Pitfalls in characterizing P450c17 mutations associated with isolated 17,20-lyase deficiency.
Pitfalls in characterizing P450c17 mutations associated with isolated 17,20-lyase deficiency.
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DOI：
10.1210/jcem.86.9.7812
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发表时间：
2001-09
期刊：
影响因子：
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通讯作者：
Manisha Gupta-;David H. Geller;R. Auchus
中科院分区：
文献类型：
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作者：
Manisha Gupta-;David H. Geller;R. Auchus
细胞色素P450 c17酶系统在人类肾上腺和性腺中进行17 α-羟化酶和17，20-裂解酶反应。这种17，20-裂解酶活性是脱氢表雄酮（性类固醇的C（19）前体）生物合成所必需的。大量证据支持这一观点，即该系统的17，20-裂解酶活性对P450 c17及其辅因子蛋白P450-氧化还原酶和细胞色素B之间相互作用的改变特别敏感（5）。我们已经描述了两名患者的临床表型孤立的17，20-裂解酶缺乏症，其中单一的氨基酸置换突变的氧化还原伴侣结合位点的P450 c17（R347 H和R358 Q）选择性地消除17，20-裂解酶的活性，同时保留大部分17 α-羟化酶的活性。我们通过计算机建模和详细的生物化学研究表明，突变R347 H和R358 Q削弱了P450 c17与P450-氧化还原酶和细胞色素B（5）（氧化还原配偶体）的相互作用。另一个突变报告导致孤立的17，20-裂解酶缺乏症（F417 C）不映射内的氧化还原伴侣结合位点，但可能会改变突变蛋白与氧化还原伴侣的相互作用。为了研究F417 C突变与P450氧化还原酶和细胞色素B的相互作用（5），我们在酵母微粒体中表达了该蛋白的cDNA，这是一种异源表达系统，其中氧化还原伴侣蛋白的组成可以系统地变化。尽管在该系统中全长蛋白质的表达量与野生型P450 c17相当，但F417 C突变没有形成经典的P450差异谱，并且缺乏17 α-羟化酶和17，20-裂解酶活性。为了确保这一结果不是酵母表达系统所独有的，我们还在COS-7细胞中表达了野生型P450 c17和F417 C突变，我们再次发现F417 C突变被表达，但没有活性。为了最终证明P450 c17中的特定突变导致孤立的17，20-裂解酶缺乏症，突变蛋白的精确酶促研究必须可重复地显示与诊断一致的活性。突变R347 H和R358 Q是在人类中发现的仅有的两种被证明引起孤立的17，20-裂解酶缺乏症的突变。
The cytochrome P450c17 enzyme system performs both the 17alpha-hydroxylase and 17,20-lyase reactions in the human adrenal glands and gonads. This 17,20-lyase activity is required for the biosynthesis of dehydroepiandrosterone, the C(19) precursor of sex steroids. Considerable evidence supports the idea that the 17,20-lyase activity of this system is particularly sensitive to alterations in the interactions between P450c17 and its cofactor proteins P450-oxidoreductase and cytochrome b(5). We have described two patients with the clinical phenotype of isolated 17,20-lyase deficiency in whom single amino acid replacement mutations in the redox partner binding site of P450c17 (R347H and R358Q) selectively ablate 17,20-lyase activity while preserving most 17alpha-hydroxylase activity. We have shown by computer modeling and detailed biochemical studies that mutations R347H and R358Q impair the interactions of P450c17 with P450-oxidoreductase and cytochrome b(5) (redox partners). Another mutation reported to cause isolated 17,20-lyase deficiency (F417C) does not map within the redox partner binding site, but might nonetheless alter the interaction of the mutant protein with redox partners. To study the interaction of the F417C mutation with P450 oxidoreductase and cytochrome b(5), we expressed the cDNA for this protein in yeast microsomes, a heterologous expression system in which the composition of redox partner proteins can be varied systematically. Although the full-length protein was expressed in quantities comparable to those of wild-type P450c17 in this system, the F417C mutation did not form a classical P450 difference spectrum and was devoid of both 17alpha-hydroxylase and 17,20-lyase activities. To ensure that this result was not unique to the yeast expression system, we also expressed wild-type P450c17 and the F417C mutation in COS-7 cells, and we again found that the F417C mutation was expressed, but was not active. To conclusively demonstrate that a particular mutation in P450c17 causes isolated 17,20-lyase deficiency, accurate enzymatic studies of the mutant protein must reproducibly show activities consistent with the diagnosis. Mutations R347H and R358Q are the only two such mutations found in humans proven to cause isolated 17,20-lyase deficiency.