Human lamin B receptor exhibits sterol C14-reductase activity in Saccharomyces cerevisiae

Sandra Silve, Pascal-Henry Dupuy, Pascual Ferrara, Gérard Loison
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引用次数: 125

Abstract

Lamin B receptor (LBR), a nuclear protein of avian and mammalian cells, contains an hydrophobic domain that shares extensive structural similarities with the members of the sterol reductase family. To test if the sterol-reductase-like domain of LBR could be enzymatically competent, several sterol reductase-defective strains of Saccharomyces cerevisiae were transformed with a human-LBR expressing vector. LBR production did not change the ergosterol biosynthesis defect in an erg4 mutant impaired in sterol C24(28) reductase. In contrast, the sterol C14 reduction step and ergosterol prototrophy were restored in LBR-producing erg24 transformants which lack endogenous sterol C14 reductase. To test the effects of C14 reductase inhibitors on LBR activity, we constructed EMY54, an ergosterol-requiring strain that is devoid of both sterol C8–C7 isomerase and sterol C14 reductase activities. EMY54 cells recovered the capability of synthesizing ergost-8-en-3β-ol upon transformation with a vector that expressed either yeast sterol C14 reductase or hLBR. In addition, growth in sterol-free medium was restored in these transformants. Sterol biosynthesis and proliferation of LBR-producing cells were found to be highly susceptible to fenpropimorph and tridemorph, but only moderately susceptible to SR 31747. Our results strongly suggest that hLBR is a sterol C14 reductase.

人纤层蛋白B受体在酿酒酵母中表现出甾醇c14还原酶活性
层粘连蛋白B受体(Lamin B receptor, LBR)是鸟类和哺乳动物细胞的一种核蛋白,它含有一个疏水结构域,该结构域与固醇还原酶家族成员具有广泛的结构相似性。为了验证LBR的甾醇还原酶样结构域是否具有酶活性,我们用人类LBR表达载体转化了几种具有甾醇还原酶缺陷的酿酒酵母菌株。在甾醇C24(28)还原酶受损的erg4突变体中,LBR的产生并没有改变麦角甾醇生物合成缺陷。相比之下,缺乏内源性甾醇C14还原酶的产生lbr的erg24转化子恢复了甾醇C14还原步骤和麦角甾醇原生营养。为了测试C14还原酶抑制剂对LBR活性的影响,我们构建了EMY54,这是一种麦角甾醇需要菌株,缺乏固醇C8-C7异构酶和固醇C14还原酶活性。EMY54细胞在表达酵母甾醇C14还原酶或hLBR的载体上转化后,恢复了合成麦角草-8-en-3β-醇的能力。此外,这些转化体在无固醇培养基中恢复了生长。研究发现,产lbr细胞的甾醇生物合成和增殖对苯丙咪唑啉和trideph高度敏感,但对SR 31747仅中等敏感。我们的结果强烈表明hLBR是一种固醇C14还原酶。
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