Evaluation of 17β-hydroxysteroid dehydrogenase activity using androgen receptor-mediated transactivation
Evaluation of 17β-hydroxysteroid dehydrogenase activity using androgen receptor-mediated transactivation
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使用雄激素受体介导的反式激活评估 17β-羟基类固醇脱氢酶活性
DOI：
10.1016/j.jsbmb.2019.105493
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发表时间：
2020
期刊：
影响因子：
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通讯作者：
Taniguchi Takano
中科院分区：
文献类型：
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作者：
Yazawa Takashi;Imamichi Yoshitaka;Uwada Junsuke;Sekiguchi Toshio;Mikami Daisuke;Kitano Takeshi;Ida Takanori;Sato Takahiro;Nemoto Takahiro;Nagata Sayaka;Islam Khan Md. Rafiqul;Takahashi Satoru;Ushikubi Fumitaka;Suzuki Nobuo;Umezawa Akihiro;Taniguchi Takano
17β-羟基类固醇脱氢酶（17β-HSDs）催化17-酮类固醇的还原和17β-羟基类固醇的氧化以调节雄激素和雌激素的产生。其中，17β-HSD 3型（HPD 17 B3）几乎仅在睾丸间质细胞中表达，并通过将雄烯二酮（A4）转化为睾酮（T）来促进男性性征的发育。HSD 17 B3基因的突变导致性发育的46，XY障碍（46，XY DSD），这是低T产生的结果。因此，17β-HSD 3酶活性的评估对于理解和诊断这种疾病是重要的。我们采用了一种方法，通过使用雄激素受体（AR）介导的反式激活定量从A4到T的转化，轻松评估17β-HSD 3的酶活性。将HEK 293细胞转导以表达人HSD 17 B3，并孵育含有A4的培养基。根据与表达HSD 17 B3的细胞的孵育时间，培养基逐渐增加了用AR表达载体和雄激素应答报告基因转染的CV-1细胞中的荧光素酶活性。来自表达HSD 17 B1和HSD 17 B5的细胞的培养基也增加荧光素酶活性。该系统也适用于检测11-酮雄烯二酮向11-酮睾酮的转化。HEK 293细胞的建立表达与46，XY DSD相关的HSD 17 B3基因中的各种错义突变，表明该系统可有效地评估突变蛋白的酶活性。
17β-Hydroxysteroid dehydrogenases (17β-HSDs) catalyze the reduction of 17-ketosteroids and the oxidation of 17β-hydroxysteroids to regulate the production of androgens and estrogens. Among them, 17β-HSD type 3 (HSD17B3) is expressed almost exclusively in testicular Leydig cells and contributes to development of male sexual characteristics by converting androstenedione (A4) to testosterone (T). Mutations of HSD17B3 genes cause a 46,XY disorder of sexual development (46,XY DSD) as a result of low T production. Therefore, the evaluation of 17β-HSD3 enzymatic activity is important for understanding and diagnosing this disorder. We adapted a method that easily evaluates enzymatic activity of 17β-HSD3 by quantifying the conversion from A4 to T using androgen receptor (AR)-mediated transactivation. HEK293 cells were transduced to express human HSD17B3, and incubated medium containing A4. Depending on the incubation time with HSD17B3-expressing cells, the culture media progressively increased luciferase activities in CV-1 cells, transfected with the AR expression vector and androgen-responsive reporter. Culture medium from HSD17B1 and HSD17B5-expressing cells also increased the luciferase activities. This system is also applicable to detect the conversion of 11-ketoandrostenedione to 11-ketotestosterone by HSD17B3. Establishment of HEK293 cells expressing various missense mutations in the HSD17B3 gene associated with 46,XY DSD revealed that this system is effective to evaluate the enzymatic activities of mutant proteins.