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

The University of Guelph

MACHTELD JOANNA BILLEN

In partial fulfilment of requirements

For the degree of

Master of Science

December, 2008

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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. 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 (CYB5B=0ug). (n=10)

Fig 10: The effect of increased expression of CYB5A on the production of 170HP and DHEA. 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 11: The effect of increased expression of CYB5B on the production of 170HP and DHEA. 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 (CYB5B=0ug). (n=10)

v Fig 12: a) The effect of lOnM OA treatment with increased expression of CYB5A on the production of ANp. * Significantly different (P <0.05) indicated when compared to the control at same transfection level treated with Norokadone. b) The effect of lOnM OA treatment with increased expression of CYB5B on the production of ANp * Significantly different (P <0.05) indicated when compared to the control at same transfection level treated with Norokadone. (n=6)

Fig 13: a) The effect of lOnM OA treatment with increased expression of CYB5A on the production of 170HP. * Significantly different (P <0.05) indicated when compared to the control at same transfection level treated with Norokadone. b) The effect of 1 OnM OA treatment with increased expression of CYB5B on the production of 170HP. * Significantly different (P <0.05) indicated when compared to the control at same transfection level treated with Norokadone. (n=6)

Fig 14: a) The effect of 1 OnM OA treatment with increased expression of CYB5A on the production of DHEA. * Significantly different (P <0.05) indicated when compared to the control at same transfection level treated with Norokadone. b) The effect of lOnM OA treatment with increased expression of CYB5B on the production of DHEA. * Significantly different (P <0.05) indicated when compared to the control at same transfection level treated with Norokadone. (n=6)

Fig 15: a) The effect of 0.25ug PP2A treatment with increased expression of CYB5A on the production of ANp. * Significantly different (P <0.05) compared to the control at same transfection level, b) The effect of 0.25ug PP2A treatment with increased expression of CYB5B on the production of AN0. * Significantly different (P <0.05) compared to the control at same transfection level. (n=6)

Fig 16: a) The effect of 0.25ug PP2A treatment with increased expression of CYB5A on the production of 170HP. * Significantly different (P <0.05) compared to the control at same transfection level, b) The effect of 0.25ug PP2A treatment with increased expression of CYB5B on the production of 170HP. * Significantly different (P <0.05) compared to the control at same transfection level. (n=6)

Fig 17: a) The effect of 0.25ug PP2A treatment with increased expression of CYB5A on the production of DHEA. * Significantly different (P <0.05) compared to the control at same transfection level, b) The effect of 0.25ug PP2A treatment with increased expression of CYB5B on the production of DHEA. * Significantly different (P <0.05) compared to the control at same transfection level. (n=6)

Fig 18: A comparison of ANp, 170HP and DHEA production between wild type CYP17A1-Ser106 and the mutant CYP17A1-Ala106 with CYB5A. (n=4)

Fig 19: A comparison of ANp, 170HP and DHEA production between wild type CYP17A1-Ser106 and the mutant CYP17A1-Ala106 with CYB5B. (n=4)

VI Appendix - Abbreviations

ANp - Andien |3 synthase

CYB5R3 - NADH CYB5 reductase

DHEA - Dehydroepiandrosterone

DMSO - Dimethyl sulfoxide

FAD - Flavinadenine dinucleotide

FMN - Flavin mononucleotide

HEK - Human embryonic kidney cells

HPLC - High performance liquid chromatography

Nor - Norokadone

OA - Okadiac Acid

PBS - Phosphate Buffered Saline

PCR - Polymerase chain reaction

POR - NADPH cytochrome P450 oxidoreductase

PP2A - Protein phosphatase 2A

PVDF - Polyvinylindene fluoride

P450ssc - P450 side chain cleavage

170HP- 17a-hydroxypregnenolone

vn Chapter I 1 Introduction

1.1 Causes and Occurrences of Boar Taint

Intact male pigs have advantageous production characteristics for both consumer and producer. Intact male pigs have improved feed conversion efficiency (Fortin et al.,

1983; Squires et al., 1993) which directly contributes to increased lean meat production

(Sather et al., 1991). However, a variable range of meat from intact males at usual slaughter weights contains boar taint, an unpleasant urine/perspiration/fecal type odour associated with boar fat when it is heated (Gunn et al., 2004; Patterson 1968).

Boar taint is the result of high concentrations of the testicular steroid 5a-androst-

16-en-3-one (5a-androstenone) (Patterson 1968) and 3-methylindole (skatole), a naturally occurring microbial metabolite of tryptophan, that accumulate in the adipose tissue of intact male pigs (Void 1970; Yokoyama and Carlson 1979). Both androstenone and skatole are lipid soluble and thus can be found in high concentrations in fat (Gunn et al., 2004). Boar taint is not present in all carcasses; however approximately five to 15 percent of market weight boars in North America have levels of androstenone high enough to be detected by consumers (Squires and Lou, 1995). The primary solution considered was castration; however this removes any of the beneficial qualities which result from anabolic effects of testicular steroids, reduces the profitability of the pork industry and also raises animal welfare concerns (Babol and Squires, 1995). Castration of pigs has been eliminated in many countries such as Australia, Ireland and the UK,

1 followed by Portugal, Spain and Denmark. The rest of the countries castrate all pigs except those used for breeding purposes (Gunn et al., 2004).

1.2 The 16-Androstene Steroids

It has long been recognized that the mammalian testis produces androgens which are required for spermatogenesis and the development of secondary sex characteristic in males. The mammalian testis is composed of 2 areas: a) the gametogenic compartment; seminiferous tubules and b) the interstitial compartment; leydig cells. The adrenal mitochrondia contain the important steroidogenic enzyme cytochrome CYP11 Al, also referred to as P450 side chain cleavage (P450scc) which converts cholesterol to pregnenolone (DuBois et al., 1981; Miller 1988). A wide range of steroids is produced within the Leydig cells, and these can be divided into three main categories: androgens, estrogens and 16-andostene steroids. The 16-androstene steroids are quantitatively the most plentiful steroid produced by the boar testis, reaching concentrations 50 to 200 times higher than that of testosterone (Booth 1975). These steroids are structurally characterized by a double bond at the CI 6 position in the D-ring of the steroid nucleus

(Booth 1975; Ruokonen 1974)

1.2.1 Biological Significance

Androstenone is a CI9 steroid produced by Leydig cells in porcine testis from precursors, pregnenolone and progesterone (Claus et al., 1994). Androstenone is then transported via the spermatic vein (Saat et al., 1972) to the submaxillary salivary glands

(Gower 1972), where it is concentrated 10-100X by the protein pheromaxein (Babol et al., 1996; Booth 1987). Once bound to pheromaxein, 5cc-androstenone is reduced to the

2 physiologically active 3a-androstenol, which is a reproductive pheromone (Booth 1984).

Sows in estrus are stimulated by the pheromone to perform a typical mating stance for the boars (Reed et al., 1974). However androstenone is also highly lipophilic and accumulates in the adipose tissue (Claus et al., 1971) leading to the disagreeable odour and flavour of meat.

1.3 Distinct Steroidogenic roles ofCYPl 7A1

CYP17A1 has three distinct steroidogenic roles all which occur in the adrenal cortex. The production of mineralcorticoids takes place in the zona glomerulosa, glucocorticoids are produced in the zona fasciculata and finally weak androgens are created in the zona reticularis. Pregnenolone can be converted to 5,16-androstadien-3p-ol via CYP17A1, CYB5 and their respective reductases, this is the primary step in androstenone synthesis (Fig 1). Likewise, CYP17A1 also catalyses the conversion of pregnenolone to androgens to promote growth and sexual development.

CYP17A1 roles begin once the precursor cholesterol is converted to pregnenolone by the mitochondrial cholesterol side chain cleavage with CYP11 Al

(Miller et al., 1997). Pregnenolone then travels to one of three different pathways. One of these three pathways occurring in the zona glomerulosa allows pregnenolone to remain a

C-21, 17-deoxy steroid and progress down the mineralcorticoid pathway toward aldosterone. Alternatively pregnenolone may undergo hydroxylation; this pathway is known as 17a-hydroxylation and produces 17a-hydroxypregnenolone. 17a- hydroxylation in the zona fasciculata proceed to the C-21,17-hydroxy pathway to the principal glucocorticoid, Cortisol. Finally, following the 17a-hydroxylation it may

3 undergo cleavage of the CI 7-20 bond to become a CI 9, 17-ketosteroid, leading to DHEA synthesis in the zona reticularis (Miller et al., 1997).

1.3.1 The andien-fiSteroidogenic Pathway The 16-androstene steroids are synthesized from steroid precursors pregnenolone and progesterone; however, human CYP17A1 has a significantly lower affinity for 17a- hydroxyprogesterone and hence in the metabolic route of testosterone formation in humans, pregnenolone is the preferred substrate with a 10 fold higher conversion efficiency (Brock and Waterman 1999; Gower 1972). The primary step in the synthesis of androstenone from pregnenolone is the irreversible formation of 5,16-androstadien-3p- ol (ANP), catalysed by the andien-P synthase (Fig 1) system in a cytochrome P450 dependent reaction (Gower 1972; Squires 1989) in the microsomal fraction of the leydig cells (Hildebrandt and Estabrook 1971). The ANP Synthase system involves CYP17A1, microsomal CYB5A, and their respective reductases; NADPH cytochrome P450 oxidoreductase (POR) and NADH CYB5 reductase (CYB5R3) (Meadus et al., 1993a;

Nakajin et al., 1985).

The P450 reductase functions by catalyzing electron transfer from NADPH to the cytochrome P450 during catalysis (Lu and West ,1978). P450 reductase is also involved in electron transfer from NADPH to heme oxygenase (Yoshida and Kikuchi, 1978) and

CYB5 (Ilan et al., 1981). The microsomal electron transport system is as follows

(Bridges et al., 1998; Meadus, 1994)

+ + RH + 02 + NADPH + H -> ROH + H20 + NADP

RH is the substrate and ROH is the oxidized product. CYB5 reductase works in a similar manner; the reaction is shown below:

4 NADH + H+ + 2 ferricytochrome CYB5 -> NAD+ + 2 ferrocytochrome CYB5

NADH-CYB5 reductase is one of the flavoproteins that catalyzes the reduction of CYB5, using FAD as a prosthetic group. Two forms of CYB5 reductase are known; CYB5R3 and CYB5R1 a membrane bound form in somatic cells and a soluble form in erythrocytes, respectively (Tomatsu et al., 1989). The membrane bound form is located mainly on the cytoplasmic side of the endoplasmic reticulum of somatic cells and functions in the desaturation and elongation of fatty

5 -03S0 SULT2A1 Pregnenolone Sulfate Sulfatase

-O3SO SULT2A1 Androstadienol Sulfate Sulfatase (5,16-androstadien-3p-ol)

Figure 1: The andien-P steroidogenic pathway

6 acids (Oshino et al., 1971), cholesterol biosynthesis (Reddy et al., 1977) and drug metabolism (Hildebrandt and Estabrook 1971). The soluble form is located in circulating erythrocytes and is involved in methemoglobin reduction (Hultquist and

Passon, 1971). Analysis of both forms indicates that the membrane-bound form consists of both the membrane binding domain and the catalytic domain, whereas the soluble form only consists of the catalytic domain (Ozols et al., 1985; Yubisui et al., 1987).

The andien P synthase reaction represents the first reaction involved in the formation of the 16-androstene steroids from the precursor pregnenolone. The primary step involves the binding of pregnenolone to CYP17A1. One electron reduction of the substrate-enzyme complex by NADPH (POR) is followed by the binding of molecular oxygen. This promotes the synthesis of 17a-hydroxypregnenolone (170HP) by receiving electrons from NADPH through the cytoplasmic flavoproteins POR. (Figure 1) The second electron is then transferred via NADPH (POR) or by CYB5 for the CI 7,20 lyase activity converting 170HP to dehydroepiandrosterone (DHEA) (Pandey et al., 2003).

Protonation of the distal oxygen atom by a hydrogen ion and cleavage of the oxygen- oxygen bond produces a reactive iron-oxo intermediate. The reaction mechanism for each activity is thought to involve formation of two distinct iron-oxygen complexes. For the hydroxylation mechanism, the oxo-intermediate, Fev=0, is considered to be the active catalytic oxygen bound CYP complex (Atkinson and Ingold, 1993), while for the acyl- carbon bond cleavage, participation of the iron-peroxo, Fem-OOH, and iron-oxo, Fev=0, species have both been suggested as possible candidates (Figure 2) (Akhtar et al., 1994;

Lee-Robichaud et al., 1995a). Figure 2 also demonstrates the Fem-OOH species is generated for conversion into the oxo-derivative that promotes hydroxylation (7-9ab in

7 figure 2). However when a suitably juxtapositioned electrophilic center is available, the iron peroxide is trapped, producing the adduct that then decomposes to promote a cleavage reaction (fragmentation path, figure 2) (Lee-Robichaud, 1995). The activated oxygen atom is then inserted into a carbon-hydrogen bond of the substrate resulting in the dissociation of product, and the regeneration of the ferric CYP17Al(Vergeres and

Waskell, 1995).

8 Fe«

iPt-450 3r° H

>-"' •< (8) OH

Fmgm

\ I. ,^ 2e Pe1in IV1 o» -* 1**50 m O-QH

Hyriroxyiatian path

\ 1JW Fa* Fe R-OH ^ 1 *^ ft»M o O C&a}

Figure 2: The Fej n -OH intermediate at the crossroad of hydroxylation and acyl-carbon bond cleavage. Hydroxylation path leads to the production of 17a-hydroxypregnenolone, Fragmentation path leads to the production of 16-steroids (Lee-Robichaud et al., 1995).

9 1.4 Role ofCYB5

It was first suggested by Estabrook (1999) that during the reductive stages of

CYP17A1, the second electron for the completion of the catalytic cycle can be derived

from CYB5, an alternative redox partner to the conventional POR. A second model

suggested that CYB5 could act allosterically to promote optimal interaction between

POR and CYP17A1, to enhance the electron flow within the system, or promote the

breakdown of the CYP-substrate intermediate in the catalytic cycle (Schenkman and

Jansson, 2003). Previous work by Lee-Robichaud (1995) established that 16-androstene

steroids arise from direct cleavage of the side chain of pregnenolone via acyl carbon

cleavage in the presence of CYB5 resulting in a delta 16 double bond (Fig 2 and 3). The

Figure 3: Acyl-Carbon cleavage reaction from pregnenolone to form 16-steroids in the presence of CYB5 (Lee-Robichaud et al., 1998).

10 importance and requirement for CYB5 in 16-androstene production has also previously been shown (Meadus et al., 1993b; Nakajin et al., 1985).

Ogishima et al., (2003) discovered that there is an outer mitochondrial membrane

CYB5B which may also be involved in steroid hormone metabolism in rats. It was determined CYB5B stimulated the CYP17A1 catalyzed reactions of 17a-hydroxylation and C17-C20 bond cleavage at a level similar to CYB5A. CYB5 is a highly conserved electron-transfer protein found in the endoplasmic reticulum and the mitochondrion;

CYB5 consists of two domains: a cytoplasmic, globular, heme-binding core domain

(approx. 100 residues) and a carboxy-terminal membrane anchor (approx. 35 residues)

(Kaderbhai MA et al., 2003). The homology in amino acid sequence between porcine microsomal CYB5A and mitochondrial CYB5B is 48%, with CYB5A and CYB5B consisting of 134 and 144 amino acids respectively (National Center for Biotechnology

Information-Blast, 2007). CYB5 as well as POR and CYB5R3 all play an integral role in the ANp Synthase pathway. CYB5 has many roles such as (a) transfer of electrons from

NADH to desaturases (Ozols J, 1976); (b) NADH-dependent reduction of methemoglobin to regenerate hemoglobin (Abe and Sugita, 1979), and (c) stimulation of

P-450 dependent oxygenation (Ogishima et al., 2003). Independent roles of porcine

CYB5A versus CYB5B in 17a-hydroxylase, CI7,20 lyase and andien (5 synthase have not previously been investigated.

1.4.1 Regulation of 17a hydroxylase and C17, 20 lyase activity Figure 4 displays the three different pathways catalyzed by CYP17A1 and how manipulation of CYP17A1 could potentially control the ANP synthase pathway and 16-

11 androstene steroid production. Three factors have been shown to manipulate the ratio of

17a-hydroxylase activity to CI7,20 lyase activity at the post translational level. Primarily higher molar ratios of POR:CYP17Al favours the CI7,20 lyase activity and DHEA production (Auchus et al., 1998; Lin et al., 1993; Nakajin and Hall, 1981). Secondly, it has been shown that CYB5 acts allostericaly to promote the interaction between

CYP17A1 and POR to stimulate the C17,20 lyase reaction. (Auchus et al., 1998; Geller et al., 1999; Nakajin and Hall, 1981). Thirdly, it has been shown in humans that phosphorylation of CYP17A1 on serine and threonine but not tyrosine residues increases

C17,20 lyase activity and the production of DHEA (Zhang et al., 1995).

A strong positive correlation between DHEA production and the co-localization of

CYB5 in the endoplasmic reticulum of the gonads and CYP17A1 in the zona reticularis of the adrenal cortex, initially implicated the involvement of CYB5 as a potential modulator of the cleavage activity of CYP17A1 (Lu et al. 1974, Lu et al. 1975). Selective stimulation of CYP17A1 cleavage activity was primarily demonstrated by Katagiri et al

(1982) who discovered the extent of side chain cleavage was dependent on the concentration of CYB5. It was later discovered that CYB5 stimulated the C17, 20 lyase activity by up to 10-fold with no effect on the 17ot-hydroxylase activity. (Katagiri et al,

1995). Lee-Robichaud et al (1998) suggest that in the CYP17A1-POR-CYB5 complex, the conformation of CYP17A1 is altered by CYB5 so as to bring the iron-oxygen intermediate, through repositioning of either the heme moiety or substrate molecule, into alignment with the C20 position of the hydroxylated precursor. A subsequent nucleophilic attack is directed on the carbonyl group of the substrate to yield the cleaved steroid substrate (DHEA) and acetic acid. This allows CYB5 to essentially be regarded as

12 a switch which promotes certain conformational changes of CYP17A1 and turn lyase

activity on and off. The exact mechanisms in which CYB5 catalyses the production of

ANp has yet to be determined.

Increase CF=O 1 Decrease G=0 OH C17-hydroxylase CYB5 <3 Pregnenolone CYP17A1 17 a- hydroxypregnenolone 3P 5,16-androstadien-3 p-ol Isomerase Dehydrogenase Isomerase 3p ^Dehydrogenase Cn-lyase

Progesterone 4,16-androstadien-3-om

5a Reductase

Dehydroepiandrosterone

Figure 4: Androstenone biosynthesis, the different pathways for production of androstenone, glucocorticoid and sex steroid synthesis all catalyzed by CYP17A1. Post- translational modifications of CYP17A1 and the interaction with CYB5A/CYB5B could contribute to the control of boar taint by increasing the production of DHEA and minimize ANP production.

13 1.5 Phosphorylation ofCYP17

Reversible phosphorylation of proteins is an important regulatory mechanism that occurs in both prokaryotic and eukaryotic organisms (Barford et al., 1998). Enzymes called kinases (phosphorylation) and phosphatases (dephosphorylation) are involved in this process. Many enzymes and receptors are switched "on" or "off" by phosphorylation and dephosphorylation. Reversible phosphorylation results in a conformational change in the structure in many enzymes and receptors, causing them to become activated or deactivated. Phosphorylation usually occurs on serine, threonine, and tyrosine residues in eukaryotic proteins. In addition, phosphorylation occurs on the basic amino acid residues histidine or arginine or lysine in prokaryotic proteins (Barford et al., 1998). The addition of a phosphate (PO4) molecule to a polar R group of an amino acid residue can turn a hydrophobic portion of a protein into a polar and extremely hydrophilic portion of the molecule. In this way it can introduce a conformational change in the structure of the protein via interaction with other hydrophobic and hydrophilic residues in the protein.

A general study investigating phosphorylation as a functional switch by Oesch-

Bartlomowicz (2003) and Oesch (2005) investigated how the decrease in monooxygenase activity occurs upon changes in cellular phosphorylation. Their research was mainly done with CYP2B1 and determined that the CYP2B1 molecule lost its ability to support monooxygenase activity rapidly due to an increase in phosphorylation.

The majority of studies investigating the effect of phosphorylation status of

CYP17A1 have used Protein phosphatase 2A (PP2A), one of the four major protein phosphatases in the cytosol of mammalian cells, to dephosphorylate serine and threonine

14 residues. Conversely, okadiac acid is used as a PP2A inhibitor to increase the level of phosphorylation. Okadiac acid is a polyether fatty acid isolated from the marine sponge

Halichondria okadaii (Cohen et al., 1990). It has been shown that the PP2A catalytic subunit is completely inhibited by InM okadiac acid (Haystead et al., 1989).

Pandey et al. (2003) treated human adrenal NCI-H295A cells with PP2A inhibitors to increase phosphorylation and found that serine and threonine phosphorylation of

CYP17A1 increases CI7,20 lyase activity and dephosphorylation of CYP17A1 decreases

CI7,20 lyase activity. In a more recent study, Pandey and Miller (2005) also concluded that serine phosphorylation of CYP17A1 increases CI7,20 lyase but had no effect on

17a-hydroxylase activity. It was proposed that the modified negatively charged phosphorylated residues served to enhance electrostatic attraction between the redox partners so as to engage a stronger interaction between POR and CYP17A1, contributing to a faster transfer of electrons and an increase in CI 7,20 lyase activity. They also determined that human CYP17A1 required either CYB5 or phosphorylation for CI7, 20 lyase activity, and that CYB5 and phosphorylation enhance CI7, 20 lyase activity independently. The effects of CYB5 and phosphorylation were not additive, since each mechanism was sufficient to achieve almost maximal induction on its own and enhanced

DHEA production was not found in samples that were both phosphorylated and contained

CYB5 when compared with the effect of each factor individually.

It was also suggested by Miller at al (1997) that perhaps the phosphorylated serine residues either increase the affinity for redox partners and/or participate in forming a modified redox partner binding site needed for binding CYB5. The effect of

15 phosphorylation status of CYP17A1 in pigs on the production of DHEA and 16- androstene steroids has not yet been investigated.

A study investigating genetic lesions and missense mutations in humans showed that the mutation Ser106—Pro in CYP17A1 caused 17a-hydroxylase deficiency in two unrelated patients (Lin et al., 1991). Site directed mutagenesis was used to construct the

Pro106 mutant and both were expressed in COS-1 cells. The Pro106 mutant did not produce

170HP or DHEA, demonstrating that this mutation diminished both 17oc-hydroxylase and C17, 20 lyase activity (Lin et al., 1991). Lin et al, (1993) subsequently investigated this further with CYP17A1 mutations with threonine or alanine at residue 106 and found that these mutants retained only 20-30% of 17a-hydroxylase and CI7,20 lyase activities.

From these experiments it is clear that the amino acid occupying position 106 greatly affects enzymatic activity, and preventing phosphorylation of this residue is important.

In summary, evidence suggests that dephosphorylation of human CYP17 eliminates almost all the CI7, 20 lyase activity without changing 17a-hydroxylase activity (Pandey and Miller, 2005). However, the effect of phosphorylation status of

CYP17 on the andien-P synthase reaction has not been investigated.

1.6 Rational and Experimental Approach

Among the numerous systems developed to characterize enzymatic activity, transfection of expression constructs into intact mammalian cells provides the chance to study the activity of the expressed proteins in the native microsomal environment and to study various combinations of enzyme, redox partner and substrate (Dufort et al., 1999;

Luu-The et al, 1995). In this report, we investigate the effects of CYB5A and CYB5B on the activities of CYP17A1 by transient transfection of human embryonic kidney (HEK-

16 293) cells with expression constructs. HEK-293 cells were chosen based on their ability to grow rapidly and transfect readily. With this system we have the capability to over- express the proteins of interest, and to vary the relative amounts of these proteins to produce a well defined and active system with a minimum interference from endogenous proteins.

The potential significance of these experiments is to allow the complete biological understanding of the andien P synthase pathway responsible for androstenone synthesis in a clearly defined in vitro system and to understand the roles of specific proteins involved in the production of both the beneficial androgenic steroids as well as androstenone synthesis. Understanding the porcine cytochrome P450s system and their appropriate reductases allows further investigation of CYP17A1 interactions with CYB5A and

CYB5B as well as post translational modifications which can increase the production of

DHEA while concurrently decrease androstenone production. The conclusions formulated can then be used to perform appropriate in vivo experiments to eventually minimize or eliminate boar taint while maintaining the advantageous qualities of intact boars.

17 Chapter II

2 Hypothesis and Research Objectives

2.1 Hypothesis

CYB5A, CYP17A1, POR and CYB5R3 are involved in the andien-p synthase,

17a-hydroxylase and CI7, 20 lyase pathways. Likewise CYB5B is also involved in 17a- hydroxylase and CI7, 20 lyase pathways while the involvement of CYB5B in the AN0 synthase pathway needs to be investigated. Phosphorylation status of CYP17A1 promotes the synthesis of sex steroids by increasing CI 7, 20 lyase activity while maintaining or decreasing 17a-hydroxylase activity and may alter the synthesis of androstenone by affecting ANp synthaseANB activity.

2.2 Research Objectives

Previous research by Squires (1989) and Gower (1972), has confirmed the role of the andien-P synthase system, CYP17A1 and microsomal CYB5A, in the conversion of pregnenolone to 5,16-androstadien-3a-ol, the primary step in androstenone synthesis.

Pregnenolone can alternatively be converted to 17a-hydroxypregnenolone via the CI 7- hydroxylase reaction and then to the androgen, DHEA, via the CI7, 20-lyase reaction.

Pandey and Miller, (2005) suggested that dephosphorylation of CYP17A1 with PP2A may eliminate almost all of the CI 7, 20 lyase activity without altering 17a-hydroxylase activity. Ogishima et al., (2003) also discovered a secondary outer mitochondrial membrane CYB5B in rats which may also potentially be involved in ANP androstenone

18 synthesis. This research will investigate the potential involvement of CYB5B in the synthesis of ANP androstenone as well as the effects of phosphorylation status of porcine

CYP17A1 on the andien-p synthase, C17, 20 lyase and 17a-hydroxylase pathways. This information may provide alternative methods to reduce the levels of androstenone stored in the adipose tissue by minimizing ANP synthesis with CYB5B without reducing the production of DHEA in intact boars.

2.2.1 Objective 1: Construction of expression vectors for POR, CYP17A1, CYB5R3, CYB5A and CYB5B

Sequences available for pig CYB5A, CYB5B, CYP17A1 and POR were obtained from the NCBI databank. The sequence of porcine CYB5R3 was assembled from pig

ESTs retrieved by BLAST searching the NCBI database using human CYB5R3

(accession number NM 000398) as a template. Sequences were amplified by PCR and cloned into pcDNA3.1/V5-His expression vector including their native stop codon. These plasmids were then over expressed in HEK-293 cell lines and used to measure activity.

A second series of expression vectors was created without the native stop codon allowing for the inclusion of the V5 epitope and the polyhistidine region of the vector. These plasmids were once again over expressed in the HEK 293 cell lines and used to measure protein expression.

2.2.2 Objective 2: Investigating the relative importance ofCYB5A and CYB5B in androgen versus 16-androstene steroid production.

Expression vectors for pig CYP17A1, POR and CYB5R3 were transfected in

HEK-293 cells along with increasing amounts of expression vectors for CYB5A and

CYB5B. This was followed by incubation with [7-3H(N)]-pregnenolone to determine the

19 effects of different forms of CYB5 on the synthesis of 17a-hydroxypregnenolone and

DHEA versus 16-androstene steroids.

2.2.3 Objective 3: Investigate the effects of phosphorylation of CYP17A1 on androgen and 16-androstene steroid production.

For this objective, we altered CYP17A1 phosphorylation status and determined its potential impact on steroid synthesis. CYP17A1, P450 reductase and CYB5R3 were over-expressed in HEK-293 cells along with increasing amounts of either CYB5A or

CYB5B. Cells were transfected with an expression vector for PP2A or treated with OA

(okadiac acid) and were incubated with [7- H(N)]-pregnenolone to determine the effects of phosphorylation on androgen and 16-androstene steroid synthesis.

2.2.4 Objective 4: The role of Serine106 on porcine CYP17A1.

This objective was accomplished by performing site directed mutagenesis to change the serine106 residue to alanine1 onthepigCYP17Al expression vector. The mutated or wild type form of CYP17A1 as well as POR, CYB5R3 along with either

CYB5A or CYB5B were over-expressed in HEK-293 cells. Cells were then transfected with an expression vector for PP2A or treated with OA and were incubated with [7-

3H(N)]-pregnenolone to determine the effects of the CYP17A1 mutation on androgen and

16-androstene steroid synthesis.

20 Chapter III

3 The role of porcine cytochrome b5A and cytochrome b5B in the regulation of cytochrome P450 CYP17A1 activities

3.1 Abstract

Male pigs are routinely castrated to prevent the accumulation of testicular 16- androstene steroids, in particular 5a-androst-16-en-3-one (5oc-androstenone), which contribute to an off-odour and off-flavour known as boar taint. Cytochrome P450 CI7

(CYP17A1) catalyses the key regulatory step in the formation of the 16-androstene steroids from pregnenolone by the andien-P synthase reaction or the synthesis of the glucocorticoid and sex steroids via 17a-hydroxylase and CI7,20 lyase pathways respectively. We have expressed CYP17A1, along with cytochrome P450 reductase

(POR), cytochrome b5 reductase (CYB5R3) and cytochrome b5 (CYB5) in HEK-293FT cells to investigate the importance of the two forms of porcine CYB5, CYB5A and

CYB5B, in both the andien-P synthase as well as the 17a-hydroxylase and CI7,20 lyase reactions. Increasing the ratio of CYB5A to CYP17A1 caused a decrease in 17a- hydroxylase (P<0.013), an increase in C17,20 lyase, and an increase in andien-P synthase activity (p<0.0001). Increasing the ratio of CYB5B to CYP17A1 also decreased 17a- hydroxylase, but did not affect the andien-p synthase activity; however, the CI7,20 lyase, was significantly increased. These results demonstrate the differential effects of two forms of CYB5 on the three activities of porcine CYP17A1 and show that CYB5B does not stimulate the andien-P synthase activity of CYP17A1.

Keywords: CYP17A1, CYB5, androstenone, C17,20 lyase, 17oc-hydroxylase

21 3.2 Introduction

Androstenone is a CI9 16-androstene steroid produced as a phermonal hormone by Leydig cells in porcine testis from C21 precursors, pregnenolone and progesterone

(Claus et al., 1994). Androstenone is transported via the blood stream to the submaxillary salivary glands (Gower, 1972), where it binds to a specific binding protein which concentrates the steroid in this area (Babol et al., 1996). Androstenone is also highly lipophilic and accumulates in the adipose tissue (Claus et al., 1971) leading to the disagreeable boar taint odour and flavour of meat from uncastrated male pigs.

The primary step in the synthesis of androstenone from pregnenolone is the formation of 5,16-androstadien-3P-ol (ANP), which is catalyzed by the andien-p synthase system in a cytochrome P450C17 (CYP17A1) dependent reaction (Gower, 1972; Squires,

1989). Alternatively, pregnenolone can be converted to 17a-hydroxypregnenolone

(170HP) via the 17a-hydroxylase reaction and then via the C17,20-lyase reaction to the androgen, dehydroepiandrosterone (DHEA). CYP17A1 is thus a key enzyme that regulates the production of androgens versus 16-androstene steroids.

The involvement of CYP17A1, microsomal cytochrome b5 (CYB5A, Type I),

NADPH cytochrome P450 reductase (POR) and cytochrome b5 reductase (CYB5R3) in the andien-P synthase system has been shown previously (Meadus et al., 1993; Nakajin et al.,1985). Ogishima et al., (2003) demonstrated that the outer mitochondrial membrane cytochrome b5 (CYB5B) is also involved in steroid hormone metabolism in rats and guinea pigs. The potential role of CYB5B in ANp synthesis in pigs has not yet been investigated.

CYB5 has many roles such as: (a) transfer of electrons from NADH to desaturase

22 (Ozols, 1976), (b) NADH-dependent reduction of methemoglobin to regenerate hemoglobin (Abe and Sugita, 1979), and (c) stimulation of cytochrome P450 dependent oxygenation (Ogishima et al., 2003). The exact mechanism of CYB5 is not yet completely understood; however, POR is the primary electron donor because the ferric state of P450 is a lower redox potential than CYB5 (Ogishima et al., 2003). Experiments with apo-CYB5, which lacks the heme moiety, and holo-CYB5 suggest that CYB5 is not responsible for direct electron transfer but exerts a saturable, allosteric effect on the

CYP17A1-POR complex (Yamazaki et al., 1998). The POR functions by catalyzing electron transfer from NADPH to cytochrome P450 during catalysis (Lu and West, 1978) and is also involved in electron transfer from NADPH to heme oxygenase (Yoshida and

Kikuchi, 1978) and CYB5 (Ilan et al., 1981).

NADH cytochrome b5 reductase (CYB5R3) works in a similar manner as shown below:

NADH + H+ + 2 ferricytochrome CYB5 -> NAD+ + 2 ferrocytochrome CY55

CYB5R3 is a flavoprotein that catalyzes the reduction of CYB5, using FAD as a prosthetic group. Two forms of CYB5R3 are known, a membrane bound form in somatic cells and a soluble form in erythrocytes (Tomatsu et al., 1989). The membrane-bound form consists of both the membrane binding domain and the catalytic domain, where as the soluble form only consists of the catalytic domain (Ozols et al., 1985; Yubisui et al.,

1987). The role of CYB5R3 on the 17a hydroxylase, CI7,20 lyase and andien-p synthase activity of CYP17A1 has previously been investigated; CYB5R3 is not necessary for the production of 170HP or DHEA, but CYB5R3 significantly increases andien-P synthase activity (Meadus et al., 1993).

23 Among the several systems developed to characterize enzymatic activity, transfection of expression constructs into intact mammalian cells provides the opportunity to study the activity of the expressed proteins in the native microsomal environment and to study various combinations of enzymes, redox partners and substrates used (Dufurt et al., 1999; Luu-The et al., 2005). In this report, we studied the effects of

CYB5A and CYB5B on the activities of CYP17A1 by transient transfection of human embryonic kidney (HEK-293FT) cells with expression constructs for CYP17A1, POR,

CYB5R3 and either CYB5A or CYB5B. With this system we have the ability to over- express the proteins of interest, and to vary the relative amounts of these proteins to produce a well defined and active system with minimal interference from endogenous proteins that would occur with hepatocytes or microsomal preparations.

Our objective was to determine how CYB5A and CYB5B modulate the three activities of porcine CYP17A1; 17a-hydroxylase, C17,20 lyase and andien-P synthase activity. We were especially interested in systems that would maintain the normal production of sex steroids (through the 17a-hydroxylase and CI7,20 lyase reactions) while decreasing the production of the 16-androstene steroids (through the andien-P synthase reaction), since this might lead to methods for controlling boar taint.

3.3 Materials and Methods

3.3.1 Construction of expression vectors for POR, CYP17A1, CYB5R3, CYB5A and CYB5B

The entire coding regions of porcine POR, CYP17A1, CYB5R3, CYB5A and CYB5B were amplified from porcine testis cDNA by PCR using platinum Pfx DNA polymerase

(Invitrogen) and primers listed in Table 1. The amplified segments were then cloned into pcDNA3.1/V5-His TOPO (Invitrogen) to produce expression vectors. Expression vectors

24 for V5-His tagged proteins were generated so that the expressed proteins could be

detected by Western blotting using anti-V5-HRP antibody; vectors expressing the

untagged protein were also generated to determine if the V5-His tag adversely affected

the activity of the protein. The PCR primers for porcine NADPH cytochrome P450

reductase (POR) were based on Genbank accession number L33893 and for porcine

CYP17A1 were based on accession number NM 214428. The primers for porcine

CYB5A and CYB5B were based accession number NM 001001770 and AY609739

respectively. The sequence of porcine CYB5R3 was assembled from pig ESTs retrieved

by BLAST searching the NCBI database using human CYB5R3 (accession number NM

000398) as a template. This sequence was used to design primers to amplify and clone

porcine CYB5R3. Figure 5 displays the nucleotide (A) and deduced amino acid (B)

sequence of porcine CYB5R3 compared to human CYB5R3. The identity of all clones

was confirmed by sequencing.

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.

Protein Forward Primer Reverse Primer Plasmid Name produced (5'-3') (5'-3') CYP17A1 gccatgtgggtgctcttggtttt cagggtggagtcaggaggta pcDNA3.1/CYP17Al CYP17A1-V5- gccatgtgggtgctcttggtttt ggaggtactcccctcagtgt pcDNA3.1/CYP17Al- His V5-His POR gccatgggggactccaacgtgg ggctggcagaagacagtgag pcDNA3.1/POR

POR-V5-His gccatgggggactccaacgtgg gctccacacgtccagggagt pcDNA3.1/POR-V5- His CYB5R3 gccatgggggcccagctgagca ccatcagaaggcgaagcag pcDNA3.1/CYB5R3

CYB5R3-V5- gccatgggggcccagctgagca gaaggcgaagcagcgctcct pcDNA3.1/CYB5R3- His V5-His CYB5A gccatggccgaacagtccgac tttcttccattggcttctgc pcDNA3.1/CYB5A

25 CYB5A-V5- gccatggccgaacagtccgac gtttccgatgtgtagaagt pcDNA3.1/CYB5A-V5- His His CYB5B gccatggcgactgtggaagcca tcaggaggatttgctctccg pcDNA3.1/CYB5B

CYB5B-V5- gccatggcgactgtggaagcca ggaggatttgctctccgccg pcDNA3.1/CYB5B-V5- His His

3.3.2 Transient expression in human embryonic kidney (HEK-293) cells

Human embryonic kidney cells (HEK-293FT; Invitrogen) were plated at 7xl05

cells per well in Falcon 6 well culture tissue plates (BD Biosciences) and grown at 37°C

in Dulbecco's modified eagle's medium (GIBCO) supplemented with 10% fetal calf

serum (PAA Laboratories, Etobicoke, ON), 1 % geneticin, penicillin/streptomycin and L-

glutamine, non-essential amino acids and sodium pyruvate. Once the cells were 90-95%

confluent, vectors expressing CYP17A1, POR, CYB5R3, and CYB5A or CYB5B were

transfected into HEK-293 cells using LipofectAMINE 2000 (Invitrogen) according to the

manufacturer's instructions. The amounts of the plasmids for expression of CYP17A1,

POR and CYB5R3 used in the transfections were adjusted to give approximately equal

levels of expression for each protein. Preliminary work was done varying transfection

levels of CYP17A1, POR and CYB5R3 until band densities from a western blot for these

three proteins were almost equal. Variable amounts of the expression plasmids for

CYB5A and CYB5B were used, with empty pcDNA3.1 vector added to bring the total

amount of DNA to 4 ug for each transfection.

3.3.3 Western analysis of protein expression Protein expression of HEK-293 cells transfected with pcDNA3.1/CYP17Al-V5-

His, pcDNA3.1/POR-V5-His, pcDNA3.1/CYB5R3-V5-His and pcDNA3.1/CYB5A-V5-

26 His or pcDNA3.2/CYB5B-V5-His was analyzed by western blotting. The cells were harvested and lysed by sonication (three times for 30s) in 300uL RIPA lysis solution (1%

Nonidet P-40, 0.1% 20% SDS and 0.5% sodium deoxycholate) including Complete protease inhibitor cocktail tablets (Roche). Samples containing 20|ng of protein were separated by electrophoresis on sodium dodecyl sulfate-16% polyacrylamide gels and transferred by semi-dry transblot to PVDF membranes. The blots were incubated overnight in 5% (w/v) dried skim milk in PBS with 0.1% Tween 20 and then incubated with a 1:5000 dilution of anti-V5-HRP antibody (Invitrogen) in PBS containing 0.1%

Tween 20 and 5% skim milk powder. Antibody antigen complexes were identified by incubating with 50uL of 68 mM p-coumaric acid (Sigma) in DMSO followed by 15uL of

3% hydrogen peroxide added into 5mL of 1.25 mM luminal (Sigma) in 0.1M Tris pH 8.5.

After 1 min the blot was exposed to film (Kodak) for 5-10 min and developed. The density of the bands on the Western blots were analysed by Northern Eclipse software

(Empix Imaging).

3.3.4 Assay of enzymatic activity

The metabolism of pregnenolone was measured in HEK-293 cells transiently transfected with vectors expressing CYP17A1, POR, CYB5R3, and CYB5A or CYB5B.

At 48 hours after transfection, [7-3H(N)]-pregnenolone (lOuM, specific activity = 33 uCi umor1) was added in fresh media to the 6 well culture plates. After incubation for 16 hours, the media was collected and the cells were harvested. Cell debris was separated at

15,000 x g for 15 min. The media and cell debris were extracted twice with 4 mL ether and the organic phases were pooled and evaporated to dryness under nitrogen. The extracts were dissolved in 85% acetonitrile:15% H20 and the radioactive steroids were

27 separated by HPLC on a Luna 5|u, 250 x 4.60 mm reverse phase C-18 column

(Phenomenex, Torrance, CA). The equipment consisted of a Spectra-Physics model

SP8880 autosampler, a Spectra Physics model SP8800 Ternary HPLC Pump (Spectra-

Physics, San Jose, CA) and a P-Ram model 2 radioactivity detector (IN/US Systems,

Tampa FL). The 16-androstene steroid product (ANP) was separated from the pregnenolone substrate and other products (170HP and DHEA) using a mobile phase of

85% acetonitrile delivered at 1 ml/min (Sinclair et al., 1995). DHEA and 170HP were separated using a 50% acetonitrile mobile phase (Bonneau et al., 1992). Substrates and metabolites were identified by comparison with the retention time of reference steroids

(Sigma).

3.3.5 Statistical analysis

All statistical analyses were made using SAS/STAT version 9.1 (SAS Institute

Inc). Treatments were compared using the general linear model procedure and a t-test was used to compare different means. A Dunnets test was used to compare transfection levels of CYB5A/CYB5B to control at a confidence level of 95%. The Pearson correlation procedure was used to check for positive or negative correlations between the amount of CYB5A or CYB5B used in the transfections and production of ANp, 170HP and DHEA.

3.4 Results

3.4.1 Cloning of porcine CYB5R3 and optimization of the expression system

The nucleotide and deduced amino acid sequence of porcine CYB5R3 compared with human CYB5R3 is shown in Fig 5. The homology between porcine and human was

90% and 93% at the nucleotide and amino acid level respectively. We first optimized the

28 HEK-293 system to obtain similar levels of protein expression and activity and to determine the optimum time for protein expression following transfection. Fig 6A displays a western blot of cells expressing V5-tagged CYP17A1, POR, CYB5R3,

CYB5A and CYB5B at 24, 48, 72 and 96 hours after transfection. The density of the bands on the Western blots were analysed by Northern Eclipse software (Empix Imaging) and the percent change in band densities are plotted in Fig 6B. The expression of the different proteins varied over time. The expressions of CYP17A1 and CYB5A were maximal at 48 hrs and then declined thereafter, while the expressions of POR, CYB5B and CYB5R3 continued to increase up to 96 hours. The 48 hr time point was subsequently chosen as the optimal time for expression and assay of enzyme activity.

29 human 61 AGTCTGCTCATGAAGCTGTTCCAGCGCTCCACGCCAGCCATCACCCTCGAGAGCCCGGAC 1 AG CTGCTCATGftAGCTGTTCCAGCG XCCAG CC GCCATCACCCTCGAGA CCC GAC Pig 61 AGCCTGCTCATGAAGCTGTTCCAGCGTTCCACCCCGGCCATCACCCTCGAGAACCCAGAC 120 human 121 ATCAAGTACCCGCTGCGGCTCATCGACCGGGAGATCATCAGCCATGACACCCGGCGCTTC 180 ATCAAGTA CCQCTG GGCTCAT OAC GGAG TC TCA CCATGACACCCGGCG TTC Pig 121 ATCAAGTATCCGCTGAGGCTCATTGACAAGGAGGTCGTCAACCATGACACCCGGCGGTTC 180 human 181 CGCTTTGCCCTGCCGTCACCCCAGCACATCCTGGGCCTCCCTGTCGGCCAGCACATCTAC 240 CGCTTTGCCCT6CCGTC CCCCAGCACATCCfGGGGCTeCCTGI1 GGCCAGCACATCTAC Pig 181 CGCTTTGCCCTGCCGTCGCCCCAGCACATCCTGGGCCTCCCTGTGGGCCAGCACATCTAC 240 human 241 CTCTCGGCTCGAATTGATGGAAACCTGGTCGTCCGGCCCTATACACCCATCTCCAGCGAT 3 00 CTCfCGGCfCG ATIGATGG AA CTGGTC f CGGCCCTA AC CCC TCTCCAG GAT Pig 241 CTCTCGGCTCGGATTGATGGGAATCTGGTCATTCGGCCCTACACGCCCGTCTCCAGTGAT 3 00 human 301 GATGACAAGGGCTTCGTGGACCTGGTCATCAAGGTTTACTTCAAGGACACCCATCCCAAG 3 60 GA GACMGGGCTI G^GSACGTGGTCATCAAGGT TACTTGAA GACACCCA CCCAAG Pig 301 GACGACAAGGGCTTTGTGGACCTGGTCATCAAGGTGTACTTCAAAGACACCCACCCCAAG 3 60 human 361 TTTCCCGCTGGAGGGAAGATGTCTCAGTACCTGGAGAGCATGCAGATTGGAGACACCATT 420 T.TTCCCGC GGAGGGAAGATGIC CAGTACCfGGAGAGCATG AGA? GGAGACACCAT Pig 3 61 TTTCCCGCCGGAGGGAAGATGTCCCAGTACCTGGAGAGCATGAAGATCGGAGACACCATC 42 0 human 421 GAGTTCCGGGGCCCCAGTGGGCTGCTGGTCTACCAGGGCAAAGGGAAGTTCGCCATCCGA 4 80 GAGITCCGGGGGCCCA GGGCfGcyGGfCfAC0A6GGCAAAGG AAG15 GCCATCCG Pig 421 GAGTTCCGGGGCCCCAACGGGCTGCTGGTCTACCAGGGCAAAGGAAAGTTTGCCATCCGC 4 80 human 481 CCTGACAAAAAGTCCAACCCTATCATCAGGACAGTGAAGTCTGTGGGCATGATCGCGGGA 540 CG GACAA AA TCCA CC tCATCA GAC GTGAAGTC$<3f GQCAfGATCGCGGGA Pig 481 CCGGACAAGAAATCCAGTCCCGTCATCAAGACGGTGAAGTCTGTTGGCATGATCGCGGGA 540 human 541 GGGACAGGCATCACCCCGATGCTGCAGGTGATCCGCGCCATCATGAAGGACCCTGATGAC 600 GG AC GGCATCACGCC ftlGCTSCAGGTGATCCG GCCATGATGA&GGACCC GATGAC Pig 541 GGAACCGGCATCACCCCAATGCTGCAGGTGATCCGAGCCATCATGAAGGACCCGGATGAC 600 human 601 CACACTGTGTGCCACCTGCTCTTTGCCAACCAGACCGAGAAGGACATCCTGCTGCGACCT 660 CACAC GTGTGCCACCTGCTCTTTGCCAACCAGACCGAGAAGGACATCCTGCTGCG CC Pig 601 CACACCGTGTGCCACCTGCTCTTTGCCAACCAGACCGAGAAGGACATCCTGCTGCGGCCC 660 Human 661 GAGCTGGAGGAACTCAGGAACAAACATTCTGCACGCTTCAAGCTCTGGTACACGCTGGAC 720 GAGCTGGAGGAACT AGGAA AACATTCTGC CGCTTCAAGCTCTGGTACACG TGGAC Pig 661 GAGCTGGAGGAACTGAGGAATGAACATTCTGCGCGCTTCAAGCTCTGGTACACGGTGGAC 720 human 721 AGAGCCCCTGAAGCCTGGGACTACGGCCAGGGCTTCGTGAATGAGGAGATGATCCGGGAC 7 80 AGA6CCCC GA&GCGtSGf&GI'&C GCCAGGGCTtSGfGAA GAGGAGA1GATCCGGGAC Pig 721 AGAGCCCCAGAAGCCTGGGACTACAGCCAGGGCTTCGTGAACGAGGAGATGATCCGGGAC 7 80 human 7 81 CACCTTCCACCCCCAGAGGAGGAGCCGCTGGTGCTGATGTGTGGCCCCCCACCCATGATC 840 CACCTTCC CCGCC GAGGASGAGCCGqfGSfGCSGATQfG §G CCCCC CCCATGATC Pig 781 CACCTTCCGCCCCCGGAGGAGGAGCCGCTGGTGCTGATGTGCGGGCCCCCGCCCATGATC 840 human 841 CAGTACGCCTGCCTTCCCAACCTGGACCACGTGGGCCACCCCACGGAGCGCTGCTTCGTC 9 00 CAGTACGCCTGCCI! CCCAACC1GQA C CGtfGGGCCAGCCCA GGAGCGCTGCTTCG C Pig 8 41 CAGTACGCCTGCCTGCCCAACCTGGAGCGCGTGGGCCACCCCAAGGAGCGCTGCTTCGCC 900 human 901 TTCTGA 906 JTjCTGA Pig 901 TTCTGA 906

30 B

human 1 MGAQLSTLGHMVLFPVWFLYSLLMKLFQRSTPAITLESPDIKYPLRLIDREIISHDTRRF 60 , MGAQJ,STLGH+Vt pyWF&YSl,W,ig

human 61 RFALPSPQHILGLPVGQHIYLSARIDGNLVVRPYTPISSDDDKGFVDLVIKVYFKDTHPK 120 RFAlPSPQaiLS&PVQQHin.SMEDQWV+RfVTP+j3SDPDIt6FVDI.VIKVyFKDTBPK Pig 61 RFALPSPQHILGLPVGQHIYLSARIDGNLVIRPYTPVSSDDDKGFVDLVIKVYFKDTHPK 120

human 121 FPAGGKMSQYLESMQIGDTIEFRGPSGLLVYQGKGKFAIRPDKKSNPIIRTVKSVGMIAG 180 FPAGS3KMaQyiB$M+I6DTJJBIWP+GiaV¥068QKffAIRPPI

Pig 121 FPAGGKMSQYLESMKIGDTIEFRGPNGLLVYQGKGKFAIRPDKKSSPVIKTVKSVGMIAG 180

human 181 GTGITPMLQVIRAIMKDPDDHTVCHLLFANQTEKDILLRPELEELRNKHSARFKLWYTLD 240

Pig 181 GTGITPMLQVIRAIMKDPDDHTVCHLLFANQTEKDILLRPELEELRNEHSARFKLWYTVD 240

human 2 41 RAPEAWDYGQGFVNEEMIRDHLPPPEEEPLVLMCGPPPMIQYACLPNLDHVGHPTERCFV 3 00 , RAPE&WDX QGFVNE;^MJR0HfcPPMBiE;PitVM?:GP.S P,Mt2XACLPNI+ VQHP ERCF Pig 2 41 RAPEAWDYSQGFVNEEMIRDHLPPPEEEPLVLMCGPPPMIQYACLPNLERVGHPKERCFA 300 human 301 F 301 F Pig 301 F 301

Figure 5: Nucleotide (A) and deduced amino acid (B) sequence of porcine CYB5R3 compared to human CYB5R3. Nucleotide "61" indicates transcriptional start, also indicated by Methionine (M) in the amino acid secuence.

31 a)

POR-78 kDa CYP17A1-59 kDa CYB5R3-33 kDa

CYB5B-16 kDa CYB5A-15 kDa

24 48 72 96

120

100

•POR -CYP17A1 •CYB5R3 -CYB5A •CYB5B

48 72 96 Time (Hours) Figure 6: a) Western blot showing the expression of CYP17A1, POR, CYB5R3, CYB5A and CYB5B at 24, 48, 72 and 96 hours after transfection. Proteins were separated on 16% SDS-PAGE, blotted and anti-V5 HRP antibody was used for detection. 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.

32 The production of the pregnenolone metabolites, AN(3, and DHEA + 170HP was

determined next over time (Fig 7). HEK-293 cells were transfected with expression

vectors for CYP17A1, POR, CYB5R3 and CYB5A and 48 hours after transfection, [7-

3H(N)]-pregnenolone substrate was added in fresh media. Cells and media were collected

after 2, 4, 8, 16 and 24 hrs of incubation with substrate and the metabolites were

extracted and analysed by HPLC with a mobile phase of 85% acetonitrile:15% H2O. The

production of AN0 and DHEA + 170HP was linear up to 16 hrs. The activity of the

proteins in the expression system was similar for both the untagged and V5-His tagged

proteins (data not shown).

100

-170HP+DHEA -Pregnenolone -ANB

Figure 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). At 48 hrs after transfection cells were incubated with 3H-pregnenolone substrate and products were analyzed by HPLC with a mobile phase of 85% acetonitrile.

33 3.4.2 Effect ofCYB5 isoforms on CI 7a-hydroxylase/Cl 7,20 lyase activities versus andien-fi synthase activity

Transfections of HEK cells were performed with 0.25 ugpcDNA3.1/CYP17Al,

0.35 ugpcDNA3.1/ POR, 0.25 ugpcDNA3.1/CYB5R3 and from 0 to 3ug pcDNA3.1/CYB5A or pcDNA3.1/CYB5B, with empty pcDNA 3.1 vector added to maintain a consistent level of 4ug of plasmid DNA in each transfection. The transfected cells were incubated with 3H-pregnenolone and the products of the combined 17a- hydroxylase/C 17,20 lyase reactions (170HP + DHEA) and andien-p synthase (ANp) reactions were analyzed via HPLC with a mobile phase of 85% acetonitrile. Increasing the expression of CYB5A (Fig 8) resulted in a significant decrease in the combinedl7a- hydroxylase/C 17,20 lyase activities (PO.013) from 97% of total products without

CYB5A, to 69% of total products formed with 3 ug of CYB5A transfected. However, increasing the expression of CYB5B had no significant effect on combined 17a- hydroxylase/C 17,20 lyase activities (Fig 9).

There was a significant positive correlation (r =0.503) between the amounts of

CYB5A plasmid transfected and ANp production (pO.OOOl), to a maximum of 31% of total products at 3 ug of CYB5A plasmid transfected (Fig 8). However, increased expression of CYB5B did not affect the production of ANp, with maximal production of

ANP not exceeding 10% of overall products formed (Fig 9).

3.4.3 Effects of CYB5 isoforms on 17a-hydroxylase versus CI 7,20 lyase activity

The effect of the CYB5 isoforms on 17a-hydroxylase versus CI 7,20 lyase activities was analyzed by separating 170HP and DHEA production for each individual transfection by HPLC using a mobile phase of 50% acetonitrile. The production of

34 170HP was negatively correlated (r = -0.779) with increasing amounts of CYB5A

(pO.OOOl, Fig 10). The production of DHEA increased at the 0.2 and 0.4 ug levels of

CYB5A transfection and then went back to base level at higher transfection levels of

CYB5A. The production of 170HP was also negatively correlated (r = -0.564) with increasing CYB5B transfection levels (p<0.004, Fig 11) while DHEA production was significantly positively correlated (r = 0.565) with increasing expression of CYB5B

(p<0.004).

35 100

90 £

^>80

- 70

U 60 3 •o 2 50 Q. V .•H 40 O "S 30 4-1 ¥ V 2 20 -170HP+DHEA -ANB 10

0 0.5 1 1.5 2.5 CYB5A(ug)

Figure 8: The effect of increased expression of CYB5A on the production of DHEA + 170HP and ANp\ CYP17A1, POR, CYB5R3 and CYB5A were transfected into HEK- 293 cells and products were analyzed by HPLC with a mobile phase of 85% acetonitrile. 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)

36 80

50 -•-170HP+DHEA 40 -*-ANB

0.5 1 1.5 2.5 CYB5B(ug)

Figure 9: The effect on increased expression of CYB5B on the production of DHEA + 170HP and ANp\ CYP17A1, POR, CYB5R3 and CYB5B were transfected into HEK- 293 cells and products were analyzed by HPLC with a mobile phase of 85% acetonitrile. 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 (CYB5B=0ug).

37 100

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 CYB5A (ug)

Figure 10: The effect of increased expression of CYB5A on the production of 170HP and DHEA. CYP17A1, POR, CYB5R3 and CYB5A were transfected into HEK-293 cells and products were analyzed by HPLC with a mobile phase of 50% acetonitrile. 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).

38 100 - 90

80 # - 70 - N. * ! 60 3 - -•-170HP 50 -B-DHFA I - ^A-ANB 40 * - I.2 30

Figure 11: The effect of increased expression of CYB5B on the production of 170HP and DHEA. CYP17A1, POR, CYB5R3 and CYB5B were transfected into HEK-293 cells and products were analyzed by HPLC with a mobile phase of 50% acetonitrile. 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 (CYB5B=0ug).

39 3.5 Discussion

The primary purpose of this study was to investigate the effect of CYB5A versus

CYB5B on the activity of porcine CYP17A1 in catalyzing the synthesis of ANp\ This is the initial step in the synthesis of 16-androstene steroids, which are a major component of boar taint in entire male pigs. A more complete understanding of this process may lead to targets for selective breeding or other methods for decreasing the production of 16- androstene steroids and thereby reduce boar taint.

It has been demonstrated previously that CYB5A stimulates the synthesis of AN0 by porcine CYP17A1, as well as decreasing 170HP production and increasing DHEA production (Meadus et al., 1993; Nakajin et al., 1985). CYB5B has been shown to stimulate the 17a-hydroxylase and CI7,20 lyase reactions of CYP17 from rat and guinea pig (Ogishima et al., 2003), but the potential role of CYB5B on synthesis of AN0 in pigs has not previously been investigated. We postulated that CYB5B may have the potential to stimulate production of 170HP and DHEA by porcine CYP17A1 at a level similar to

CYB5A, while significantly reducing the production of 16-androstene steroids. Thus, increasing the expression of CYB5B and decreasing the expression of CYB5A would maintain the production of androgens, while decreasing the production of 16-androstene steroids. This would allow entire male pigs to be produced that have the improved lean growth and feed efficiency due to the anabolic effects of testicular steroids, but do not have boar taint.

In this experiment, we investigated the interactions between CYP17A1, POR,

CYB5R3 and CYB5A/CYB5B by over-expressing various amounts of these proteins in

HEK-293 cells. CYP17A1 catalyzes 2 types of reactions; hydroxylation and the cleavage

40 process also referred to as the fragmentation path by Lee-Robichaud et al (Lee-

Robichaud et al., 1995). These authors demonstrated that when porcine CYP17A1 is challenged with a substrate analogue in which the C-20 position contains a highly electrophilic aldehyde group, the iron peroxide is trapped to alter the entire course of enzymatic reactions towards the exclusive formation of 16-androstene steroids (Lee-

Robichaud et al., 1995).

CYP17A1 catalyzes the production of 170HP and DHEA from pregnenolone via the 17a-hydroxylase and CI7,20 lyase reactions (Nakajin and Hall, 1981), as well as the production of AN0 by the andien-(3 synthase pathway. Consistent with our results, it has been previously observed in porcine microsomes that the production of ANP required the presence of CYB5A (Lee-Robichaud et al., 1995; Meadus et al., 1993; Nakajin et al.,

1985). Our results show that the increased expression of CYB5A is negatively correlated with the production of 170HP, while concurrently positively correlated with the production of AN(3 . CYB5A thus switches the activity of CYP17A1 from 17a- hydroxylation of pregnenolone to removal of the carbon side chain and formation of the delta 16 double bond.

The potential role of the outer mitochondrial CYB5B in 16-androstene steroid production has not been previously investigated; however, the ability of CYB5B to modulate 17a-hydroxylase and CI 7,20 lyase activity has been investigated in humans

(Soucy and Luu-The, 2002). Our present results show that porcine CYB5A and CYB5B increase the production of DHEA by CYP17A1, while decreasing the production of

170HP. However, CYB5B had no effect on the production of ANp by CYP17A1. Thus,

CYB5A stimulates both the CI7,20 lyase and andien-P synthase pathways, while CYB5B

41 only stimulates the CI7,20 lyase pathway and has no effect on the andien-P synthase pathway.

The role of CYB5A as a primary electron donor was investigated with

"humanized" yeast strains and it was observed that when the amount of POR was low, maximal stimulation of C17,20 lyase activity occurred at a CYB5:CYP17A1 ratio of 3:1, with higher ratios inhibitory (Auchus et al., 1998). These results were consistent with our observations of an inhibitory effect on CI 7,20 lyase activity at higher ratios of CYB5A but not CYB5B. This suggests that CYB5 may not function as an electron donor, but perhaps facilitates the transfer of electrons from POR to CYP17A1. Our results also suggest that CYB5A and CYB5B work by different mechanisms and likely interact differently with POR and CYP17A1. The stimulatory effect of CYB5A on androgen and

16-androstene steroid production was previously investigated and it was hypothesized that a) CYB5 is involved in the second electron transfer, since participation in the first electron transfer is unlikely because the redox potential of the ferric state of POR is lower than that of the ferrous state of CYB5 or b) POR undergoes conformational changes through complex formation between POR and CYB5 (Ogishima et al., 2003).

We have shown that 170HP production was negatively correlated with increasing expression of CYB5A and CYB5B, while DHEA production was positively correlated with expression of CYB5B. The decrease in 170HP production and the concurrent increase in DHEA production suggests that 170HP may be a substrate for the CI7,20 lyase activity. Previous studies by Soucy and Van Luu-The (2000) also suggest that the reactions catalyzed by human CYP17A1 follow 2 distinct steps; with a dissociation of the

42 intermediate from the active site, the increase in DHEA would not necessarily correspond with a decreased accumulation of 170HP.

The homology of the amino acid sequences between porcine CYB5A and CYB5B is 48%, with CYB5A and CYB5B consisting of 134 and 144 amino acids respectively.

Perhaps the lack of an increase in ANP production at higher expression of CYB5B can be explained by conformational differences between CYB5 isoforms A and B. Lee-

Robichaud et al. (1997) concluded in a study of human CYP17A1 that the interaction between CYP17A1 and CYB5 is governed by the presence of a membrane insertable hydrophobic region on CYB5, but also by the defined spatial orientation of the exposed globular domain at the C-terminus. Residues 1 -92 are folded to create a compact globular domain, whereas the C-terminal sequence residues from 93-onwards form a hydrophobic tail, which protrudes through the membrane bilayer (Lee-Robichaud et al.,

1997). Perhaps the extra 10 amino acids on CYB5B change the interaction of the globular domain with CYP17A1. This conformational difference may allostericaly alter the conformational change of POR once bound to CYP17Aland favour the CI7,20 lyase activity over the andien-p synthase pathway, reducing the production of ANP . This may be related to conformational changes that allow the 17a-hydroxylase intermediate to remain bound to the active site of CYP17A1 more than when CYB5A is present, thus increasing DHEA synthesis.

In summary, we have found that CYB5B and CYB5A interact with porcine

CYP17A1 to produce increased amounts of 170HP and DHEA, but CYB5B does not stimulate the production of ANp. Increased expression of CYB5A first increases and then decreases the production of DHEA, but this was not seen with CYB5B. It is likely that

43 the mechanisms by which CYB5A and CYB5B interact with the CY17A1-P0R complex are different. These results point to the different isoforms of CYB5 as selective targets for producing entire male pigs with a normal production of sex steroids and decreased production of the 16-androstene steroids that contribute to boar taint.

44 Chapter 4 4 The effect of phosphorylation of cytochrome P45017A1 on 17a- hydroxylase, CI7,20 lyase and the andien-p synthase activities

4.1 Abstract

In this study, we investigated the effect of phosphorylation status of porcine

CYP17A1 on 17a-hydroxylase activity, CI7,20 lyase activity and the andien-|3 synthase pathway to produce 16-androstene steroids. HEK-293 cells that were over expressing

CYP17A1, cytochrome P450 reductase (POR), cytochrome b5 reductase (CYB5R3) and cytochrome b5A (CYB5A) or cytochrome b5B (CYB5B) were used. Cells were treated with either the phosphatase inhibitor, okadiac acid (OA) or transfected with an expression vector for protein phosphatase 2A (PP2A). In cells over expressing CYB5A, treatment with OA increased CI7,20 lyase and andien-P synthase activity and decreased 17a- hydroxylase activity, while treatment with PP2A increased 17a-hydroxylase and decreased CI7,20 lyase activity as well as andien-P synthase activities. In cells overexpressing CYB5B, there was no effect of OA or PP2A treatment on CI 7, 20 lyase,

17a-hydroxylase or andien-p synthase activities. Using site directed mutagenesis to change wild type CYP17A1-Ser106 to CYP17A1-Ala106 resulted in an increase in 17a- hydroxylase activity and decrease in CI7,20 lyase activities in the presence of CYB5A.

We conclude that phosphorylation of porcine CYP17A1 at Ser106 increases C17,20 lyase activity and decreases 17a-hydroxylase and andien-P synthase activities stimulated by

CYB5A.

Keywords: CYP17A1, phosphorylation, CYB5, 17a-hydroxylase, C17,20 lyase

45 4.2 Introduction

Intact male pigs have beneficial production characteristics, since higher levels of androgens produced by intact male pigs result in improved feed conversion and lean meat production. Boar taint, caused in part by high concentrations of the testicular 16- androstene steroid 5a-androst-16 en-3-one (5ot-androstenone) in backfat, is the key factor that hinders the use of intact male pigs for pork production. Cytochrome P450 CI 7

(CYP17A1) catalyses the key regulatory step in the formation of the 16-androstene steroids by the andien-P synthase reaction, as well as the synthesis of the glucocorticoid and sex steroids via the 17a-hydroxylase and CI7,20 lyase pathways respectively with pregnenolone as the substrate (Pandey et al., 2003). Other components of this system include NADPH cytochrome P450 reductase (POR), NADH cytochrome b5 reductase

(CYB5R3) and cytochrome b5 (CYB5).

Human CYP17A1 first catalyzes the synthesis of 17a-hydroxypregnenolone

(170HP) and the CI7, 20 lyase activity subsequently converts 170HP to dehydroepiandrosterone (DHEA) (Pandey et al., 2003). Three factors have been shown to manipulate the ratio of 17a-hydroxylase activity to CI7, 20 lyase activity at the post translation level. Higher molar ratios of POR:CYP17Al favours the C17,20 lyase activity and DHEA production (Auchus et al., 1998; Lin et al., 1993; Nakajin and Hall, 1981).

Secondly, it has been shown that CYB5 acts allosterically to promote the interaction between CYP17A1 and POR to stimulate the CI7,20 lyase reaction, although it is likely that CYB5 is not an electron donor (Auchus et al., 1998; Geller et al., 1999; Nakajin and

Hall, 1981). Thirdly, it has been shown that phosphorylation of human CYP17A1 on

46 serine and threonine but not tyrosine residues increases CI7,20 lyase activity (Zhang L et al., 1995).

The majority of studies on the effect of phosphorylation status of CYP17A1 have used protein phosphatase 2 A (PP2A), one of the four major protein phosphatases in the cytosol of mammalian cells, to dephosphorylate serine and threonine residues.

Conversely, okadiac acid, a polyether fatty acid isolated from the marine sponge

Halichondria okadaii (Cohen et al., 1990), is used as a PP2A inhibitor to increase the level of phosphorylation. It has been shown that the PP2A catalytic subunit is completely inhibited by InM okadiac acid (Haystead et al., 1989). Pandey et al. (2003) treated human adrenal NCI-H295A cells with PP2A inhibitors and found that serine and threonine phosphorylation of CYP17A1 increases CI7,20 lyase activity and dephosphorylation of CYP17A1 decreases C17,20 lyase activity. In a more recent study

Pandey et al (2005), also concluded that serine phosphorylation of CYP17A1 increases

CI7, 20 lyase but had no effect of 17a-hydroxylase activity. The effect of phosphorylation status of CYP17A1 in pigs on the production of sex steroids and 16- androstene steroids has not yet been investigated.

Pro106 mutant and both were expressed in COS-1 cells. The Pro mutant did not produce

170HP or DHEA, demonstrating that this mutation diminished both 17a-hydroxylase and C17, 20 lyase activity (Lin et al., 1991). Lin et al, (1993) subsequently investigated this further on CYP17A1 mutations with threonine or alanine at residue 106 and found

47 that these mutants retained only 20-30% of 17a-hydroxylase and CI7,20 lyase activities.

From these experiments, it is clear that the amino acid occupying position 106 greatly affects enzymatic activity.

In this report, we studied the effects of phosphorylation status of porcine CYP17A1 as well as the involvement of CYB5 isoforms on 17a-hydroxylase, CI7,20 lyase and andien-p pathways. Human embryonic kidney (HEK-293) cells were transfected with expression constructs for CYP17A1, POR, CYB5R3 and either CYB5A or CYB5B and

OA or PP2A treatments were then applied to the system. We also tested an expression construct containing a mutant of porcine CYP17A1 with alanine instead of serine at residue 106 in this system. Our objectives were to investigate whether the phosphorylation status of CYP17A1 influences the allosteric actions of CYB5A and

CYB5B on the 17a-hydroxylase, C17,20 lyase and andien-P synthase activities and to identify the potential site of phosphorylation of CYP17A1.

4.3 Materials andMetholds

4.3.1 Construction of expression vectors

The expression vectors for porcine POR, CYP17A1, CYB5R3, CYB5A and

CYB5B were constructed as described previously in chapter 3 (Billen and Squires, 2008).

Briefly, the entire coding regions were amplified from porcine testis cDNA by PCR and the amplified segments were cloned into pcDNA3.1/V5-His TOPO (Invitrogen) to produce expression vectors. Expression vectors for V5-His tagged proteins were generated so that the expressed proteins could be detected by Western blotting using anti-

V5-HRP antibody; vectors expressing the untagged protein were previously used to demonstrate that the V5-His tag did not adversely affect the activity of the expressed

48 proteins (Billen and Squires, 2008). The expression vector pcDNA3.1/PP2A-V5-His which expresses the catalytic subunit of human protein phosphatase 2A was kindly provided by Dr. Mashiko Negishi, National Institute of Environmental Health Sciences,

USA.

In order to create a mutant of CYP17A1 with alanine instead of serine at residue

106, the Quikchange site directed mutagenesis kit (Stratagene) was used to produce

CYP17A1-Ala106. pcDNA3.1/CYP17Al-Ser106-V5-His was used as a template and primers 5'-3' actctagacatcctggcagacaaccaaaaggggand ccccttttggttgtctgccaggatgtctagagt were used. The template DNA was digested with Dpn I, which cleaves only Dam methylated DNA, leaving only the mutated strand for cloning with pcDNA3.1 to create pcDNA3.1 /CYP17-Ala106-V5-His. The identity of all clones was confirmed by sequencing.

4.3.2 Transient expression in human embryonic kidney (HEK-293) cells.

Human embryonic kidney (HEK-293) cells were plated at 7xl05 cells per well in

Falcon 6 well culture tissue plates (BD Biosciences) and grown in Dulbecco's Modified

Eagle's Medium (GIBCO) supplemented with 10% fetal calf serum (PAA Laboratories,

Etobicoke, ON) at 37°C. Once cells were 90-95% confluent, vectors expressing

CYP17A1, POR, CYB5R3, CYB5A and CYB5B, and in some experiments PP2A, were transfected into HEK-293 cells using LipofectAMiNE 2000 (Invitrogen) according to the manufacturer's instructions. The amounts of the plasmids for expression of CYP17A1,

POR and CYB5R3 used in the transfections were adjusted to give an approximately equal amount of expression of each of these proteins as described previously (Billen and

Squires, 2008). Variable amounts of the expression plasmids for CYB5A and CYB5B

49 were used, Oug, 0.2ug, 0.8ug and 1.5ug as the maximum amount of DNA transfected, results seen in chapter 3 indicate that a ration of CYPB5:CYP17A1 any higher results in an inhibitory effect. Cells were transfected with empty pcDNA3.1 vector added to bring the total amount of DNA to 4ug for each transfection.

4.3.3 Assay of enzym atic activity

The metabolism of pregnenolone was measured in intact cells transiently transfected and treated accordingly:

a) Increased phosphorylation of CYP17A1 via PP2A inhibition Okadiac acid:

pcDNA3.1/CYP17Al-V5-His, pcDNA3.1/ POR-V5-His, pcDNA3.1/CYB5R3-

V5-His, and pcDNA3.1/CYB5A-V5-His or pcDNA3.1/CYB5B-V5-His and

treated with lOnM OA or lOnM Norokadone control at 21 hours prior to the

addition of pregnenolone substrate.

b) Dephosphorylation of CYP17A1 with PP2A: pcDNA3.1/CYP17Al-V5-His.

pcDNA3.1/ POR-V5-His, pcDNA3.1/CYB5R3-V5-His, pcDNA3.1/PP2A-V5-His

and pcDNA3.1/CYB5A-V5-His or pcDNA3.1/CYB5B-V5-His

c) Site of phosphorylation on CYP17A1: pcDNA3.1/CYP17Al-V5-His or

pcDNA3.1/CYP17Al-Ala106-V5-His, pcDNA3.1/ POR-V5-His,

pcDNA3.1/CYB5R3-V5-His and pcDNA3.1/CYB5A-V5-His or

pcDNA3.1/CYB5B-V5-His.

For all treatments, at 48 hours post transfection [7-3H(N)]-pregnenolone (lOuM, specific activity = 33 uCi umol"1) was added with fresh media to the 6 well culture plates. After incubation for 16 hours, the media was collected and the cells were harvested (Billen and

Squires, 2008). Cell debris was separated at 15,000 x g for 15 min. The media and cells

50 were extracted twice with 4 mL ether and the organic phases were pooled and evaporated to dryness. The extracts were dissolved in 85% acetonitrile and the radioactive steroids were quantified by reverse phase HPLC as described previously (Billen and Squires,

2008). The 16-androstene steroid product (5,16 androstadien-3p-ol, AN0) was separated from the pregnenolone substrate and other products (170HP and DHEA) using a mobile phase of 85% acetonitrile delivered at 1 ml/min. DHEA and 170HP were then separated using a 50% acetonitrile mobile phase.

4.3.4 Statistical analysis

All statistical analyses were made by the general linear model procedures and mixed model procedures of SAS/STAT version 9.1 (SAS Institute Inc). The t-test was used to compare different means. A Dunnets test was used to compare transfection levels of CYB5A/CYB5B to control at a confidence level of 95%. The Pearson correlation procedure was used to check for positive or negative correlations between the amount of

CYB5A or CYB5B transfected and the production of ANp, 170HP and DHEA after OA or PP2A treatments. Levene tests were used to compare variances among treatments, B5 types and transfection levels.

4.4 Results

4.4.1 Effect ofCYB5 isoform and treatment with OA on 17a-hydroxylase, CI 7,20 lyase and andien-p synthase activities.

Transfections of HEK cells were performed with the following expression vectors:

0.25ug of CYP17A1, 0.35ug of POR, 0.25ug of CYB5R3, and 0 - 1.5ug of CYB5A or

CYB5B and empty pcDNA 3.1 vector to maintain a consistent level of 4ug of plasmid

DNA in each transfection. The transfected cells were treated with lOnM OA, 21 hours

51 prior to being incubated with 3H-pregnenolone (lOuM, specific activity = 33 uCi umol"1) and the products of the 17a-hydroxylase/C 17,20 lyase (170HP + DHEA) and andien-|3 synthase (ANp) reactions were analyzed via HPLC. Controls were transfected identically, however were treated with inactive OA analog Norokadone control 21 hours prior to being incubated with 3H-pregnenolone (10uM, specific activity = 33 uQ amor1).

Norokadone was used as a control to remove any effects which may have been seen due to cytotoxicity when compared to non treated controls. Figure 12a displays the effect of

OA treatment and CYB5A expression on andien-f} activity. OA treatment increased the production of ANp in the presence of CYB5A, however no significance was noted.

52 a)

12 £ O 10 "-8 8 3 1 6 Q. m Norokadone •a 4 BOA ae 2

4.0 3.5 5 3.0 i i 111 i i i 2.5 SB Norokadone 2.0 0 OA Q. 1.5 '5 1.0 0.5 0.0 0 0.2 0.8 1.5 CYB5B Transfection Level (ug)

Figure 12: a) The effect of lOnM OA treatment with increased expression of CYB5A on the production of AN0. Expression vectors for CYP17A1, POR, CYB5R3 and CYB5A were transfected into HEK-293 cells and products were analyzed by HPLC with a mobile phase of 85% acetonitrile. * Significantly different (P <0.05) indicated when compared to the control at same transfection level treated with Norokadone. b) The effect of lOnM OA treatment with increased expression of CYB5B on the production of ANp\ Expression vectors for CYP17A1, POR, CYB5R3 and CYB5B were transfected into HEK-293 cells and products were analyzed by HPLC with a mobile phase of 85% acetonitrile. * Significantly different (P <0.05) indicated when compared to the control at same transfection level treated with Norokadone. (n=6)

53 a) y 80 70 IWiifolil

ion ( 60 -S 50 uc t m Norokadone *i* i * i' ik g 40 '. .' 1' H aOA £ 30 20 10 70H P T" 0 0 0.2 0.8 1.5 CYB5A Transfection Level (ug)

90 80 s 70 60 50 m Norokadone 40 BOA 30 0. 20 10 s 0 0 0.2 0.8 1.5 CYB5B Transfection Levels (ug)

Figure 13: a) The effect of lOnM OA treatment with increased expression of CYB5A on the production of 170HP. Expression vectors for CYP17A1, POR, CYB5R3 and CYB5A were transfected into HEK-293 cells and products were analyzed by HPLC with a mobile phase of 50% acetonitrile. * Significantly different (P <0.05) indicated when compared to the control at same transfection level treated with Norokadone. b) The effect of lOnM OA treatment with increased expression of CYB5B on the production of 170HP. Expression vectors for CYP17A1, POR, CYB5R3 and CYB5B were transfected into HEK-293 cells and products were analyzed by HPLC with a mobile phase of 50% acetonitrile. * Significantly different (P <0.05) indicated when compared to the control at same transfection level treated with Norokadone. (n=6)

54 a)

C 50 o 40 3 l Norokadone 30 lOA 0. 20 10 £ 0 0 0.2 0.8 1.5 CYB5A Transfection Levels

35 ST 30 5 25

20 I Norokadone 15 lOA 10 5 0 0 0.2 0.8 1.5 CYB5B Transfection Levels (ug)

Figure 14: a) The effect of lOnM OA treatment with increased expression of CYB5A on the production of DHEA. Expression vector for CYP17A1, POR, CYB5R3 and CYB5A were transfected into HEK-293 cells and products were analyzed by HPLC with a mobile phase of 50% acetonitrile. * Significantly different (P <0.05) indicated when compared to the control at same transfection level treated with Norokadone. b) The effect of lOnM OA treatment with increased expression of CYB5B on the production of DHEA. Expression vector for CYP17A1, POR, CYB5R3 and CYB5B were transfected into HEK-293 cells and products were analyzed by HPLC with a mobile phase of 50% acetonitrile. * Significantly different (P <0.05) indicated when compared to the control at same transfection level treated with Norokadone. (n=6)

55 The effect of OA treatment and CYB5A on 17a-hydroxylase activity and CI7,20 lyase activity is shown in Fig 13a and 14a respectively. OA treatment significantly decreased

170HP production and significantly increased DHEA (p<0.05) at CYB5A transfection levels of 1.5ug compared to controls treated with Norokadone. 17oc-hydroxylase activity was also negatively correlated with levels of CYB5A (r = -0.629).

When CYB5B expression was increased with OA treatment, there was no significant difference between ANP production compared to control with Norokadone

(Fig 12b). However similar to results seen with CYB5A, 17a-hydroxylase activity (Fig

13b) decrease significantly (p<0.05) and CI7,20 lyase activity (Fig 14b) increased significantly (P<0.05) with increased expression of CYB5B and OA treatment. Thus, the effects of CYB5B on 17a-hydroxylase and CI7,20 lyase activities are also found with increased phosphorylation of CYP17A1 via OA treatment. Table 2 summarizes the general trends of 170HP, DHEA and ANP productions treated with OA and both isoforms of CYB5.

4.4.2 Effect ofCYBS isoform and treatment with PP2A on 17a-hydroxylase, CI 7,20 lyase and andien-fi synthase activities.

HEK cells were transfected with the following expression vectors: 0.25ug of

CYP17A1, 0.35ugofPOR, 0.25ugof CYB5R3, 0.25ugof PP2A and 0- 1.5ugof

CYB5A or CYB5B and empty pcDNA 3.1 vector to maintain a consistent level of 4ug of plasmid DNA in each transfection. The transfected cells were incubated with H- pregnenolone and the 170HP, DHEA and ANP products were analyzed via HPLC.

Controls were transfected identically however they were not treated with PP2A. Figure

15a displays the effects of PP2A treatment and CYB5A on ANp activity. ANp production significantly decreased for PP2A treatment compared to control across

56 CYB5A transfection levels (p<0.05). Figure 16a and 17a display the effects of PP2A treatment and increased levels of CYB5A on 170HP and DHEA production respectively.

There was no significant change in 170HP or DHEA productions under PP2A.

When similar experiments were conducted using the CYB5B isoform, it was found that there was no significant change in ANP production under PP2A treatment and increasing levels of CYB5B (Figure 15b). Production of 170HP was lower than controls without PP2A treatment at low levels of CYB5B yet production increased significantly

(p<0.05) at higher levels of CYB5B (Fig 16b). DHEA production was exactly opposite to 170HP in that production was significantly higher (p<0.05) than controls at lower levels of CYB5B and then significantly decreased (p<0.05) compared to controls as levels of CYB5B increased (Fig 17b). Table 2 summarizes the general trends of 170HP,

DHEA and ANP productions treated with PP2A and both isoforms of CYB5.

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.

OA-B5 OA+CYB5A OA+CYB5B 17ahydroxylase - 17ahydroxylase 4 17ahydroxylase i 17, 20 lyase t 17, 20 lyase t 17, 20 lyase t ANp - ANP - ANp - PP2A-B5 PP2A+CYB5A PP2A+CYB5B 17ahydroxylase - 17ahydroxylase 1 17ahydroxylase i 17, 20 lyase t 17, 20 lyase t 17, 20 lyase t ANp - ANp i ANp 4,

57 a)

100

60 T3 m Control 2 40 ePP2A aQ. x 20 o 1^ 0 0 0.2 0.8 1.5 CYB5A Transfection Level (ug) b) C? 120 c 100 o 80 '-8 m Control 60 HPP2A I 40 Q. Q. 20 5 0 Baa 0 0.2 0.8 1.5 CYB5B Transfection Level (ug)

Figure 16: a) The effect of 0.25ug PP2A treatment with increased expression of CYB5A on the production of 170HP. Expression vectors for CYP17A1, POR, CYB5R3 and CYB5A were transfected into HEK-293 cells and products were analyzed by HPLC with a mobile phase of 50% acetonitrile. * Significantly different (P <0.05) compared to the control at same transfection level, b) The effect of 0.25ug PP2A treatment with increased expression of CYB5B on the production of 170HP. Expression vectors for CYP17A1, POR, CYB5R3 and CYB5B were transfected into HEK-293 cells and products were analyzed by HPLC with a mobile phase of 50% acetonitrile. * Significantly different (P <0.05) compared to the control at same transfection level. (n=6)

59 JO. 40 35 C |i i i i -r-i 0 30 h I' I HI'!' II 25 m Control 20 B PP2A 15 10 X 5 Q 0 0 0.2 0.8 1.5 CYB5A Transfection Level (ug) b) 35 30 C o 25 • ••• H'l'l'l'll 3 20 M Control ? 15 HPP2A Q. S 10 X 5 Q 0 0 0.2 0.8 1.5 CYB5B Transfection Level (ug)

Figure 17: a) The effect of 0.25ug PP2A treatment with increased expression of CYB5A on the production of DHEA. Expression vector for CYP17A1, POR, CYB5R3 and CYB5A were transfected into HEK-293 cells and products were analyzed by HPLC with a mobile phase of 50% acetonitrile. * Significantly different (P <0.05) compared to the control at same transfection level, b) The effect of 0.25ug PP2A treatment with increased expression of CYB5B on the production of DHEA. Expression vector for CYP17A1, POR, CYB5R3 and CYB5B were transfected into HEK-293 cells and products were analyzed by HPLC with a mobile phase of 50% acetonitrile. * Significantly different (P <0.05) compared to the control at same transfection level. (n=6)

60 4.4.3 Effect of phosphorylation status and site of phosphorylation on CYP17A1

Transfections of HEK-293 cells were performed with the following expression vectors:

a) 0.35ug POR, 0.25ug CYB5R3 and lug CYB5A and 0.25ug CYP17A1-Ser106 or

CYP17A1-Ala106.

b) 0.35ug POR, 0.25ug CYB5R3 and lug CYB5B and 0.25ug CYP17A1-Ser106 or

CYP17A1-Ala106. and empty pcDNA 3.1 vector to maintain a consistent level of 4ug of plasmid DNA in each transfection. The 170HP, DHEA and AN0 products were analyzed via HPLC.

Figure 18 compares ANp\ 170HP and DHEA production for CYP17A1-Ser106 and

CYP17A1-Ala106 when co-transfected with CYB5A. Figure 19 displays ANp\ 170HP and DHEA production for CYP17A1-Ser106 and CYP17A1-Ala106 when co-transfected with CYB5B. It is also important to note that CYP17A1 expression was compared via western blot and equal expression was seen for both wildtype CYP17A1-Ser106 and mutant CYP17A1-Ala106. Overall CYP17A1-Ala106 had increased 170HP production and decreased DHEA production compared to CYP17-Ser106 irrespective of the CYB5 type, however no significance was noted. To ensure that CYP17A1-Ser106 was phosphorylated, similar phosphorylation experiments were conducted with CYP17A1-Ser106 and

CYP17A1-Ala106. It is important to note that any effects that were seen due to phosphorylation with the native serine residue were no longer seen when serine was mutated to alanine at residue 106. This procedure helped us ensure that out native

61 CYP17A1-Ser106 was being phosphorylated and dephosphorylated with OA and PP2A respectively. Production (%) -*N3G>)£»U10)->IOO ooooooooo

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« 4.5 Discussion

The purpose of this study was to further investigate potential mechanisms available to minimize the production of 16-steroids in pigs while simultaneously maintain the androgenic characteristics of an intact boar. Producers commonly castrate boars in order to obliterate the potential of producing pork containing boar taint. Post- translational mechanisms such as phosphorylation of CYP17A1 were investigated with the anticipation that there may be methods of controlling the production of 16-steroids without concurrently controlling sex steroid production.

In this report, we investigated the interactions between phosphorylation status of

CYP17A1 and CYB5A/CYB5B treatments in an over-expression system in HEK-293 cells. It was previously shown using a defined system with CYP17A1, CYB5A or

CYB5B and their appropriate reductases that CYP17A1 interacts with both CYB5A and

CYB5B to produce similar levels of 170HP and DHEA; however CYB5B did not stimulate ANp production compared to CYB5A. These results support our recent findings and control values shown in our previous paper, chapter 3 (Billen and Squires,

2008). It was also concluded that OA treatment of porcine CYP17A1 co-transfected with

CYB5A decreased 170HP production and increased DHEA and ANp production.

Similarly, dephosphorylation of CYP17A1 by PP2A treatment in the presence of CYB5A decreased 170HP and ANp production and increased DHEA production levels. When isoform CYB5B was used along with OA and PP2A treatments, there were no significant effects on ANP production, however 170HP production decrease and DHEA production increased irrespective of the type of phosphorylation treatment.

When alanine occupied position 106 of CYP17A1, there was an increase in

65 170HP production and decreased in DHEA production, whether it was cotransfected with CYB5A or CYB5B. The results determined that Ser106 on porcine CYP17A1 is also the site of phosphorylation and therefore greatly affects enzymatic activity. From these results we can conclude that phosphorylation status and CYB5 can both influence the production of 170HP, DHEA and ANp however there effects were not additive and therefore phosphorylation status and CYB5 influence these productions via a common mechanism.

It has previously been shown that an abundance of a redox partner will favour the

CI7,20 lyase activity following 17oc-hydroxylation. Studies have concluded that POR is the primary electron donor because the redox potential of the ferric state of P450 is lower than that of CYB5 (Ogishima et al., 2003). The POR functions by catalyzing electron transfer from NADPH to the cytochrome P450 during catalysis (Lu and West, 1978) and is also involved in electron transfer from NADPH to heme oxygenase (Yoshida and

Kikuchi, 1978) and CYB5 (Ilan et al., 1981).

There have been numerous studies which demonstrate that CYB5 may stimulate

CI7, 20 lyase activity. Experiments with apo- CYB5, which lacks the heme moiety, and holo-CYB5 suggest that CYB5 is not responsible for direct electron transfer but exerts a saturable, allosteric effect on the CYP17A1-POR complex (Yamazaki et al., 1998). It is suggested that CYB5 causes a conformational change in CYP17A1 that is brought about by binding of 170HP which makes a CYB5 binding site available, which is originally not available when CYP17A1 binds pregnenolone (Miller et al., 1997).

Miller et al (1997) also suggested that perhaps the phosphorylated serine106 residue of CYP17A1 either increases the affinity for redox partners and/or participates in

66 forming a modified redox partner binding site needed for binding CYB5. We found that phosphorylation increase overall DHEA production, and the opposite was true for 170HP production, which was observed to be negatively correlated to increasing amounts of

CYB5A. It has previously been observed that serine phosphorylation increases CI7, 20 lyase activity yet does not change 17a-hydroxylation (Pandey and Miller, 2005). The effects of phosphorylation on ANP production in pigs has not previously been studied.

We found that phosphorylation of CYP17A1 increased the production of ANP compared to control, and the production was positively correlated with CYB5A levels. Overall, our evidence shows that serine dephosphorylation of CYP17A1 and increasing amounts of

CYB5A can maximize CI7, 20 lyase activity and sex steroid production while simultaneously reducing ANP production. Serine phosphorylation also increases DHEA production, however it simultaneous increases ANP production. These results are supported by those found in a study with human NCI-H295A cells, which concluded that human CYP17A1 required either CYB5 or phosphorylation for C17, 20 lyase activity, and that the combination was not additive, CYB5 and phosphorylation enhance CI7, 20 lyase activity independently via a common mechanism (Pandey and Miller, 2005).

The effects of phosphorylation of CYP17A1 combined with the different levels of the CYB5B isoform have not previously been investigated. From our previous work

(Billen and Squires, 2008), we conclude that CYB5B reduces ANP production while increasing DHEA production. However, in this investigation phosphorylation status of

CYP17A1 in the presence of CYB5B had no significant effect on DHEA, 170HP or

ANP production. Our results indicate that the interaction between CYB5B and post- translationaly modified CYP17A1 no longer interact, suggesting that phosphorylated or

67 dephosphorylated CYP17A1 causes a change in binding site for CYB5 which continues to function with CYB5A but not CYB5B.

Although many studies focus on the ability of kinases to phosphorylate proteins, recent data suggests that phosphatases are equally as important components of reversible regulatory cycles of phosphorylation and dephosphorylation (Cohen, 1989; Virshup,

2000). Studies on the dephosphorylation of CYP17A1 have concluded that in vitro treatment with alkaline phosphatases decreased CI7, 20 lyase activity, while PP2A treatment inhibited CI7, 20 lyase activity in a dose dependant manner to the same extent as alkaline phosphatases, but had no effect on the conversion of progesterone/pregnenolone to 170HP (Pandey et al., 2003). Pandey et al., (2003) also concluded that PP2A interacts directly with CYP17A1 to dephosphorylate CYP17A1 itself and not a protein which may affect the activity of CYP17A1. In this study it was also concluded that OA has the ability to reactivate CI 7,20 lyase activity by inhibiting the dephosphorylation of CYP17A1 in NCI-H295A microsomes treated with PP2A

(Pandey et al., 2003).

In summary, we have found that the serine residue at position 106 is important in controlling the enzymatic activity of porcine CYP17A1 as well as humans. OA treatment increase the phosphorylation of porcine CYP17A1 and increases CI 7, 20 lyase activity and DHEA as well as ANP production, while decreasing 17a-hydroxylation activity and

170HP production. PP2A treatment of porcine CYP17A1 increases C17, 20 lyase activity and DHEA production while maintaining 17a-hydroxylation activity and 170HP production and decreasing ANP production. We can conclude that post-translational mechanisms such as phosphorylation of CYP17A1 have proven to be an effective method

68 of controlling the production of 16-steroids without concurrently controlling sex steroid production. Overall results support the hypothesis that CYB5A and CYB5B work via different mechanisms and that post translational modifications such as phosphorylation and dephosphorylation affect the binding site for CYB5 on the CYP17A1/ POR complex.

Further research investigating conformational difference between the two CYB5 isoforms and the exact mechanisms of interaction with phosphorylated porcine CYP17A1 are warranted.

69 5 General Discussion and Conclusions

Androstenone is a major component of boar taint (Gunn et al., 2004; Patterson,

1968). The involvement of CYP17A1, microsomal CYB5A, NADPH cytochrome POR and CYB5R3 NADH reductase in the andien-p synthase system has been confirmed

(Meadus et al., 1993a; Nakajin et al., 1985). Ogishima et al., (2003) discovered an outer mitochondrial membrane CYB5B which may also be involved in steroid hormone metabolism. For this reason both isoforms of CYB5 were investigated in this work to determine their relative importance in the production of 16-androstene steroids.

CYP17A1 is involved in the catalysis of 3 different pathways. CYP17A1 is important in the ANP pathway, and it has been shown in humans that CYP17A1 primarily promotes the synthesis of 17a-hydroxypregnenolone (170HP) and the CI7,20 lyase activity converts 170HP to dehydroepiandrosterone (DHEA) (Pandey et al., 2003).

Recent evidence suggests that dephosphorylation of CYP17 with protein phosphatase 2A

(PP2A) may eliminate almost all the CI7, 20 lyase activity without changing 17a- hydroxylase activity (Pandey and Miller, 2005). This lead to the hypothesis that phosphatase inhibitors may potentially have the ability to increase CI 7, 20 lyase activity and sex steroid production. Serine phosphorylation also increases DHEA production, however it simultaneous increases ANP production. Similar results were found in a study with human NCI-H295A cells, which concluded that human CYP17A1 required either

CYB5 or phosphorylation for CI7, 20 lyase activity, and that the combination was not additive, CYB5 and phosphorylation enhance CI7, 20 lyase activity independently via a common mechanism (Pandey and Miller, 2005).

70 The goals of this research were to investigate the involvement of both isoforms of

CYB5 and their importance in 16-androstene steroid production as well as their involvement with CYP17A1 in 17oc-hydroxylase and 17, 20 lyase activity in pigs.

Phosphorylation status of CYP17A1 and interaction with isoforms of CYB5 was further investigated with the objective of increasing sex steroid production while simultaneously decreasing 16-steroid production and minimizing boar taint.

5.1 Thesis Conclusions

Upon investigation of CYB5 isoforms it was concluded that in a defined HEK-

293 cell line system in which P450 cytochromes of interest were over expressed, CYB5A stimulates both, CI7, 20 lyase and AN|3 pathways, while CYB5B only stimulates CI7, 20 lyase pathway and has no effect on the ANP synthase pathway. These results purposed that isoform CYB5B works via a different mechanism then CYB5A thereby minimizing

ANP production. It is likely that CYB5A and CYB5B interact differently with CYP17A1 and POR and therefore affect the allosteric exchange of electrons. However when comparing isoforms and the production of 170HP and DHEA, CYB5A and CYB5B produce 170HP at a comparable level, while CYB5B increases DHEA production.

Conclusions again may be related to the conformational change between POR and

CYP17A1 which allows the 17a-hydroxylase intermediate to remain bound to the active site of CYP17A1 more than when CYB5A is present, maximizing DHEA synthesis.

These results open the door to potentially minimize or eliminate boar taint by minimizing

16-steroids produced in the ANP pathway while maintaining DHEA production and the beneficial androgenic characteristic of the boar, allowing them to maintain intact.

71 The effects of post-translational modifications such as phosphorylation status also contribute to our goal of increasing DHEA production while minimizing the synthesis of

ANp production. Our results conclude that phosphorylation of CYP17A1 with a phosphatase inhibitor; okadiac acid increases the production of DHEA and ANp, and decreased 170HP production with isoform CYB5A. When CYB5A and CYP17A1 was dephosphorylated with PP2A, production of 16-steroid was reduced and DHEA production increased. CYB5B and phosphorylated / dephosphorylated CYP17A1 had no overall significant effect on DHEA, 170HP or ANP production.

A potential site of phosphorylation was also identified in the CYP17A1 amino acid sequence at serine106. Mutating Serine106 to Alanine106, removes the hydroxyl group making it no longer available to be phosphorylated. Ala106 was incubated with CYB5 and the appropriate reductases in an identical defined systems and any effect that was due to phosphorylation status was no longer seen. These results conclude that the Ser106 residue on CYP17A1 is a site of phosphorylation.

Based on these findings we have determined that CYB5B, and dephosphorylation of CYP17A1 are potential in vitro mechanisms which minimize 16-steroid production and control boar taint. It is purposed that both CYB5B and serine phosphorylation effect the affinity for redox partners and serine phosphorylation may also participate in forming modified redox partner binding site needed to bind CYB5. This experiment was performed by using a cell line to over express the cytochromes of interest, ensuring that the only proteins expressed were those needed for the ANB, 17a hydroxylase and 17, 20 lyase activity. An in vivo study using Leydig cells would be beneficial to observe the results of phosphorylation treatment in vivo.

72 5.2 Future research ofisoforms ofCYB5 and phosphorylation status in vivo.

This study has further characterized the in vitro metabolic profile of ANp\ 170HP and DHEA production in HEK-293 cells that were over expressing CYP17A1,

CYB5A/CYB5B and their appropriate reductases POR and CYB5R3. The results from this study are important for further regulation of boar taint; however experiments were done in a highly controlled and defined environment expressing only those cytochrome and reductases of interest. In vivo experiments would extend our results into Leydig cells which naturally express the cytochromes of interest, as well as others and at a level controlled by the boar. Isolation of Leydig cells is a simple process which can be done and treated with a phosphatase or a phosphatase inhibitor to compare results. The observations would allow us to determine if phosphorylation of CYP17A1 naturally occurring in the Leydig cells has the same effects in vivo as were observed in vitro. These results would contribute to the ongoing research to eliminate boar taint in boars.

5.3 Future research in the potential production of a knockout pis only expressing CYB5B

As our results have clearly indicated that CYB5B has the potential to increase

170HP and DHEA in a manner similar to the levels produced with CYB5A, it would be interesting to investigate the potential of creating a knockout pig. The knockout pig would contain both CYB5A and CYB5B, however CYB5A activity would be inoperative. The production of chimeras may be beneficial as it allows the mixing of species DNA, and promotes the production of a knockout species. This would permit

CYB5B isoform to interact with CYP17A1 and it would tolerate further investigation on mechanisms of interaction while in a system that is getting closer to that of a live animal.

73 Production of a knockout pig allows us to identify if there is a difference between

CYB5A and CYB5B, and whether or not CYB5A is an essential component of AN|3 synthase activity and the cytochrome P450 system in the pig. If results conclude that

CYB5A is not necessary for 170HP and DHEA production, we can start to create genetically modified knockout pigs in which only CYB5B is active minimizing and perhaps eliminating the production of 16-steroids.

5.4 Future research in the development boar taint: Skatole metabolism and its relationship with Androstenone

Along with androstenone, skatole (3-methyl-indole) is the other compound responsible for boar taint. Skatole is produced by bacterial degradation of tryptophan in the hindgut. It is absorbed from the gut, metabolized in the liver, partially excreted in urine and partially deposited in adipose tissue (Abergaard and Laue, 1993). The two main metabolites of skatole are 6-sulfatoxyskatole (Mil) and 3-hydroxy-3-methyloxindole

(Mill) (Beak et al., 1995). Diaz et al., (1999) also reported 3-hydroxy-3-methyloxindole

(HMOI), 3-methyloxindole (3MOI) and 2-aminoacetophenone (2AAP) as metabolites of

3-methylindole in porcine microsomes.

Studies conducted by Squires and Lundstrom (1997), show that CYP2E1 may be responsible for the metabolism of skatole in pigs. Studies by Diaz and Squires (2000) confirmed the role of CYP2E1 as well as CYP2A on skatole metabolism by using various inhibitors; more specifically it was shown that the activity of CYP2A6 was negatively correlated to the accumulation of skatole in adipose tissue of pigs. Terner et al., (2006) also discovered that incubation of primary hepatocytes with 4-methylpyrazole, a CYP2E1 inhibitor, resulted in a decrease is the production of skatole metabolites. However the

74 specificity of substrates and inhibitors are questionable because of the numerous interactions between P450s and particular CYP isoforms within a species, and even further between species.

Lewis (2003) produced a compilation of substrates and inhibitors of the CYP1,

CYP2 and CYP3 families. It was observed that substrate and inhibitor specificity is generally determined by the size and shape of the P450 active site, but especially by the spatial disposition of hydrogen bond donor/acceptor amino acid residues, together with the presence and location of aromatic residues within the heme environment. Therefore some compounds can be substrates of more than one P450, and this is reflected in their physiochemical and structural properties which indicate overlapping selectivity. In another study conducted by Tassaneeyakul et al., (1993) it was also stated that CYP1A xenobiotic inhibitor and substrate probes are nonspecific in their recognition of CYP1A1 and CYP1A2. Due to the overlapping in selectivity of P450 isoforms it may be beneficial to investigate the action of each CYP450 individually without the potential interference of others being present by expressing individual CYPs in HEK-293 cell lines (Soucy et al., 2003; Soucy and Luu-The, 2002).

The involvement of several other P450s in skatole metabolism was investigated by Thorton-Manning et al., (1996) and it was discovered that human CYP1A2 produced great amounts of the skatole metabolite 3-methyloxindole (3MOI). It was also observed that mouse CYP1A2, rabbit CYP4B1 as well as human CYP1A2 and CYP2A6 produced detectable levels of the skatole metabolite indole-3-carbinol (ICOH); however this research was done with recombinant vaccinia-expressed viruses containing human, mouse and rabbit P450s. Preliminary data from our lab has also shown that CYP1A is

75 higher in pigs with lower skatole levels and CYP1A activity was inhibited by menadione and quinicrine in isolated hepatocytes, leading to an increase in skatole levels in hepatocytes. There is also data which suggests the potential involvement of CYP3A in skatole metabolism; however CYP3 A is associated with high levels of skatole which is opposite to CYP1 A, CYP2A and CYP2E1 which are shown to be linked to low levels of skatole in adipose tissues of pigs. Further research is needed to determine and confirm whether various isoforms of P450s, particularly CYP1A and CYP3A metabolize skatole in pigs as well as test the specificity of substrates and inhibitors without the interference of other P450s by expressing them in HEK293 cell lines.

The potential relationship between levels of androstenone and skatole in adipose tissue was examined by Babol et al., (1999). They discovered that the level of androstenone is closely related to the synthesis of sex steroids; this indicated that levels of androstenone and estrogens increase simultaneously. It was hypothesized that androstenone accumulates in adipose tissue while elevated levels of sex steroids inhibit the metabolism and clearance of skatole from the body, resulting in increased levels of skatole in adipose tissue as well. Doran et al.,(2002) investigated the effects of androstenone on CYP2E1 expression in pig hepatocytes and concluded that high androstenone levels antagonize the induction of CYP2E1 by skatole, leading to low

CYP2E1 expression and high skatole accumulation. Doran et al.,(2004; 2002) suggested that skatole may potentially induce CYP2E1 expression by binding to a specific transcription factor which interacts with the CYP2E1 promotor and that androstenone may have the ability to antagonize the binding of skatole to this transcription factor.

Sinclair et al., (2006) researched phase II metabolism of androstenone by

76 SULT2A1 and discovered that nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are involved in the regulation of SULT2A1, which metabolize androstenone and skatole. Peacock's work also showed that CAR regulated a number of genes involved in androstenone and skatole metabolism (Peacock, 2005). This research also suggests the metabolites from androstenone may potentially interact with nuclear receptors and in turn affect the expression of the genes responsible for metabolizing skatole.

Duarte et al.,(2007) examined the expression and bioactivation of CYP1A2,

CYP2A6 and CYP2E1 with the co-expression of human CYB5 through the development of a new mutagenicity tester strain; BTC-bs-CYP. They showed that the co-expression of

CYB5 decreased the expression of CYP1A2 and CYP2E1, yet increased the expression of CYP2A6. The bioactivation of the CYPs was evaluated using procarcinogens and the results show that the co-expression of human CYB5 increased the bioactivation of

CYP2A6 and CYP2E1; however bioactivation of CYP1A2 could not be confirmed. Not much research has been done to clarify if porcine CYB5A and CYB5B has the same potential to activate CYP2A1, CYP2E1 and CYP2A; however this would help clarify the synthesis of androstenone and the metabolism of skatole and the potential importance of

CYB5A and CYB5B on skatole metabolism as well as androstenone synthesis.

5.5 Final Conclusions

The potential significance of these experiments is to allow the complete biological understanding of the andien p synthase pathway responsible for androstenone synthesis in a clearly defined in vitro system and to understand the roles of specific proteins involved in the production of both the beneficial androgenic steroids as well as androstenone

77 synthesis. Understanding the porcine cytochrome P450s system and their appropriate reductases allows further investigation of CYP17A1 interactions with CYB5A and

CYB5B as well as post translational modifications which can increase the production of

DHEA while concurrently decrease androstenone production. The conclusions formulated can be used to potentially replace CYB5A activity with CYB5B and permanently alter the phophrylated state of CYP17A1. These modifications may have the ability to maintain DHEA production and androgenic characteristics of the boar, yet minimize AN|3 synthesis and eventually eliminiate boar taint.

78 Reference List

Abe K, and Sugita Y. 1979. Properties of cytochrome b5 and methemoglobin reduction in human erythrocytes. European Journal of Biochem 101: 423-428.

Abergaard N, and Laue A (Editors). 1993. Absorption from gastrointestinal tract and liver turnover of skatole. Measurement and prevention of boar taint in entire male pigs. INRA Editions, Paris, 107-112 pp.

Akhtar M, Corina D, Miller S, Shyadehi AZ, and Wright JN. 1994. Mechanism of the acyl-carbon clevage and related reactions catalyzed by multifunctional p450s:Studies on cytochrome p45017a. Biochemistry 33: 4410-4418.

Atkinson JK, and Ingold KU. 1993. Cytochrome p450 hydroxylation of hydrocarbons: Variation in the rate of oxygen rebound using cyclopropyl radical clocks including two new ultrafast probes. Biochemistry 32: 9209-9214.

Auchus RJ, Lee TC, and Miller WL. 1998. Cytochrome b5 augments the 17,20-lyase activity of human p450cl7 without direct electron transfer. The Journal of Biological Chemistry 273: 3158-3165.

Babol J, and Squires EJ. 1995. Quality of meat from entire male pigs. Food Research International 28: 201-212.

Babol J, Squires EJ, and Gullet EA. 1996. Investigation of factors responsible for the development of boar taint. Food Research International 28: 573-581.

Babol J, Squires EJ, and Lundstorm K. 1999. Relationship between metabolism of androstenone and skatole in intact male pigs. Journal of Animal Science 77: 84- 92.

Barford D., Das A.K., and Egloff MP. 1998. The structure and mechanism of protein phosphatases: Insights into catalysis and Regulation Annu Rev Biophys Biomol Struct. 27:133-164.

Biason-Lauber A, Kempken B, Werder E, Forest MG, Einaudi S, Ranke MB, Matsuo N, Brunelli V, Schonle EJ & Zachmann M. 2000. 17a-hydroxylase/ 17,20-lyase deficiency as a model to study enzymatic activity regulation: Role of phosphorylation. The Journal of Clinical Endocrinology and Metabolism 85: 1226-1231.

Billen MJ & Squires EJ. (2008) The role of porcine cytochrome b5A and cytochrome b5B in the regulation of cytochrome P45017A1 activities, Submitted for publication.

Booth WD. 1975. Changes with age in the occurrence of cl9 steroids in testis and submaxillary gland of the boar. J. Reprod. Fert 42: 459-472.

79 Booth WD. 1984. Sexual dimorphism involving steroidal phermones and their binding protein in the submaxillary glands of the gottingen minature pig. J. Endocrinol 100: 195-202.

Booth WD. 1987. Factors affecting the phermone composition of voided boar saliva. J. Reprod. Fert 81: 427-431.

Bridges A et al. 1998. Identification of the binding site of cytochrome p450 2b4 for cytochrome b5 and cytochrome p450 reductase. The Journal of Biological Chemistry 273: 17036-17049.

Brock BJ, and Waterman MR. 1999. Biochemical differences between rat and human cyochrome p450cl7 support the different steroidogenic needs of these two species. Biochemistry 83: 1598-1606.

Claus R, Hoffman B, and Karg H. 1971. Determination of 5-androst-16-ene-3-one, a boar taint steroid in pigs, with reference to relationship to testosterone. Journal of Animal Science 33: 1293-1297.

Claus R, Weiler U, and Herzog A. 1994. Physiological aspects of androstenone and skatole formation in boar-a review with experimental data. Meat Science 38: 289- 305.

Cohen P. 1989. The structure and regulation of protein phosphatases. Annual Review of Biochemistry 58: 453-508.

Cohen P, Holmes CFB, and Tsukitani Y. 1990. Okadiac acid a new probe for the study of cellular regulation. TIBS 15.

Diaz GJ, Skordos KW, Yost GS, and Squires EJ. 1999. Identification of phase 1 metabolites of 3-methylindole produced by pig liver microsomes. Drug Metabolism and Disposition 27: 1150-1156.

Diaz GJ, and Squires EJ. 2000. Metabolsim of 3-methylindole by porcine liver microsomes: Responsible cytochrome p450 enzymes. Toxicological Science 55: 284-292.

Doran E, Whittington FW, Wood JD, and McGivan JD. 2002. Cytochrome p450iiel (cyp2el) is induced by skatole and this induction is blocked by androstenone in isolated pig hepatocytes. Chemico-Biological Interactions 140: 81-92.

Doran E, Whittington FW, Wood JD, and McGivan JD. 2004. Characterisation of androstenone metabolism in pig liver microsomes. Chemico-Biological Interactions 147: 141-149.

Duarte MP et al. 2007. The stimulatory role of human cytochrome b5 in the bioactivation of human cypla2, 2a6 and 2el: A new cell expression system to study

80 cytochrome p450-mediated biotransformation (a corrigendum report on Duarte et al (2005) mutagenesis 20:93-100).. Mutagenesis 20: 75-81.

DuBois RN, Simpson ER, Kramer RE, and Waterman MR. 1981. Induction of synthesis of cholesterol side chain clevage cytochrome p450 by adrenocorticotropin in cultured bovine adrenocortical cells. J. Biol. Chem. 256: 7000-7005.

Dufort I, Rheault p, Huang XF, Soucy P, and Luu-The V. 1999. Characteristics of a highly labile human type 5 17P-hydroxysteroid dehydrogenase. Endocrinology 140: 568-574.

Estabrook RW. 1999. Discovering the function of cytochrome p450 in drug metabolism: The role of alfred hildebrandt. Drug metabolism reviews 31:317-331.

Fortin A, Friend WD, and Sarkar NK. 1983. A note on the carcass composition of yorkshire boars and barrows. Canadian Journal of Animal Science 63: 711.

Geller DH, Auchus RJ, and Miller WL. 1999. P450cl7 mutations r347h and r358q selectively disrupt 17,20-lyase activity by disrupting interactions with p450 oxidoreductase and cytochrome b5. Molecular Endocrinology 13: 167-175.

Gower DB. 1972. 16-unsaturated cl9 steroids: A review of their chemistry, biochemistry and possible physiological role. J. Steroid. Biochem. 3: 45-103.

Gunn M et al. 2004. Welfare aspects of the castration of piglets scientific report of the scientific panel for animal health and welfare. The European Food Safety Authority Journal 91:1-18.

Haystead TAJ, Sim AT, Carling D, Honnor RC, Tsukitani Y, Cohen P & Hardie DG. 1989. Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism, Nature. 337, 78-81.

Hildebrandt A, and Estabrook RW. 1971. Evidence for the participation of cytochrome b5 in hepatic microsomal mixed function oxidation reactions. Arch Biochem Ciophys 143: 66-79.

Hultquist DE, and Passon PG. 1971. Reduction of methemoglobin catalysed by erythrocyte cytochrome b5 and cytochrome b5 reductase. Nature New Biol 299: 252-254.

Ilan Z, Ilan R, and Cinit DL. 1981. Evidence for a new physiological role of hepatic nadph: Ferricytochrome (p-450) oxidoreductase. J. Biol. Chem. 256: 10066- 10072.

Kaderbhai MA, Morgan R, and Kaderbhai NN. 2003. The membrane interactive tail of cytochrome b(5) can function as a stop-transfer sequence in concert with a signal sequence to give inversion of protein topology in the endoplasmic reticulum. Archives of Biochemistry and Biophysics 412: 259-266.

81 Katagiri M, Suhara K, Shiroo M and Fujimura Y. 1982. Role of cytochrome b5 in the cytochrome P-450-mediated C21-steroid 17, 20 lyase reaction. Biochemical and Biophysical Research Communications 108: 379-384

Katagiri M, Kagawa N and Waterman MR. 1995. The role of cytochrome b5 in the biosynthesis of androgens by human P450cl7. Archives of Biochemistry and Biophysics 317: 343-347.

Lee-Robichaud P, Kaderbhai MA, Kaderbhai N, Wright JN, and Akhtar M. 1997. Interaction of human cypl7 (p-45017a, 17a-hydroxylase-17,20-lyase) with cytochrome b5: Importance of the orientation of the hydrophobic domain of cytochrome b5. Biochem. J. 321:857-863.

Lee-Robichaud P, Shyadehi AZ, Wright JN, Akhtar ME, and Akhtar M. 1995a. Mechanistic kinship between hydroxylation and desaturation reactions: Acyl- carbon bond clevage promoted by pig and human cypl7 (p-450(17)a; 17a- hydroxylase-17,20-lyase). Biochemistry 34: 14104-14113.

Lee-Robichaud P, Wright JN, Akhtar ME, and Akhtar M. 1995b. Modulation of the activity of human 17a-hydroxylase-17,20-lyase (cypl7) by cytochrome b5:Endocrinological and mechanistic implications. Biochem. J. 308: 901-908.

Lee-Robichaud P, Akhtar ME and Akhtar M. 1998. Control of androgen biosynthesis in the human through the interaction of Arg347 and Arg358 of CYP17 with cytochrome b5. Biochemical Journal 332: 293-296.

Lewis DFV. 2003. Human cytochrome p450 associated with phase 1 metabolism of drugs and other xenobiotics: A compilation of substrates and inhibitors of the cypl, cyp2 and cyp3 families. Current Medical Chemistry 10: 1955-1972.

Lin D, Black SM, Nagahama Y, and Miller WL. 1993. Steroid 17a-hydroxylase and 17,20-lyase activities of p450cl7: Contributions of serine106 and p450 reductase. Endocrinology 132: 2498-2506.

Lin D, Harikrishna JA, Moore CD, Jones KL, and Miller WL. 1991. Missense mutation serine106~*proline causes 17a-hydroxylase deficiency. The Journal of Biological Chemistry 266: 15992-15998.

Lu AYH, and West SB. 1978. Reconstituted mammalian mixed-function oxidases: Requirements, specificities and other properties. Pharmacol. Ther. Part A 2: 337- 359.

Luu-The V, Zhang L, Poirier D, and Labrie F. 1995. Characteristics of human types 1, 2 and 3 17p-hydroxysteroid dehydrogenase activities: Oxidation/reduction and inhibition. J. Steroid Biochem. Mol. Biol 55: 581-587.

82 Meadus WJ. 1994. An investigation of androstenone synthesis regulation and purification of cytochrome p450cl7, University of Guelph, Guelph.

Meadus WJ, Mason JI, and Squires EJ. 1993a. Cytochrome p450cl7 from porcine and bovine adrenal catalyses the formation of 5,16-androstadien-3J3-ol from pregnenolone in the presence of cytochrome b5. J. Steroid. Biochem. Molec. Biol. 46: 565-572.

Meadus WJ, mason JI, and Squires EJ. 1993b. Cytochrome p450cl7 from porcine and bovine adrenal catalyses the formation of 5,16-androstadien-3P-ol from pregnenolone in the presence of cytochrome b5. Journal of Steroid Biochemistry and Molecular Biology. 46.

Miller WL. 1988. Molecular biology of steroid hormone synthesis. Endocr. Rev. 9: 295- 318.

Miller WL, Auchus RJ, and Geller DH. 1997. The regulation of 17,20 lyase activity. Steroids 62: 133-142.

Nakajin S, and Hall PF. 1981. Microsomal cytochrome p-450 from neonatal pig testis. The Journal of Biological Chemistry 256: 3871-3876.

Nakajin S, Takahashi M, Shinoda M, and Hall PF. 1985. Cytochrome b5 promotes the synthesis A16-cl9 steroids by homogenous cytochrome p-450 c21 side-chain clevage from pig testis. Biochemical and biophysical research communications 132: 708-713.

National Center of Biotechnlogy Information - Blast.(1988), viewed November 11, 2007. http://blast.ncbi.nlm.nih.gov/Blast.cgi

Oesch-Bartlomowicz B, and Oesch F. 2003. Cytochrome-p450 phosphorylation as a functional switch. Archives of Biochemistry and Biophysics 409: 228-234.

Oesch-Bartlomowicz B, and Oesch F. 2005. Phosphorylation of cytochromes p450: First discovery of a posttranslational modification of a drug-metabolizing enzyme. Biochemical and Biophysical Research Communications 338: 446-449.

Ogishima T, Kinoshita J, Mitani F, Suematsu M, and Ito A. 2003. Identification of outer mitochondrial membrane cytochrome b5 as a modulator for androgen synthesis in leydig cells. The Journal of Biological Chemistry 278: 21204-21211.

Oshino N, Imai Y, and Sato R. 1971. A function of cytochrome b5 in fatty acid desaturation by rat liver microsomes. Journal of Biochemistry 69: 155-167.

Ozols J. 1976. The role of microsomal cytochrome b5 in the metabolism of ethanol, drugs and the desaturation of fatty acids. Ann. Clin. Res. 8: 182-192.

83 Ozols J, Korza G, Heinemann FS, Hediger MA, and Strittmatter P. 1985. Complete amino acid sequence of steer liver microsomal nadh-cytochrome b5 reductase. The Journal of Biological Chemisrty 260: 11953-11961.

Pandey AV, Mellon SH, and Miller WL. 2003. Protein phosphatase 2a and phosphoprotein set regulate androgen production by p450cl7. The Journal of Biological Chemistry 278: 2837-2844.

Pandey AV, and Miller WL. 2005. Regulation of 17,20 lyase activity by cytochrome b5 and by serine phosphorylation of p450cl7. . The Journal of Biological Chemistry 280: 13265-13271.

Patterson RLS. 1968. 5a androst-16-ene-3-one: Compound responsible for taint in boar fat. J. Sci. Food. Agric. 19: 31-41.

Peacock JN. 2005. Characterization of the pig nuclear receptor car and its role in boar taint, University of Guelph, Guelph.

Reddy VV, Kupfer D, and Caspi E. 1977. Mechanism of c-5 double bond introduction in the biosynthesis of cholesterol by rat liver microsomes. Journal of Biological Chemistry 252: 2797-2801.

Reed HC, Melrose DR, and Patterson RLS. 1974. Androgen steroids as an aid to the detection of oestrus in pig artificial insemination. Br. Vet. J. 130: 61-67.

Ruokonen A, and V. R. 1974. Steroid metabolism in testis tissues: Concentrations of unconjugated and sulfated neutral steroids in boar testis. J. Steroid Biochem. 5: 33-38.

Saat YA, Gower DB, Harrison FA, and Heap RB. 1972. Studies on the biosynthesis in vivo and excretion of 16-unsaturated c 19 steroids in the boar. Journal of Biochemistry 129: 657-663.

Sather AP, Jones SDM, and J. S. 1991. Feedlot performance, carcass composition and pork quality from entire male and female landrace and large white market-weight pigs. Canadian Journal of Animal Science 71: 29-42.

Schenkman JB, and Jansson I. 2003. The many roles of cytochrome b5. Pharmacology and Therapeutics 96: 139-152.

Sinclair PA, Gimore WJ, Lin Z, Lou Y, and Squires EJ. 2006. Molecular cloning and regulation of porcine sult2al: Relationship between sult2al expression and sulfoconjugation of androstenone. Journal of Molecular Endocrinology 36: 301- 311.

Soucy P, Lacoste L, and Luu-The V. 2003. Assessment of the porcine and human 16-ene- synthase, a third activity of p450cl7, in the formation of an androstenol precursor.

84 Role of recombinant cytochrome b5 and p450 reductase. European Journal of Biochem270: 1349-1355.

Soucy P, and Luu-The V. 2000. Conversion of pregnenolone to dhea by human 17a- hydroxylase/17,20-lyase (p450cl7). European Journal of Biochem 267: 3243- 3247.

Soucy P, and Luu-The V. 2002. Assessment of the ability of type 2 cytochrome b5 to modulate 17,20-lyase activity of human p450cl7. Journal of Steroid Biochemistry and Molecular Biology. 80: 71-75.

Squires EJ. 1989. Involvement of cytochrome p450 in the synthesis of 5,16, androstadien-3p-ol from pregnenolone in pig testis microsomes. J. Steroid Biochem. 33: 621-626.

Squires EJ, Adeola O, Young LG, and Hacker RR. 1993. The role of growth hormones, beta-adrenergic agents and intact male in pork production-a review. Canadian Journal of Animal Science 73: 1-23.

Squires EJ, and Lou Y. 1995. Levels of boar taint in purebred entire male pigs, University of Guelph, Guelph.

Squires EJ, and Lundstorm K. 1997. Relationship between cytochrome p450iiel in liver and levels of skatole and its metabolites in intact male pigs. Journal of Animal Science 75: 2506-2511.

Tassaneeyakul W et al. 1993. Specificity of substrate and inhibitor probes for huma cytochrome p450 lal an la2. The Journal of Pharmacology and Experimental Therapeutics 265: 401-407.

Terner MA, Gilmore WJ, Lou Y, and Squires EJ. 2006. The role of cyp2a and cyp 2el in the metabolism of 3-methylindole in primary cultured porcine hepatocytes. Drug Metabolism and Disposition 38: 848-854.

Thorton-Manning J, Appleton ML, Gonzalez J, and Yost GS. 1996. Metabolism of 3- methylondole by vaccinia-expressed p450 enzymes: Correlation of 3- methyleneindolenine formation and protein binding. The Journal of Pharmacology and Experimental Therapeutics 276: 21-29.

Tomatsu S et al. 1989. The organization and complete nucleotide sequence of the human nadh-cytochrome b5 reductase gene. Gene 80: 353-361.

Vergeres G, and Waskell L. 1995. Cytochrome b5, its function, structure and membrane topology. Biochimie 77: 604-620.

Virshup DM. 2000. Protein phosphatase 2a: A panoply of enzymes. Current Opinion in Cell Biology 12: 180-185.

85 Void E. 1970. Fleishproduktionseigenschaften bei ebern und kastraten. Iv. Organoleptisch und gaschromatografische untersuchungen wasserdampffluchtiger stoffe des ruckenspeckes von ebern. Meldinger fra norges landbrukshogskalo 49: 1-25.

Yamazaki T, Ohno T, Sakaki T, Akiyoshi-Shibata M, Yabusaki Y, Imai T & Kominami S. 1998. Kinetic analysis of successive reactions catalyzed by bovine cytochrome p45017a,iyase- Biochemistry 37: 2800-2806.

Yokoyama MT, and Carlson JR. 1979. Microbial metabolites of tryptophan in the intestinal tract with specific reference to skatole. Am. J. Clin. Nutr. 32: 173-178.

Yoshida T, and Kikuchi G. 1978. Features of the reaction of heme degradation catalyzed by the reconstituted microsomal heme oxygenase system. J. Biol. Chem. 253: 4230-4236.

Yubisui T et al. 1987. Molecular cloning of cdnas of human liver and placenta nadh- cytochromeb5 reductase. Proc. Natl. Acad. Sci. 84: 3609-3613.

Zhang L, Rodriguez H, Ohno S, and Miller WL. 1995. Serine phosphorylation of human p450cl7 increases 17,20-lyase activity:Implication for adrenarche and the polycystic ovary syndrome. Proc. Natl. Acad. Sci. 92: 10619-10623.