH), cholesterol, and 25-hydroxycholesterol were obtained from Sigma. Mevalonolactone, GOH, FOH, and GGOH were dissolved in ethanol as 250ϫ stock solutions. A 10 mM simvastatin stock solution was prepared by dissolving the prodrug in pure ethanol, followed by hydrolysis of the lactone by adding 0.1 N NaOH. Quantitative PCR—The relative expression levels of glyceraldehyde3-phosphate dehydrogenase (GAPDH) and HMGR mRNAs were determined using the Lightcycler system (Roche Molecular Biochemicals). To this end, total RNA (free of genomic DNA) was isolated with the SV total RNA isolation system (Promega) after which first-strand cDNA was prepared as described by IJlst et al [21]. The relative mRNA expression levels of HMGR and GAPDH were determined using a plasmid containing the corresponding gene as the standard. The fibroblast pellets were dissolved in HMGR assay buffer containing 100 mM KPi, 200 mM KCl, 5 mM EGTA, 5 mM EDTA, 10 mM DTT, and 10 g/ml
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