Shaheena Parween1, Florence Roucher-Boulez1, Christa E Flück1, Anne Lienhardt-Roussie1, Delphine Mallet1, Yves Morel1, Amit V Pandey1. 1. Pediatric Endocrinology, Diabetology, and Metabolism (S.P., C.E.F., A.V.P.), University Children's Hospital, and Department of Clinical Research, University of Bern, CH-3010 Bern, Switzerland; Service d'Endocrinologie Moléculaire et Maladies Rares (F.R.-B., D.M., Y.M.), Centre de Biologie et Pathologie Est, Hospices Civils de Lyon, Université Lyon 1, 69002 Bron-Lyon, France; and Endocrinologie Pédiatrique Département de Pédiatrie Médicale (A.L.-R.), Hôpital de la Mère et de l'Enfant, 87042 Limoges, France.
Abstract
CONTEXT: P450 oxidoreductase (POR) is required for the activities of steroid-metabolizing cytochrome P450 enzymes in the endoplasmic reticulum. POR deficiency (PORD) is a form of congenital adrenal hyperplasia. Objective and Aim: Enzymatic and structural analysis of a novel L374H POR mutation from a patient with 46,XX disorder of sexual development. Design, Setting, Patient, and Intervention: The patient was a 46,XX girl with nonconsanguineous Turkish parents. She had virilized external genitalia at birth, a uterus and ovaries, and no sign of Antley-Bixler syndrome. The initial diagnosis was CYP21A2 deficiency with no mutations in CYP21A2, but POR mutations were found. Functional testing was done after producing recombinant POR proteins for analyzing enzymatic and structural properties. MAIN OUTCOME: Novel mutations were causing severe loss of POR activities for metabolism of steroids and small molecules. RESULTS: The L374H mutation reduced activities by 80% in cytochrome c, 97% in thiazolyl blue tetrazolium bromide, and 86% in ferricyanide reduction assays. The catalytic efficiency of the 17 α-hydroxylation of progesterone and the 17,20-lyase reaction of 17-OH pregnenolone was decreased by 87 and 90%, respectively; 21-hydroxylation of progesterone was decreased by 96%, and androstenedione aromatization was decreased by 90%. Analysis of the mutant structure by molecular dynamics simulations revealed structural instability. Flavin release and fast proteolysis assays showed that the L374H mutant is less stable than wild-type POR, confirming the bioinformatics prediction. CONCLUSIONS: This is the first report of a mutation causing PORD by affecting protein stability that causes severe reduction in POR activities. Detailed characterization of individual mutations in POR is required for understanding novel molecular mechanisms causing PORD.
CONTEXT: P450 oxidoreductase (POR) is required for the activities of steroid-metabolizing cytochrome P450 enzymes in the endoplasmic reticulum. POR deficiency (PORD) is a form of congenital adrenal hyperplasia. Objective and Aim: Enzymatic and structural analysis of a novel L374H POR mutation from a patient with 46,XX disorder of sexual development. Design, Setting, Patient, and Intervention: The patient was a 46,XX girl with nonconsanguineous Turkish parents. She had virilized external genitalia at birth, a uterus and ovaries, and no sign of Antley-Bixler syndrome. The initial diagnosis was CYP21A2 deficiency with no mutations in CYP21A2, but POR mutations were found. Functional testing was done after producing recombinant POR proteins for analyzing enzymatic and structural properties. MAIN OUTCOME: Novel mutations were causing severe loss of POR activities for metabolism of steroids and small molecules. RESULTS: The L374H mutation reduced activities by 80% in cytochrome c, 97% in thiazolyl blue tetrazolium bromide, and 86% in ferricyanide reduction assays. The catalytic efficiency of the 17 α-hydroxylation of progesterone and the 17,20-lyase reaction of 17-OH pregnenolone was decreased by 87 and 90%, respectively; 21-hydroxylation of progesterone was decreased by 96%, and androstenedione aromatization was decreased by 90%. Analysis of the mutant structure by molecular dynamics simulations revealed structural instability. Flavin release and fast proteolysis assays showed that the L374H mutant is less stable than wild-type POR, confirming the bioinformatics prediction. CONCLUSIONS: This is the first report of a mutation causing PORD by affecting protein stability that causes severe reduction in POR activities. Detailed characterization of individual mutations in POR is required for understanding novel molecular mechanisms causing PORD.
Authors: C Gusmano; R Cannarella; A Crafa; F Barbagallo; S La Vignera; R A Condorelli; A E Calogero Journal: J Endocrinol Invest Date: 2022-07-17 Impact factor: 5.467
Authors: Mónica Fernández-Cancio; Núria Camats; Christa E Flück; Adam Zalewski; Bernhard Dick; Brigitte M Frey; Raquel Monné; Núria Torán; Laura Audí; Amit V Pandey Journal: Pharmaceuticals (Basel) Date: 2018-04-29
Authors: Shaheena Parween; Maria Natalia Rojas Velazquez; Sameer S Udhane; Norio Kagawa; Amit V Pandey Journal: Front Pharmacol Date: 2019-10-15 Impact factor: 5.810