Francine Durocher

Structure-function relationships and molecular genetics of the 3β-hydroxysteroid dehydrogenase gene family

The isoenzymes of the 3 beta-hydroxysteroid dehydrogenase/5-ene-4-ene-isomerase (3 beta-HSD) gene family catalyse the transformation of all 5-ene-3 beta-hydroxysteroids into the corresponding 4-ene-3-keto-steroids and are responsible for the interconversion of 3 beta-hydroxy- and 3-keto-5 alpha-androstane steroids. The two human 3 beta-HSD genes and the three related pseudogenes are located on the chromosome 1p13.1 region, close to the centromeric marker D1Z5. The 3 beta-HSD isoenzymes prefer NAD+ to NADP+ as cofactor with the exception of the rat liver type III and mouse kidney type IV, which both prefer NADPH as cofactor for their specific 3-ketosteroid reductase activity due to the presence of Tyr36 in the rat type III and of Phe36 in mouse type IV enzymes instead of Asp36 found in other 3 beta-HSD isoenzymes. The rat types I and IV, bovine and guinea pig 3 beta-HSD proteins possess an intrinsic 17 beta-HSD activity specific to 5 alpha-androstane 17 beta-ol steroids, thus suggesting that such secondary activity is specifically responsible for controlling the bioavailability of the active androgen DHT. To elucidate the molecular basis of classical form of 3 beta-HSD deficiency, the structures of the types I and II 3 beta-HSD genes in 12 male pseudohermaphrodite 3 beta-HSD deficient patients as well as in four female patients were analyzed. The 14 different point mutations characterized were all detected in the type II 3 beta-HSD gene, which is the gene predominantly expressed in the adrenals and gonads, while no mutation was detected in the type I 3 beta-HSD gene predominantly expressed in the placenta and peripheral tissues. The mutant type II 3 beta-HSD enzymes carrying mutations detected in patients affected by the salt-losing form exhibit no detectable activity in intact transfected cells, at the exception of L108W and P186L proteins, which have some residual activity (approximately 1%). Mutations found in nonsalt-loser patients have some residual activity ranging from approximately 1 to approximately 10% compared to the wild-type enzyme. Characterization of mutant proteins provides unique information on the structure-function relationships of the 3 beta-HSD superfamily.

Localization of BRCA1 Gene Expression in Adult Cynomolgus Monkey Tissues

The breast and ovarian cancer susceptibility gene BRCA1 encodes a phosphoprotein of 1863 amino acids containing a highly conserved N-terminal RING finger domain and a C-terminal acidic region typical of several transcription factors. BRCA1 acts as a tumor suppressor that may inhibit the proliferation of breast and ovarian cancer cells. To gain knowledge and to further understand the biological function of BRCA1, we examined its localization and expression in various tissues from 20-year-old male and female cynomolgus monkeys (Macaca fascicularis) by in situ hybridization using a 35S-labeled human BRCA1 DNA probe fragment derived from exon 11. In mammary glands, BRCA1 expression was primarily located in the duct and acinar epithelial cells. In the ovary, strong BRCA1 expression was detected in granulosa cells in maturing follicles and in luteal cells of the corpus luteum, as well as in the epithelial cells overlying the tunica albuginea. Specific signal was also observed in epithelial cells of the oviduct, endometrium, cervix, and vagina. Moreover, BRCA1 was strongly expressed in the germinal epithelium of the seminiferous tubules as well as over interstitial cells of the testis, in the epithelium of the epididymis, and in epithelial cells bordering the glandular lumen of the seminal vesicles. Signal was also detected in both the anterior and posterior lobes of the pituitary. In the adrenal glands, the signal was greater in the zona glomerulosa compared to the two other cortical zones, whereas the medullary cells were weakly labeled. In the stomach, and in small and large intestine, epithelial cells of the crypts usually exhibited stronger positive reaction than that observed over surface epithelial lining cells. BRCA1 expression was also found in diverse types of epithelial cells of the thyroid, pancreas, salivary glands, trachea, urinary bladder, and kidneys. In addition to demonstrating widespread tissue- and cell-specific expression of the BRCA1 gene in primate tissues, primarily in the epithelia, we observed a weaker but specific signal in various other cell types, suggesting a generalized biological function of BRCA1.

Formation and degradation of dihydrotestosterone by recombinant members of the rat 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase family

Abstract The structures of cDNA clones encoding four members of the rat 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD) family were characterized. The rat type I, type II and the novel type IV are genuine NAD+/H-dependent 3β-HSD isoenzymes. On the other hand, the liver-specific type III protein is a specific 3-keto-reductase (3-KSR) that catalyzes the conversion of 5α-androstane-3-one-17β-ol (DHT) and 5α-androstane 3,17-dione (A-dione) into their 3β-hydroxy metabolites. The aim of the present study was to further characterize the enzymatic properties of rat types I, III and IV, especially their role in the formation and degradation of DHT after transient expression in intact human HeLa cervical carcinoma, JEG-3 choriocarcinoma or SW-13 adrenal cortex adenocarcinoma cells in culture. The expressed type III 3-KSR in intact HeLa cells catalyzed the reduction of DHT into 3β-diol, whereas expression of type I 3β-HSD in these cell lines had no significant effect on the basal conversion of DHT into 3β-diol, but it did increase the formation of DHT from 3β-diol. A-dione is the predominant product obtained when DHT and 5α-androstane-3β,17β-diol (3β-diol) are used as substrates in intact JEG-3 and SW-13 cells transfected with rat type I 3β-HSD. Furthermore, this predominant 17β-HSD activity was also observed in SW-13 cells transfected with the novel rat type IV 3β-HSD. The predominance of this ‘secondary’ 17β-HSD activity is also reflected in HeLa cells transfected with type I 3β-HSD by the deduced predominant pathway 3β-diol → DHT → 5α-androstane- 3α, 17β-diol (3α-diol) → androsterone (ADT), in which formation of 3α-diol was due to endogenous 3α-HSD activity of HeLa cells, whereas the other reactions are catalyzed by the type I 3β-HSD isoenzyme. This observation thus demonstrates that rat type I 3β-HSD may also catalyze the conversion of 3α-diol into ADT through its intrinsic 17β-HSD activity. The predominant metabolic pathways observed in the present study could be attributed to preponderant bioavailability of NAD+ and NADPH in the intact transfected cells used.