Lin H K, Jez J M, Schlegel B P, Peehl D M, Pachter J A, Penning T M
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104-6084, USA.
Mol Endocrinol. 1997 Dec;11(13):1971-84. doi: 10.1210/mend.11.13.0026.
In androgen target tissues, 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) may regulate occupancy of the androgen receptor (AR) by catalyzing the interconversion of 5alpha-dihydrotestosterone (5alpha-DHT) (a potent androgen) and 3alpha-androstanediol (a weak androgen). In this study, a 3alpha-HSD cDNA (1170 bp) was isolated from a human prostate cDNA library. The human prostatic 3alpha-HSD cDNA encodes a 323-amino acid protein with 69.9%, 84.1%, 99.4%, and 87.9% sequence identity to rat liver 3alpha-HSD and human type 1, type 2, and type 3 3alpha-HSDs, respectively, and is a member of the aldo-keto reductase superfamily. The close homology with human type 2 3alpha-HSD suggests that it is either identical to this enzyme or a structural allele. Surprisingly, when the recombinant protein was expressed and purified from Escherichia coli, the enzyme did not oxidize androsterone when measured spectrophotometrically, an activity previously assigned to recombinant type 2 3alpha-HSD using this assay. Complete kinetic characterization of the purified protein using spectrophotometric, fluorometric, and radiometric assays showed that the catalytic efficiency favored 3alpha-androstanediol oxidation over 5alpha-DHT reduction. Using [14C]-5alpha-DHT as substrate, TLC analysis confirmed that the reaction product was [14C]-3alpha-androstanediol. However, in the reverse reaction, [3H]-3alpha-androstanediol was oxidized first to [3H]-androsterone and then to [3H]-androstanedione, revealing that the expressed protein possessed both 3alpha- and 17beta-HSD activities. The 17beta-HSD activity accounted for the higher catalytic efficiency observed with 3alpha-androstanediol. These findings indicate that, in the prostate, type 2 3alpha-HSD does not interconvert 5alpha-DHT and 3alpha-androstanediol but inactivates 5alpha-DHT through its 3-ketosteroid reductase activity. Levels of 3alpha-HSD mRNA were measured in primary cultures of human prostatic cells and were higher in epithelial cells than stromal cells. In addition, elevated levels of 3alpha-HSD mRNA were observed in epithelial cells derived from benign prostatic hyperplasia and prostate carcinoma tissues. Expression of 3alpha-HSD was not prostate specific, since high levels of mRNA were also found in liver, small intestine, colon, lung, and kidney. This study is the first complete characterization of recombinant type 2 3alpha-HSD demonstrating dual activity and cellular distribution in the human prostate.
在雄激素靶组织中，3α-羟基类固醇脱氢酶（3α-HSD）可通过催化5α-二氢睾酮（一种强效雄激素）和3α-雄甾二醇（一种弱雄激素）的相互转化来调节雄激素受体（AR）的占有率。在本研究中，从人前列腺cDNA文库中分离出一个3α-HSD cDNA（1170 bp）。人前列腺3α-HSD cDNA编码一种323个氨基酸的蛋白质，与大鼠肝脏3α-HSD以及人1型、2型和3型3α-HSD的序列同一性分别为69.9%、84.1%、99.4%和87.9%，它是醛酮还原酶超家族的成员。与人类2型3α-HSD的高度同源性表明它要么与该酶相同，要么是一个结构等位基因。令人惊讶的是，当从大肠杆菌中表达并纯化重组蛋白时，用分光光度法测量发现该酶不会氧化雄甾酮，而此前使用该检测方法对重组2型3α-HSD赋予了这种活性。使用分光光度法、荧光法和放射性检测法对纯化蛋白进行完整的动力学表征表明，催化效率更倾向于3α-雄甾二醇氧化而非5α-二氢睾酮还原。以[14C]-5α-二氢睾酮为底物，薄层色谱分析证实反应产物是[14C]-3α-雄甾二醇。然而，在逆向反应中，[3H]-3α-雄甾二醇首先被氧化为[3H]-雄甾酮，然后再氧化为[3H]-雄甾二酮，这表明表达的蛋白同时具有3α-和17β-HSD活性。17β-HSD活性解释了观察到的3α-雄甾二醇具有较高催化效率的原因。这些发现表明，在前列腺中，2型3α-HSD不会使5α-二氢睾酮和3α-雄甾二醇相互转化，而是通过其3-酮类固醇还原酶活性使5α-二氢睾酮失活。在人前列腺细胞原代培养物中测量了3α-HSD mRNA的水平，上皮细胞中的水平高于基质细胞。此外，在源自良性前列腺增生和前列腺癌组织的上皮细胞中观察到3α-HSD mRNA水平升高。3α-HSD的表达并非前列腺特异性的，因为在肝脏、小肠、结肠、肺和肾脏中也发现了高水平的mRNA。本研究首次对重组2型3α-HSD进行了完整表征，证明了其在人前列腺中的双重活性和细胞分布。
