Catalytically relevant electrostatic interactions of cytochrome P450c17 (CYP17A1) and cytochrome b5.

Two acidic residues, Glu-48 and Glu-49, of cytochrome b5 (b5) are essential for stimulating the 17,20-lyase activity of cytochrome P450c17 (CYP17A1). Substitution of Ala, Gly, Cys, or Gln for these two glutamic acid residues abrogated all capacity to stimulate 17,20-lyase activity. Mutations E49D and E48D/E49D retained 23 and 38% of wild-type activity, respectively. Using the zero-length cross-linker ethyl-3-(3-dimethylaminopropyl)carbodiimide, we obtained cross-linked heterodimers of b5 and CYP17A1, wild-type, or mutations R347K and R358K. In sharp contrast, the b5 double mutation E48G/E49G did not form cross-linked complexes with wild-type CYP17A1. Mass spectrometric analysis of the CYP17A1-b5 complexes identified two cross-linked peptide pairs as follows: CYP17A1-WT: (84)EVLIKK(89)-b5: (53)EQAGGDATENFEDVGHSTDAR(73) and CYP17A1-R347K: (341)TPTISDKNR(349)-b5: (40)FLEEHPGGEEVLR(52). Using these two sites of interaction and Glu-48/Glu-49 in b5 as constraints, protein docking calculations based on the crystal structures of the two proteins yielded a structural model of the CYP17A1-b5 complex. The appositional surfaces include Lys-88, Arg-347, and Arg-358/Arg-449 of CYP17A1, which interact with Glu-61, Glu-42, and Glu-48/Glu-49 of b5, respectively. Our data reveal the structural basis of the electrostatic interactions between these two proteins, which is critical for 17,20-lyase activity and androgen biosynthesis.

Isolation and characterization of the CYP17A1 gene and its processed pseudogene in Rana rugosa.

CYP17A1 expression is up-regulated in the gonad in Rana (Glandirana) rugosa tadpoles treated with androgens to induce female-to-male sex-reversal. In this study, we isolated the CYP17A1 gene and its processed pseudogene from R. rugosa. The former was found to consist of 8 exons, and the latter a single-exon gene, designated CYP17A1P. The sequence of the promoter region of CYP17A1 differed from that of CYP17A1P. We found several consensus binding-sites for candidate transcription factors including androgen receptor (AR), Sox and FoxL2 in the CYP17A1 promoter region, but an AR-binding site was absent from CYP17A1P. When AR was over-expressed in Xenopus A6 cells, it did not increase CYP17A1 transcription in luciferase assays. CYP17A1 was strongly expressed in indifferent male gonads during sex determination and exclusively in testis, among eight adult tissues of R. rugosa. By contrast, CYP17A1P was expressed at very low, and similar levels in the adult tissues of both sexes. Fluorescent In-Situ Hybridization (FISH) analysis showed that CYP17A1P is localized to chromosome 4, while CYP17A1 is on chromosome 9. These results collectively suggest that CYP17A1, but not CYP17A1P is involved in male sex-determination in R. rugosa, and that androgens may not have a direct effect on the CYP17A1 transcription.

A role for cytochrome P450 17alpha-hydroxylase/c17-20 lyase during shift in steroidogenesis occurring in ovarian follicles prior to oocyte maturation.

Cytochrome P450 17alpha-hydroxylase/c17-20 lyase (P450c17) is regarded as one of the key enzymes involved in the steroidogenic shift that occurs prior to oocyte maturation in teleosts. Role of P450c17 in the shift in steroidogenesis during oocyte maturation is a contentious issue even after identification of a novel type of P450c17 that lacks lyase activity. To understand the role of P450c17 in steroidogenic shift explicitly, a full length cDNA encoding p450c17 from ovary of air-breathing catfish, Clarias gariepinus was cloned. p450c17 transiently expressed in COS-7 cells converted progesterone to androstenedione through 17alpha-hydroxyprogesterone and catfish p450c17 was found to be expressed ubiquitously with relatively higher levels in gonads, brain, kidney and gills. Immunocytochemical analysis revealed the presence of P450C17 in follicular layer of ovarian follicle, interstitial cells and spermatocytes of testis. p450c17 expression and ratio of lyase to hydroxylase was high in preparatory and pre-spawning phases of ovary and low in spawning phase. Expression of p450c17 correlated well with testicular recrudescence with maximum expression in preparatory and spawning phases. Neither protein expression nor lyase/hydroxylase activity changed significantly during hCG-induced oocyte maturation, in vitro and in vivo though mRNA levels increased. These results tend to suggest that the ovarian follicles attains capacity to produce maximum precursor steroid levels before spawning that might contribute to the shift in steroidogenesis.

Alternative splicing and differential expression of P450c17 (CYP17) in gonads during sex transformation in the rice field eel.

Several mechanisms were used in determination of the development of the male or female of vertebrates. The genes for determination of sequential hermaphrodite sex are unknown. Here, we reported cloning, alternative splicing, and expression patterns of the CYP17 gene of the rice field eel, a teleost fish with a characteristic of nature sex reversal. The CYP17 gene of the rice field eel was clustered into the CYP17 gene group of all the other vertebrates, especially into the fish subgroup. Four isoforms of the CYP17 were generated in gonads by alternative splicing and polyadenylation. Alternative splicing events of all these isoforms occurred in 3(') regions, which encoded three different sizes (517, 512, and 159aa) of proteins. RT-PCR results indicate specific expression in gonads of these isoforms. Northern blot analysis shows that expression patterns of the CYP17 (dominantly expressed in testis, less in ovary, and the least in ovotestis) are consistent with the sex reversal process of the rice field eel. In situ hybridization further shows its specific expression in germinal lamellae, the gonadal epithelium of the gonads. These findings indicate that CYP17 is differentially regulated in a sex- and developmentally specific manner, suggesting that the CYP17 potentially has important roles in gonad differentiation during sex reversal of the rice field eel.