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Regulation of Cyp17a1 Activity and Its Potential

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Regulation of Cyp17a1 Activity and Its Potential REGULATION OF CYP17A1 ACTIVITY AND ITS POTENTIAL IMPLICATIONS ON THE DEVELOPMENT OF BOAR TAINT. A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph by MACHTELD JOANNA BILLEN In partial fulfilment of requirements For the degree of Master of Science December, 2008 © Machteld J Billen, 2008 Library and Bibliotheque et 1*1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-47751-9 Our file Notre reference ISBN: 978-0-494-47751-9 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library permettant a la Bibliotheque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par Plntemet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans loan, distribute and sell theses le monde, a des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, electronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in et des droits moraux qui protege cette these. this thesis. Neither the thesis Ni la these ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent etre imprimes ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission. In compliance with the Canadian Conformement a la loi canadienne Privacy Act some supporting sur la protection de la vie privee, forms may have been removed quelques formulaires secondaires from this thesis. ont ete enleves de cette these. While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. Canada ABSTRACT REGULATION OF CYP17A1 ACTIVITY AND ITS POTENTIAL IMPLICATIONS ON THE DEVELOPMENT OF BOAR TAINT. Machteld J Billen Advisor: University of Guelph, 2008 Professor E. J. Squires Testicular 16-androstene steroids, in particular 5a-androstenone, contribute to an off-odour and off-flavour known as boar taint. Cytochrome P450 C17 (CYP17A1) catalyses the key regulatory step in the formation of the 16-androstene steroids from pregnenolone via the andien-P synthase reaction, or the synthesis of the glucocorticoid and sex steroids via 17a-hydroxylase and CI7,20 lyase pathways respectively. The goals of this research were to investigate the effect of both isoforms of CYB5 and the phosphorylation status of CYP17A1 on these different activities of CYP17A1, by overexpressing these proteins in HEK-293 cells. CYB5A increased andien-P synthase and CI7,20 lyase activities while CYB5B increased CI7,20 lyase but had no effect on andien-P synthase activity. Phosphorylation of CYP17A1 at Ser 106 in the presence of CYB5A increased CI7,20 lyase and andien-P synthase activity. This suggests that decreased expression of CYB5A and increased expression of CYB5B would maintain sex steroid production and decrease boar taint. Key Words: CYP17A1, CYB5, Androstenone, C17, 20 lyase, 17a-hydroxylase Acknowledgments First and foremost I would like to thank my advisor, Dr. Jim Squires for his guidance, patience, support and enthusiasm through the course of this degree. It was his passion for research that made my results exciting and made it that much easier to persevere through my graduate career. I would also like to thank Yanping Lou for her never-ending support, advice and service with a smile. To date she is the most impressive multi-tasker I have ever seen and nothing is impossible for this woman. A great thanks goes out to my committee members, Dr. Gordon Kirby and Dr. Julang Li for their support and input throughout this journey. I would also like to remind my peers in the lab just how important there weekly input was and how impossible some of the long days would have been without them. They have defined the true meaning of peer-input and team work. My appreciation is extended to my family, friends and especially my roommate, Erin Edwards who never failed to listen the numerous times I tried to explain boar taint and the moments I needed to go on a scientific rant to remind myself that I was smart and nothing is impossible. Last but not least I would like to thank my boyfriend for his never ending support and encouragement and our constant ability to remind one another that ambition, determination and perseverance lead to success. 1 Table of Contents 1 Introduction 1 1.1 Causes and Occurrences of Boar Taint 1 1.2 The 16-Androstene Steroids 2 1.2.1 Biological Significance 2 1.3 Distinct Steroidogenic roles of CYP17A1 3 1.3.1 The andien-P Steroidogenic Pathway 4 1.4 RoleofCYB5 10 1.4.1 Regulation of 17a hydroxylase and C17, 20 lyase activity 11 1.5 Phosphorylation of CYP17 14 1.6 Rational and Experimental Approach 16 2 Hypothesis and Research Objectives 18 2.1 Hypothesis 18 2.2 Research Objectives 18 2.2.1 Objective 1: Construction of expression vectors for POR, CYP17A1, CYB5R3, CYB5A and CYB5B 19 2.2.2 Objective 2: Investigating the relative importance of CYB5A and CYB5B in androgen versus 16-androstene steroid production. 19 2.2.3 Objective 3: Investigate the effects of phosphorylation of CYP17A1 on androgen and 16- androstene steroid production. 20 2.2.4 Objective 4: The role of Serine106 on porcine CYP17A1. 20 3 The role of porcine cytochrome b5A and cytochrome b5B in the regulation of cytochrome P450 CYP17A1 activities 21 3.1 Abstract 21 3.2 Introduction 22 3.3 Materials and Methods 24 3.3.1 Construction of expression vectors for POR, CYP17A1, CYB5R3, CYB5A and CYB5B 24 3.3.2 Transient expression in human embryonic kidney (HEK-293) cells 26 3.3.3 Western analysis of protein expression 26 3.3.4 Assay of enzymatic activity 27 3.3.5 Statistical analysis 28 3.4 Results 28 3.4.1 Cloning of porcine CYB5R3 and optimization of the expression system 28 3.4.2 Effect of CYB5 isoforms on C17a-hydroxylase/C 17,20 lyase activities versus andien-P synthase activity 34 3.4.3 Effects of CYB5 isoforms on 17a-hydroxylase versus CI7,20 lyase activity 34 3.5 Discussion 40 4 The effect of phosphorylation of cytochrome P45017A1 on 17a-hydroxylase, CI 7,20 lyase and the andien-P synthase activities 45 4.1 Abstract 45 ii 4.2 Introduction 46 4.3 Materials and Metholds 48 4.3.1 Construction of expression vectors 48 4.3.2 Transient expression in human embryonic kidney (HEK-293) cells. 49 4.3.3 Assay of enzymatic activity 50 4.3.4 Statistical analysis 51 4.4 Results 51 4.4.1 Effect of CYB5 isoform and treatment with OA on 17a-hydroxylase, C17,20 lyase and andien-p synthase activities. 51 4.4.2 Effect of CYB5 isoform and treatment with PP2A on 17a-hydroxylase, CI7,20 lyase and andien-p synthase activities. 56 4.4.3 Effect of phosphorylation status and site of phosphorylation on CYP17A1 61 4.5 Discussion 65 5 General Discussion and Conclusions 70 5.1 Thesis Conclusions 71 5.2 Future research of isoforms of CYB5 and phosphorylation status in vivo. 73 5.3 Future research in the potential production of a knockout pig only expressing CYB5B 73 5.4 Future research in the development boar taint: Skatole metabolism and its relationship with Androstenone 74 5.5 Final Conclusions 77 in List of Tables Table 1: Forward and reverse primers used to amplify sequences of tagged and untagged CYP17A1, POR, CYB5R3, CYB5A and CYB5B and the complete names of the plasmids produced. Table 2: A summary of the trends of 170HP, DHEA and ANp production when cells incubated with CYP17A1, POR, CYB5R3 and CYB5A or CYB5B are treated with phosphatase inhibitor, OA and protein phosphatase 2A. IV List of Figures Fig 1: The andien-P steroidogenic pathways Fig 2: The Fem-OH intermediate at the crossroad of hydroxylation and acyl-carbon bond cleavage. (Lee-Robichaud P et al., 1995). Fig 3: Acyl-Carbon cleavage reaction from pregnenolone to form 16-steroids in the presence of CYB5 (Lee-Robichaud P et al., 1998). Fig 4: Androstenone biosynthesis, the different pathways for production of androstenone, glucocorticoid and sex steroid synthesis all catalyzed by CYP17A1. Fig 5: The nucleotide (A) and deduced amino acid (B) sequence of porcine CYB5R3 compared to human CYB5R3. Fig 6: a) Western blot showing the expression of CYP17A1, POR, CYB5R3, CYB5A and CYB5B at 24, 48, 72 and 96 hours after transfection. CYP17A1 (59 kDa), POR (78 kDa), CYB5R3 (33 kDa), CYB5A (15 kDa) and CYB5B (16 kDa). b) Band densities determined by scanning the western blots. Fig 7: The production of pregnenolone metabolites ANp, DHEA + 170HP over time. HEK-293 cells were transfected with expression vectors for POR (0.35ug), CYP17A1 (0.25ug), CYB5R3 (0.25 ug) and CYB5A (1 ug). Fig 8: The effect of increased expression of CYB5A on the production of DHEA + 170HP and ANp. The results are expressed as the mean ± S.E of 5 replicate experiments with duplicate transfections in each experiment. * Significantly different (P <0.05) compared to the control (CYB5A=0ug). (n=10) Fig 9: The effect on increased expression of CYB5B on the production of DHEA + 170HP and ANp.
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