Browsing by Subject "Steroid 17-alpha-Hydroxylase"
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Item Mechanistic Studies of Two Steroidogenic Cytochromes P450, CYP17A1 and CYP21A2(2012-07-17) Yoshimoto, Francis Kiichi Iida; Auchus, Richard J.Human CYP17A1 (P450c17 or 17alpha-hydroxylase/17,20-lyase) is a member of the P450 superfamily of proteins. This enzyme is responsible for hydroxylating the 17-position of pregnenolone or progesterone and cleaving the 17,20-carbon,carbon-bond of its hydroxylated products, which leads to the formation of androgens. The latter activity makes the inhibition of this enzyme a target for the treatment of prostate cancer. Human CYP21A2 (P450c21 or 21-hydroxylase) hydroxylates the 21-position of progesterone and 17-hydroxyprogesterone – deficiency in this enzyme leads to congenital adrenal hyperplasia. Intermolecular and intramolecular kinetic isotope effects were determined for both enzymes, which required the synthesis of regioselectively deuterated steroids: 17alpha-[2H]-progesterone, 17alpha-[2H]-pregnenolone, 21,21,21-[2H3]-progesterone, 21-[2H]-progesterone, 16alpha-[2H]-progesterone and 21,21,21-[2H3]-17-hydroxyprogesterone. Based on the calculated isotope effects the C-H abstraction step was determined to be partially rate-limiting in the overall hydroxylation process. Moreover, novel 21-hydroxylase activity and 16-hydroxylase activity on progesterone was observed for CYP17A1 and CYP21A2, respectively. CYP17A1 catalyzed hydroxylation of progesterone on the 21:17:16 positions in a 3.4:76.3:20.3 ratio, and this ratio changed to 5.5:87.4:7.1 when 16alpha-[2H]-progesterone was used as the substrate. Meanwhile, CYP21A2 hydroxylated the 21:16 positions in a 99.6:0.4 ratio and this ratio changed to 94.3:5.7 when 21,21,21-[2H3]-progesterone was used as the substrate. Kinetic isotope effects with 17alpha-[3H]-pregnenolone were calculated for CYP17A1. Olefinated progesterone analogs were synthesized to probe possible epoxidase activity of these steroidogenic enzymes. CYP17A1 hydroxylated the 21-position and epoxidized the 16alpha,17alpha-position of 16,17-dehydroprogesterone while CYP21A2 was found to only hydroxylate the 21-position of the same substrate. Both CYP17A1 and CYP21A2 reduced the 21,22-alkene of 21-homo-21,22-dehydroprogesterone and further hydroxylated the metabolite. Other potential substrates such as cyclopropyl and halogenated progesterone analogs were also synthesized to study the enzymatic reactivity towards these substrates. CYP17A1 was found to metabolize 17beta-cyclopropylmethylandrostenone and CYP21A2 was found to metabolize 17-fluoroprogesterone. Both enzymes were found to metabolize 20-desoxoprogesterone – each enzyme yielded a different product with this substrate. Exploring the mechanistic behavior of these enzymes towards various steroid analogs enhances our understanding of their reactivities and structural properties. This research ultimately provides insights on more detailed roles of these enzymes in human disease and may help us to design better inhibitors.Item The Search for the Endogenous RORg Ligand and Investigating the Role of Cytochrome B5 in Steroidogenesis and Regulation of the Cyp17a1 Lyase Reaction(2013-07-18) Sondhi, Varun; Russell, David W.; Mangelsdorf, David J.; Kliewer, Steven A.; Hobbs, Helen H.; Auchus, Richard J.RORg is an orphan nuclear receptor important in the regulation of immune development and function. RORg regulated TH17 cells have been implicated in the pathology of various autoimmune diseases including multiple sclerosis and rheumatoid arthritis. Targeting RORg through antagonists has emerged as a novel therapeutic tool in the treatment of autoimmune diseases. Identification of the RORg endogenous ligand would offer insight into RORg regulation. It is currently believed that sterols are the endogenous RORg ligands. In these studies I will show that while sterols can bind RORg, they fail to modulate its activity in-vivo or in-vitro. Endogenously extracted lysophospholipids on the other hand, such as 22:4 LPE, can activate RORg in co-transfection assays and may be RORg ligands. Additionally, 22:4 LPE can increase IL17 production in TH17 cells. My second project involved investigating the role of cytochrome B5 in steroidogenesis. Cyb5 is involved in regulating electron transfer to numerous P450-mono-oxygenases Cyb5's role in human physiology has been confirmed by the discovery of patients with mutations in Cyb5 that present with isolated 17, 20-lyase deficiency, characterized by low plasma androgens and ambiguous external genitalia. To study the consequences of Cyb5 deficiency in an intact animal and in steroidogenic tissue, I generated mice lacking Cyb5 in the Leydig cell by crossing Cyb5fl/fl and CreSF1 animals. I show that the Cyb5-/- animals were born in a normal Mendelian ratio and had normal fertility with no overt phenotype. Testicular histology revealed no differences between Cyb5-/- and WT animals. Homogenates from Cyb5-/- testes had normal progesterone (P)-to-17α-hydroxyprogesterone (17-OHP) conversion but low 17-OHP-to-androstenedione (A) and testosterone (T) metabolism. The ratio of the hydroxylase to lyase activity was observed to be 1.7 in the WT and 4.5 (3-fold higher) in the Cyb5-/- testes due to deficient lyase activity in the knockout animals. However, steroid production was found to be normal in these animals. Exogenous hCG administration gave a large increase in serum steroids for both the Cyb5-/- and WT animals. In the Cyb5-/- animals, this rise was accompanied by the accumulation of 17-OHP in serum, which led to a 17-OHP/(A+T) ratio that was 44-fold in the KOs. Thus these data demonstrate the physiological significance of Cyb5 in the Cyp17a1 lyase reaction.