Hogenboom Sietske, Wanders Ronald J A, Waterham Hans R
Laboratory Genetic Metabolic Diseases, Department of Pediatrics, Emma Children's Hospital, Academic Medical Center, Amsterdam, The Netherlands.
Mol Genet Metab. 2003 Nov;80(3):290-5. doi: 10.1016/S1096-7192(03)00143-4.
To evaluate the presumed peroxisomal involvement in cholesterol/isoprenoid biosynthesis, we determined the protein levels and activities of five different enzymes of the presqualene segment of the cholesterol/isoprenoid biosynthetic pathway in primary skin fibroblasts of selected patients with a peroxisomal biogenesis disorder (PBD). These five enzymes all have been reported to be partly or exclusively peroxisomal and include HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, and isopentenyl pyrophosphate isomerase. To exclude that genetic differences, resulting in different defects in peroxisomal biogenesis, have differential effects on the activity of the cholesterol biosynthetic enzymes and on de novo cholesterol biosynthesis, we chose fibroblasts of patients with defined defects in one of four different PEX genes leading to Zellweger syndrome (PEX1, PEX5, PEX16 or PEX19). We found that all enzymes measured are at least as active in the peroxisome-deficient cells cultured in cholesterol-depleted medium as in identically cultured control cells. This indicates that if these presumed peroxisomal proteins are mislocalized to the cytosol they do not loose their activity, nor get degraded unlike most other authentic peroxisomal proteins. We also measured de novo cholesterol synthesis from radio-labeled acetate in all cell lines and found similar or even elevated rates for the PBD cells when compared to controls. Our results imply that functional peroxisomes are not a prerequisite for the functioning of enzymes involved in cholesterol/isoprenoid biosynthesis and as such raise doubts about the true involvement of peroxisomes therein.
为了评估过氧化物酶体在胆固醇/类异戊二烯生物合成中可能的参与情况，我们测定了选定的过氧化物酶体生物发生障碍（PBD）患者原代表皮成纤维细胞中胆固醇/类异戊二烯生物合成途径中鲨烯前体片段的五种不同酶的蛋白水平和活性。据报道，这五种酶部分或全部定位于过氧化物酶体，包括HMG-CoA还原酶、甲羟戊酸激酶、磷酸甲羟戊酸激酶、甲羟戊酸焦磷酸脱羧酶和异戊烯基焦磷酸异构酶。为了排除导致过氧化物酶体生物发生不同缺陷的基因差异对胆固醇生物合成酶活性和从头胆固醇生物合成有不同影响，我们选择了患有由四种不同的PEX基因（导致泽尔韦格综合征，即PEX1、PEX5、PEX16或PEX19）之一的特定缺陷的患者的成纤维细胞。我们发现，在胆固醇缺乏培养基中培养的过氧化物酶体缺陷细胞中，所有检测的酶的活性至少与在相同培养条件下的对照细胞中一样高。这表明，如果这些推测的过氧化物酶体蛋白错误定位于细胞质中，它们不会失去活性，也不会像大多数其他真正的过氧化物酶体蛋白那样被降解。我们还测定了所有细胞系中从放射性标记的乙酸盐从头合成胆固醇的情况，发现与对照相比，PBD细胞的合成速率相似甚至更高。我们的结果表明，功能性过氧化物酶体不是胆固醇/类异戊二烯生物合成所涉及酶发挥功能的先决条件，因此对过氧化物酶体在其中的真正参与情况提出了质疑。
