Functional characterization of the G162R and D216H genetic variants of human CYP17A1.
Functional characterization of the G162R and D216H genetic variants of human CYP17A1.
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人类 CYP17A1 的 G162R 和 D216H 遗传变异的功能表征。
DOI：
10.1016/j.jsbmb.2017.12.002
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Rae,JM
中科院分区：
文献类型：
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作者：
Capper,CP;Liu,J;McIntosh,LR;Larios,JM;Johnson,MD;Hollenberg,PF;Osawa,Y;Auchus,RJ;Rae,JM
细胞色素P450 17 A1（CYP 17 A1）是一种双功能酶，催化皮质醇和雄激素生物合成所必需的反应。CYP 17 A1是经验证的前列腺癌药物靶标，因为CYP 17 A1抑制剂可显著降低循环雄激素并改善去势抵抗性前列腺癌的生存率。表现为各种内分泌疾病的具有改变的CYP 17 A1活性的种系CYP 17 A1遗传变体是极其罕见的;然而，表征这些变体提供了对CYP 17 A1蛋白质结构和功能的重要见解。通过查询dbSNP在线数据库和来自1000个基因组计划（http：//www.example.com）的可生物学获得的数据，我们鉴定了两种对酶活性和稳定性具有未知后果的CYP 17 A1非同义遗传变体。browser.1000genomes.org我们假设由此产生的氨基酸变化会改变CYP 17 A1的稳定性或活性。为了验证这一假设，我们利用基于HEK-293 T细胞的表达系统来表征两种CYP 17 A1变体D216 H（rs 200063521）和G162 R（rs 141821705）的功能后果。与野生型（WT）CYP 17 A1相比，瞬时表达D216 H变体的细胞表现出对16α-羟孕酮合成的选择性损害为2.1倍，而对17α-羟孕酮合成没有观察到影响。这些数据表明，在活性位点的底物取向可能会改变这种氨基酸取代。相比之下，G162 R取代显示与WT相比CYP 17 A1蛋白稳定性降低，通过免疫印迹分析测定蛋白水平降低近70%。该变体优先被泛素化并过早降解，酶半衰期计算为约2.5小时，蛋白酶体抑制剂治疗使G162 R蛋白表达恢复至WT水平。总之，这些数据为CYP 17 A1的结构-功能和稳定性机制提供了新的见解。
Cytochrome P450 17A1 (CYP17A1) is a dual-function enzyme catalyzing reactions necessary for cortisol and androgen biosynthesis. CYP17A1 is a validated drug target for prostate cancer as CYP17A1 inhibition significantly reduces circulating androgens and improves survival in castration-resistant prostate cancer. GermlineCYP17A1genetic variants with altered CYP17A1 activity manifesting as various endocrinopathies are extremely rare; however, characterizing these variants provides critical insights into CYP17A1 protein structure and function. By querying the dbSNP online database and publically available data from the 1000 genomes project (http://browser.1000genomes.org), we identified two CYP17A1 nonsynonymous genetic variants with unknown consequences for enzymatic activity and stability. We hypothesized that the resultant amino acid changes would alter CYP17A1 stability or activity. To test this hypothesis, we utilized a HEK-293T cell-based expression system to characterize the functional consequences of two CYP17A1 variants, D216H (rs200063521) and G162R (rs141821705). Cells transiently expressing the D216H variant demonstrate a selective impairment of 16α-hydroxyprogesterone synthesis by 2.1-fold compared to wild-type (WT) CYP17A1, while no effect on 17α-hydroxyprogesterone synthesis was observed. These data suggest that substrate orientations in the active site might be altered with this amino acid substitution. In contrast, the G162R substitution exhibits decreased CYP17A1 protein stability compared to WT with a near 70% reduction in protein levels as determined by immunoblot analysis. This variant is preferentially ubiquitinated and degraded prematurely, with an enzyme half-life calculated to be ∼2.5 h, and proteasome inhibitor treatment recovers G162R protein expression to WT levels. Together, these data provide new insights into CYP17A1 structure-function and stability mechanisms.