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Review

Cytochrome b5 modulates multiple reactions in steroidogenesis by diverse mechanisms Karl-Heinz Storbeck, Amanda C. Swart, Cheryl L. Fox, Pieter Swart * Department of Biochemistry, University of Stellenbosch, Stellenbosch 7600, South Africa

A R T I C L E I N F O

A B S T R A C T

Article history: Received 14 May 2014 Received in revised form 2 November 2014 Accepted 21 November 2014 Available online xxx

Cytochrome b5 (cyt-b5) is a relatively small haemoprotein which plays an important role in the regulation of mammalian steroidogenesis. This unique protein has the ability to modulate the activity of key steroidogenic enzymes via a number of diverse reaction mechanisms. Cyt-b5 can augment the 17,20-lyase activity of CYP17A1 by promoting the interaction of CYP17A1 and POR; enhance the 16-enesynthase activity of CYP17A1 by acting as an electron donor; and enhance the activity of 3bHSD by increasing the affinity of 3bHSD for its cofactor NAD+. We review the modulation of CYP17A1 and 3bHSD activity by cyt-b5 and discuss the reaction mechanisms associated with each activity. The physiological importance of cyt-b5 in regulating mammalian steroidogenesis is presented and the impact of inactivating cyt-b5 mutations are reviewed. This article is part of a Special Issue entitled 'Steroid/Sterol signaling'. ã 2014 Elsevier Ltd. All rights reserved.

Keywords: Cytochrome b5 Cytochrome P450 17-hydroxylase/17,20lyase CYP17A1 3b-hydroxysteroid dehydrogenase 3bHSD 16-ene-synthase

Contents 1. 2.

3. 4. 5. 6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cyt-b5 and CYP17A1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CYP17A1 lyase activity is modulated by cyt-b5 in a species and substrate dependent manner 2.1. Mechanisms required for 17,20-lyase stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. 16-ene-synthase: a different mechanism for a different activity? . . . . . . . . . . . . . . . . . . . . . . . 2.3. Cyt-b5 increased the affinity of 3bHSD towards its cofactor, NAD+ . . . . . . . . . . . . . . . . . . . . . . . . . . . Different structural features of cyt-b5 are required for its functional promiscuity . . . . . . . . . . . . . . . The influence of cyt-b5 on steroidogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inactivating cyt-b5 mutations cause true 17,20-lyase deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Cytochrome b5 (cyt-b5), discovered in 1942 and named b5 in 1952, is unique amongst the cytochrome family of haemoproteins, as this relatively small ubiquitous protein participates in a wide range of biochemical transformations in a number of

* Corresponding author. Department of Biochemistry, University of Stellenbosch, Private Bag X1, Matieland, 7602, South Africa. Tel.: +27 21 8085862; fax: +27 21 8085863. E-mail address: [email protected] (P. Swart).

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different tissue types [1,2]. These bio-transformations include the reduction of methemoglobin to haemoglobin [3], fatty acid desaturation and elongation [4], plasmalogen and cholesterol biosynthesis [5,6],N-glycolylneuraminic acid biosynthesis [7] and interactions with xenobiotic as well as steroid metabolising cytochromes P450 [8,9]. Mammalian cyt-b5 occurs in three distinct forms – a soluble cytosolic form and two amphipatic membrane-bound forms. The soluble cytosolic form (98 amino acids) is expressed mainly in the erythrocytes where it is involved in the reduction of methaemoglobin to haemoglobin [3]. The two membrane bound forms of cyt-b5 are found in the endoplasmic reticulum (134 amino acids)

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and the outer mitochondrial membrane (146 amino acids) [10,11]. The membrane bound isoforms of cyt-b5 are expressed in a wide range of tissue types which include the adrenals, gonads, liver, kidneys, spleen, lungs, the brain and fat tissue [12–14]. The ability of cyt-b5 to augment cytochrome P450-catlysed reactions is well established [8,9]. Recent studies have shown that cyt-b5 can also influence the activity of 3b-hydroxysteroid dehydrogenase (3bHSD) [15,16]. Although the mechanisms whereby cyt-b5 influences these different reactions are not fully understood, it is apparent that the haemoprotein stimulates, or inhibits, catalytic activity in an enzyme and substrate dependent manner [17,18]. The two most prominent and well accepted mechanisms for the influence of cyt-b5 on cytochrome P450-dependent reactions are enhanced second electron transfer, requiring an intact heme group, and protein– protein interaction, causing allosteric effects, which can be affected by apocytochrome-b5 [9,19]. The role of cyt-b5 in steroidogenic enzyme promiscuity was first recognised when, in 1982, Katagiri et al. reported on the influence of cyt-b5 on pig testicular cytochrome P450 17-hydroxylase/17,20-lyase (CYP17A1) [20]. This paper was followed in 1985 by a report by Shinzawa et al. on the influence of cyt-b5 on the dual function of CYP17A1 in guinea pig adrenal microsomes [21]. In the same year results obtained by Nakajin et al. indicated that a purified preparation of pig testicular CYP17A1 could synthesise D16-C19-steroids under the influence of cyt-b5 [22], prompting the authors to remark, “the ability of various purified cytochromes P450 to catalyse numerous reactions is well known but the apparent specificity of the steroidogenic cytochromes P450 makes the present findings surprising”. The important role cyt-b5 plays in the so called promiscuity of enzymes involved in adrenal and gonadal steroidogenesis will be discussed in more detail. 2. Cyt-b5 and CYP17A1 2.1. CYP17A1 lyase activity is modulated by cyt-b5 in a species and substrate dependent manner CYP17A1 is a multifunctional enzyme, the expression and activity of which plays a pivotal role in determining the output of steroidogenic tissue. The absence of CYP17A1 in the zona glomerulosa of the adrenal allows for the production of

mineralocorticoids through the actions of 3bHSD, CYP21A2 and CYP11B2. On the other hand, in the zona fasciculata the 17a-hydroxylase activity towards both pregnenolone and progesterone directs steroidogenesis in the direction of glucocorticoid production. Both the 17a-hydroxylase activity, and the subsequent 17,20-lyase activity of CYP17A1 towards the resulting 17a-hydroxylated products, are required for the production of androgen and oestrogen precursors in the zona reticularis, testes and ovaries (though not in the corpus leutem, which produces progesterone) [23]. CYP17A1 was the first steroidogenic enzyme shown to be influenced by cyt-b5 [20,21,24,25] and it has since been well established that cyt-b5 selectively regulates the 17,20-lyase activity of CYP17A1, with minimal effect (
2-fold) on the 17a-hydroxylase function (Table 1) [26]. The up-regulation of cyt-b5 expression in the zona reticularis is linked to the increase in C19 steroid production associated with adrenarche [27–30] and recent clinical studies have confirmed that true isolated 17,20-lyase deficiency is caused by inactivating cyt-b5 mutations [31,32]. Both the preferred substrate for the 17,20-lyase reaction as well as the degree of cyt-b5 stimulation differ greatly between species. The human, primate, bovine, caprine, ovine and feline enzymes favour the D5-substrate, 17a-hydroxypregnenolone; the guinea pig enzyme favours the D4-substrate, 17a-hydroxyprogesterone; and the enzymes from the pig, hamster, horse and rat can accept either D5- or D4-substrates [33]. Studies have shown that the degree of cyt-b5 stimulation is not the same, even among enzymes from different species with the same substrate preference [33,34]. In addition, differences in the stimulation of the 17,20-lyase activity towards different substrates can occur within the same species. For example cyt-b5 has a minimal effect on the 17,20-lyase activity of rat CYP17A1 towards 17a-hydroxypregnenolone (1.3-fold), but substantially enhances the enzymes 17,20-lyase activity towards 17a-hydroxyprogesterone (4.4-fold) [34]. Another example can be gleaned from the human enzyme. It has conventionally been accepted that 17a-hydroxypregnenolone is the substrate for the 17,20-lyase activity of the human enzyme. Gupta et al. (2003) have, however, shown that the enzyme is also able to efficiently catalyse both the 17a-hydroxylation and 17,20-lyase of 5a-reduced steroids. The steroid, 5a-pregnan-3a,17a-diol-20one, is in fact a better substrate for the 17,20-lyase reaction than 17a-hydroxypregnenolone. The 17,20-lyase activity

Table 1 Influence of cyt-b5 on reactions catalysed by human and porcine CYP17A1. Enzyme

Reaction

Substrate

Effect

References

Human CYP17A1

17a-hydroxylase

Pregnenolone Progesterone 5a-pregnan-3,20-dione 5a-pregnan-3a-ol-20-one Progesterone 17a-hydroxypregnenolone 17a-hydroxyprogesterone 5a-pregnan-3a,17a-diol-20-one Pregnenolone

0 to 2-fold " No effect Not determined Not determined No effect 5 to 10-fold " 5 to 10-fold "a 3-fold " Cyt-b5 dependentb

[26,41,47,62] [26,47] [35] [35] [54] [26,33,41,47] [26,45] [35] [62,63]

Pregnenolone Progesterone 5a-pregnan-3,20-dione 5a-pregnan-3a-ol-20-one Progesterone 17a-hydroxypregnenolone 17a-hydroxyprogesterone 5a-pregnan-3a,17a-diol-20-one Pregnenolone

1.8-fold " No effect Unknown Unknown No effect 2 to 5-fold " 2 to 3-fold " Unknown Cyt-b5 dependent

[62] [62]

16a-hydroxylase 17,20-lyase

16-ene synthase Porcine CYP17A1

17a-hydroxylase

16a-hydroxylase 17,20-lyase

16-ene synthase a b

[54] [34,62] [34,54,62] [62,63]

17a-hydroxyprogesterone is a poor substrate for 17,20-lyase activity of human CYP17A1 [26,45]. The maximal reported 16-ene synthase activity human CYP17A1 is only 12% of total activity towards pregnenolone [63].

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towards 5a-pregnan-3a,17a-diol-20-one, in the absence of cyt-b5, was reported to be similar to the 17,20-lyase activity towards 17a-hydroxypregnenolone in the presence of cyt-b5. In addition, while cyt-b5's stimulation of the 17,20-lyase activity towards 17a-hydroxypregnenolone was 10-fold, stimulation of the activity towards 5a-pregnan-3a,17a-diol-20-one was only 3-fold (Table 1) [35]. This activity of CYP17A1 has been implicated in the so-called backdoor pathway, which leads to the production of DHT, in which testosterone is bypassed [35–37]. It is therefore apparent that cyt-b5 influences the activity of CYP17A1 in a species and substrate dependent manner. The mechanism by which cyt-b5 modulates CYP17A1 17,20-lyase activity will be discussed in detail. 2.2. Mechanisms required for 17,20-lyase stimulation At present, the direct interaction between cyt-b5 and CYP17A1, and the resulting increase in 17,20-lyase activity, is well documented. Numerous studies have confirmed that specific cationic residues, found on the proximal surface of CYP17A1 (R347, R358 and R449), are required for cyt-b5 to bind through interactions with anionic cyt-b5 residues (E48 or E49) [38–44]. A number of studies has confirmed that the stimulation of CYP17A1 17,20-lyase activity does not require electron transfer from cyt-b5. Auchus et al., 1998 demonstrated that apocytochrome-b5 exhibited a similar stimulatory profile to cyt-b5, while Lee-Robichaud et al. (1998) reported similar observations with Mn2+-substituted cyt-b5 which cannot transfer electrons. In support of these findings, Brock and Waterman (1999) showed that cyt-b5 did not alter H2O2 production by CYP17A1, also indicating the absence of electron transfer [45]. Our group recently showed that the introduction of the H68A mutation, which abolishes cyt-b5's ability to coordinate heme, does not eliminate its ability to stimulate the 17,20-lyase activity of CYP17A1 (Fig. 1) [46]. The exact mechanism by which cyt-b5 acts to increase the 17,20-lyase activity of CYP17A1 is, however, unclear. Two general mechanisms have been proposed to date. The first suggests that cyt-b5 enhances the complex formation between CYP17A1 and cytochrome P450 reductase (POR), thereby facilitating more efficient electron transfer required for the 17,20-lyase reaction [47,48]. This mechanism accounts for the observations that cyt-b5

Fig. 1. Cyt-b5 structural domains influence its ability to augment the 17,20-lyase activity of CYP17A1. COS-1 cells were cotransfected with caprine CYP17A1 and the following caprine cyt-b5 constructs; full length cyt-b5 (134 residues), apo-b5 (H68A mutation), truncated-b5 (residues 1–93) or b5-C-ter (residues 95–134). The fold increase in DHEA production from pregnenolone was calculated relative to the activity of caprine CYP17A1 in the absence of cyt-b5. Modified from [46].

3

has no effect on the Km of CYP17A1, but significantly increases the Vmax of the 17,20-lyase reaction [40,47,48], and that increased ratios of POR to CYP17A1 have been shown to increase 17,20-lyase activity in the absence of cyt-b5 [49,50]. Geller et al. (1999) further demonstrated the importance of the interaction between POR and CYP17A1 when they showed that mutations in the POR binding site of CYP17A1 selectively reduced 17,20-lyase activity. Partial 17,20-lyase activity could, however, be restored by the addition of cyt-b5 [40] supporting the idea that cyt-b5 enhances the binding of POR to CYP17A1. Using fixed molar ratios of cyt-b5 to CYP17A1, and increasing quantities of POR, Pandey and Miller (2005) later demonstrated a dose response for the 17,20-lyase activity of CYP17A1. While a dose response was also observed in the absence of cyt-b5, the 17,20-lyase activity was always higher in the presence of cyt-b5, confirming that cyt-b5 can augment 17,20-lyase activity by facilitating electron transfer from POR [48]. The second mechanism proposed for the stimulation of the 17,20-lyase activity by cyt-b5, is that the binding of cyt-b5 to CYP17A1 induces a conformational change in CYP17A1. This interaction is suggested to favour the repositioning of the steroid substrate so that the C20, rather than C17, is aligned with the iron-oxygen complex [39]. Indeed, cyt-b5 induces conformational changes in a number of other cytochromes P450 with resulting changes in activity [51–53]. Studies from our laboratory have confirmed that cyt-b5 induces a subtle conformational change in the active pocket of CYP17A1 [54]. We have shown that the ability of human and baboon CYP17A1 to catalyse the 16a-hydroxylation of progesterone is due to the small amino acid residue A105, located in the B’-C domain. This residue allows for the repositioning of the progesterone substrate required for the 16a-hydroxylation, while the larger L105, found in the CYP17A1s of all other mammalian species, prevents the repositioning of the substrate through sterical hindrance [54,55]. The addition of cyt-b5 significantly increases the 16a-hydroxylase activity of baboon and goat CYP17A1 (both L105) indicating that cyt-b5 induces a conformational change which allows for the repositioning of progesterone [54]. We have previously suggested that there is validity for both mechanisms and that cyt-b5 enhances both the interaction between POR and CYP17A1 for efficient electron transfer and also induces a conformational change in CYP17A1 [56]. Recent NMR chemical shift mapping studies have confirmed that cyt-b5 induces conformational changes within CYP17A1, which include changes to the I, F and G helices, perceivably altering substrate orientation [43,57]. Substrate binding in the absence of cyt-b5 also induced conformational changes within CYP17A1, however, the resulting interactions between CYP17A1 and cyt-b5 were stronger when the hydroxylase substrate pregnenolone was bound, than when the 17,20-lyase substrate 17a-hydroxypregnenolone was present [43,57]. While this result was unexpected, the data nevertheless imply direct communication between the redox binding site and the active site of CYP17A1 [43]. Interestingly, CYP17A1-b5 complex formation was disrupted by the addition of rat POR, suggesting a mutually exclusive mechanism for cyt-b5 and POR binding, most likely due to overlapping or partially overlapping binding sites on the proximal surface of CYP17A1 [43]. These observations first led Estrada et al. (2013) to suggest a second order model in which cyt-b5 acts to lessen POR binding when pregnenolone is bound, reducing electron transfer, while the weaker CYP17A1-b5 interaction in the presence of 17a-hydroxypregnenolone promotes POR binding, thereby ensuring efficient electron transfer for the 17,20-lyase reaction [43]. This model did not, however, address findings which demonstrate that cyt-b5 binding to CYP17A1 is essential for its function [40,42,48]. Furthermore, it should be noted that while the addition of POR disrupted the CYP17A1-b5 complex in the absence of substrate, the

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inclusion of pregnenolone or 17a-hydroxypregnenolone strengthened the CYP17A1-b5 complex substantially, requiring significantly more POR to disrupt cyt-b5 binding [43]. In addition, these studies were conducted with soluble cyt-b5, which lacks the membrane binding domain previously shown to contribute towards the interaction with CYP17A1 (Fig. 1) [46,58,59]. The idea that cyt-b5 and POR binding is definitively mutually exclusive therefore requires further investigation. Recent evidence has since emerged which supports the argument that cyt-b5 acts to increase the efficiency of the 17,20-lyase reaction [60] presumably by influencing the interaction between CYP17A1 and POR. While investigating the mechanisms of the 17a-hydroxylase and 17,20lyase reactions, Khatri et al. (2014) showed that the 17ahydroxylation of pregnenolone is highly coupled (97%), but the 17,20-lyase of 17a-hydroxypregnenolone is not (4%) [60]. Based on this data together with the cyt-b5 induced conformational changes observed in their own NMR studies, Estrada et al. (2014) postulated that cyt-b5 may well drive subtle modulations in the conformation of CYP17A1, leading to a decrease in 17,20-lyase uncoupling [57]. Indeed cyt-b5 has been shown to stimulate catalysis of other cytochromes P450 by decreased uncoupling, albeit in these cases cyt-b5 itself serves as an electron donor [9]. In the case of CYP17A1 such a mechanism would account for the importance of POR [40,48–50] as well as an allosterically induced conformational change [39,54]. The degree of uncoupling, associated with the 17,20-lyase activity towards 5a-pregnan-3a,17a-diol-20-one, has yet to be determined. It is entirely likely that this reaction demonstrates less uncoupling than the 17,20-lyase reaction towards 17a-hydroxypregnenolone, thereby accounting for its greater activity and the diminished effect of cyt-b5 [35]. In the light of recent data it is clear that cyt-b5 binds to CYP17A1, thereby inducing a conformational change, which may allow for the optimal orientation of the substrate, but certainly enhances the binding and electron transfer from POR. In addition, there appears to be two way communication between the active site and the redox binding site as substrate binding appears to play a previously unidentified role in the recruitment of cyt-b5. The weaker association between cyt-b5 and CYP17A1 in the presence of the 17,20-lyase substrate, 17a-hydroxypregnenolone, than the 17ahydroxylase substrate, pregnenolone, together with the apparent competition between cyt-b5 and POR for the same binding site [43,57] suggests that cyt-b5 might dissociate from CYP17A1 prior to POR binding, but only after cyt-b5 has induced a conformational change within CYP17A1. Conversely, a more recent study by Peng et al. (2014) demonstrated that cross-linking between CYP17A1 and cyt-b5 was influenced by the substrate and was most efficient in the presence of the 17,20-lyase substrate, 17ahydroxypregnenolone [44]. While the results obtained by these studies differed in terms of which substrates have the biggest effect on cyt-b5 binding, both confirmed that substrate binding plays a role in CYP17A1-b5 complex formation [44,57]. The observed differences in substrate effects are likely due to experimental differences. Notably the study by Peng et al. (2014) used full-length cyt-b5, while that of Estrada et al. (2014) used truncated cyt-b5 which is not necessarily reflective of the in vivo reality [44,57]. Our group has demonstrated that, the co-expression of CYP17A1 with a cyt-b5 construct in which the globular head domain had been removed, was able to stimulate 17,20-lyase activity, albeit less efficiently than full length cyt-b5 (Fig. 1). This finding demonstrated that regions of the C-terminal linker and membrane binding domains are able to associate with CYP17A1 and likely contribute to the conformational change induced by cyt-b5 binding [46]. Similarly, although the interaction of POR with cytochrome P450 is largely dependent on electrostatic interactions, the removal of the N-terminal hydrophobic domain abolishes the ability of POR to reduce cytochrome P450, but not in the case of cytochrome c [33].

The importance of the membrane binding domains should therefore not be underestimated when investigating these complex systems. The proposal that cyt-b5 and POR are able to bind to CYP17A1 at the same time, therefore remains a possibility, although the structure of such a quaternary complex is yet to be determined and remains a significant challenge due to the hydrophobic nature of the proteins. 2.3. 16-ene-synthase: a different mechanism for a different activity? Human, bovine and porcine CYP17A1 all possess 16-enesynthase activity, though this activity is most pronounced in the porcine enzyme. Cyt-b5 is essential for the 16-ene-synthase activity of CYP17A1 and has a greater effect on this reaction than on the 17,20-lyase reaction [22,61–63]. A number of observations suggest that the mechanism by which cyt-b5 stimulates the 16-ene activity of CYP17A1 might well be different to that of the mechanism involved in the 17,20-lyase reaction. The 16-enesynthase reaction is not sensitive to POR, but appears to be completely dependent on cyt-b5 [22,61–64]. This observation suggests that in the case of 16-ene-synthase activity, cyt-b5 may well function as an electron donor. The observation that 16-enesynthase activity is significantly increased in the presence of cyt-b5 by the addition of cyt-b5 reductase, supports this theory [61]. These findings suggest that, instead of facilitating the interaction between POR and CYP17A1 for efficient electron transfer, as is the case for the 17,20-lyase activity, the 16-ene-synthase activity requires cyt-b5 to act as an electron shuttle between cyt-b5 reductase and CYP17A1. 3. Cyt-b5 increased the affinity of 3bHSD towards its cofactor, NAD+ A novel role was recently reported for cyt-b5 when our investigations into caprine and ovine steroidogenesis revealed the interaction of cyt-b5 with adrenal 3bHSD. We demonstrated that cyt-b5 could significantly increase the D5-hydroxysteroid to D4-ketosteroid conversion in non-steroidogenic COS-1 cells co-expressing either ovine or caprine 3bHSD and cyt-b5 (Fig. 2) [15]. Our study also showed that the degree of stimulation varied between the two species and between the respective substrates, indicating that augmentation occurred in a substrate and species specific manner.

Fig. 2. Cyt-b5 augments the activity of 3bHSD. COS-1 cells were cotransfected with ovine 3bHSD and ovine cyt-b5 or apo-b5 (H68A mutation) at a ratio of 4:1 (cytb5:3bHSD). The fold increase in progesterone production from pregnenolone was calculated relative to the activity of ovine 3bHSD in the absence of cyt-b5. Modified from [15].

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Unlike the cytochromes P450, 3bHSD does not require electrons from an external electron donor for catalytic activity [65–67] thus eliminating the role of electron transfer in the augmentation of 3bHSD activity by cyt-b5. In addition, our group showed that apocytochrome-b5 could stimulate 3bHSD-activity, indicating that stimulation occurs via an allosteric mechanism (Fig. 2). Investigating the influence of cyt-b5 on the kinetic parameters of the 3bHSD catalysed reactions, it was shown that the apparent Km values for the different D5-hydroxysteroids did not change, but that the apparent Vmax values were significantly increased [15]. Structural homology modelling and theoretical computer modelling led to the deduction that the apparent Vmax for the 3bHSD-catalysed reactions could be increased by decreasing the KNAD+ value without altering the Km value for the respective steroid substrates. Subsequent studies with purified ovine 3bHSD and cyt-b5 confirmed that cyt-b5 significantly (3.5-fold) decreased the Km of 3bHSD for NAD+ [16]. The cyt-b5-induced increase in affinity for its cofactor is an elegant mechanism by which 3bHSD activity can be regulated, especially since the activity of hydroxysteroid dehydrogenases (HSDs) are believed to be modulated by the abundance of co-factor as well as the relative affinity of these enzymes towards their co-factors [68]. A comprehensive review of the mechanism by which cyt-b5 modulates 3bHSD activity can be found in Storbeck et al., 2013 [56]. 4. Different structural features of cyt-b5 are required for its functional promiscuity Cyt-b5 consists of three distinct domains: an N-terminal hemecontaining soluble domain, a C-terminal membrane anchor and a linker region which connects the head and membrane binding domains [69,70]. Anionic cyt-b5 residues in the heme containing globular head domain are essential for protein–protein interaction [41,71]. However, the exact residues required differ between P450s supporting the multifunctional role of cyt-b5 [9,72]. Intriguingly, the cyt-b5 residues involved in binding a specific P450 do not necessarily correlate to a specific mechanism [72]. Cyt-b5 enhances the activity of CYP17A1 and CYP2C19 without electron transfer, while CYP2E1 requires direct electron transfer from cyt-b5 [47,71,72]. The interaction of cyt-b5 with CYP2E1 and CYP2C19, however, requires residues D58 and D65, while E48 or E49 are required for binding CYP17A1. It remains to be determined if anionic cyt-b5 residues are involved in cyt-b5's interaction with 3bHSD. While the identity of the specific conserved anionic cyt-b5 residues required for interaction with CYP17A1 is well established, a factor which is often over looked is the influence of other non-conserved residues which differ between species. Studies with CYP17A1 often make use of POR and/or cyt-b5 from other species, however, the results may well be skewed. We recently demonstrated this by investigating the ability of cyt-b5 from four different species to enhance the 17,20-lyase activity of porcine CYP17A1 (Fig. 3). A significant decrease in 17,20-lyase stimulation correlated well to decreased sequence identity in relation to porcine cyt-b5. Caprine cyt-b5 (90.2% identity) only elicited a 1.5-fold increase in 17,20-lyase activity compared to porcine cyt-b5's 5.3-fold increase (unpublished data). It is therefore clear that the overall fold of cyt-b5 is important for its function. The importance of the structural fold is especially not difficult to imagine given the proposed mechanism by which cyt-b5 induces a structural change in CYP17A1. The C-terminal tail membrane anchoring domain and the linker domain have been shown to play an important role in the stimulation of CYP17A1 17,20-lyase activity, but not of 3bHSD

5

Fig. 3. Cyt-b5 sequence homology influences the degree of CYP17A1 17,20-lyase stimulation. COS-1 cells were cotransfected with porcine CYP17A1 and either pig, rat, goat or human cyt-b5. Sequence identity was compared to pig cyt-b5 which was set at 100%. The fold increase in androstenedione production from progesterone was calculated relative to the activity of porcine CYP17A1 in the absence of cyt-b5. All experiments were performed as previously described [55].

activity – 3bHSD activity was stimulated equally in the presence of full-length cyt-b5 (134 residues) or truncated cyt-b5 (84 residues) [15]. Conversely, similar studies with CYP17A1 showed that truncated cyt-b5 (93 residues) could only partially stimulate 17,20-lyase activity (Fig. 1) [46], confirming previous evidence for the importance of this domain [33,58,73]. The linker domain has also been implicated in the ability of cyt-b5 to form homomeric complexes in vivo and also contributes to the stimulation of CYP17A1 17,20-lyase activity [46]. The ability of cyt-b5 to modulate a vast array of enzymatic activities is therefore clearly not due to a “one size fits all” approach. Instead conserved anionic residues, the presence of heme, the overall structural fold of all three domains as well as the orientation of the protein relative to the membrane, all contribute to cyt-b5's versatility and ability to interact with cytochrome P450 and HSD enzymes in an enzyme specific manner. 5. The influence of cyt-b5 on steroidogenesis The most well defined role of cyt-b5 within adrenal steroidogenesis is the augmentation of the 17,20-lyase reaction catalysed by CYP17A1. This role is particularly evident in humans and higher primates during adrenarche, in which a significant increase in adrenal DHEA and DHEAS production results due to the expansion and differentiation of the adrenal zona reticularis [30]. Two of the key factors allowing for the dramatic increase in the production of the D5-steroids by the zona reticularis are the up-regulation of cyt-b5 expression and the down-regulation of 3bHSD expression [28,74,75]. These changes promote the production of C19 steroids through the 17,20-lyase activity of CYP17A1, while preventing the conversion of D5-steroids to D4-steroids, respectively. Although the primary C19 steroids produced by the human adrenal are DHEA and DHEAS, substantial amounts of the D4-steroids, androstenedione and 11b-hydroxyandrostenedione, are also produced [76–78]. Together with DHEA and DHEAS, these steroids contribute to the pool of circulating androgen precursors [79,80]. The production of all C19 steroids requires the 17,20-lyase activity of CYP17A1, which is enhanced in the zona reticularis by the expression of cyt-b5 [28,75,81]. In addition, D4-steroid production requires the conversion of DHEA to androstenedione by the activity 3bHSD, with 11b-hydroxyandrostenedione requiring a further hydroxylation by CYP11B1 [77,82,83]. The expression of 3bHSD in the zona reticularis is, however, very low [28,74]. The stimulation of 3bHSD by cyt-b5 is seemingly small (less than 2-fold) when expressed at a 1:1 ratio. Stimulation is, however, increased up to

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3-fold at higher cyt-b5:3bHSD ratios (Fig. 2) [15], which are representative of conditions within the zona reticularis. Cyt-b5 may therefore play a dual role in the production of C19 steroids by the zona reticularis. Apart from ensuring optimal 17,20-lyase activity, the co-expression of cyt-b5 with low levels of 3bHSD likely ensures optimal 3bHSD activity, thereby accounting for adrenal D4-steroid production. Unlike the zona reticularis, the Leydig and theca cells produce androstenedione as a precursor to testosterone and oestrogens, respectively. 3bHSD expression is therefore higher in these cells and co-expression with cyt-b5 may further ensure the optimal CYP17A1 and 3bHSD activity [56]. The expression of cyt-b5 in the Leydig cells of porcine testes has a profound effect on steroidogenesis. The absence of cyt-b5 in boar testes favours androgen production via the 17,20-lyase activity of CYP17A1, while high levels of cyt-b5 result in androstadienol production by the 16-enesynthase activity of porcine CYP17A1 [22,63,64,84,85]. Androstadienol is a precursor to the pheromone androstenone, which accumulates in the fat tissue of uncastrated boars and results in an unpleasant odour in meat known as boar taint [85]. Studies have shown that cyt-b5 expression in the Leydig cells of pigs is strongly associated with androstenone production and accumulation [64,86,87] due to the dependency of CYP17A1's 16-ene-synthase activity on cyt-b5 [61,63]. Androstadienol production has also been reported in human testis, albeit at significantly lower levels than produced by the pig [63,88]. It is clearly evident that cyt-b5 plays an essential, yet diverse role in the regulation of mammalian steroidogenesis, yet further insight into the importance of cyt-b5 can nevertheless be gleaned from clinical studies involving inactivating cyt-b5 mutations. A number of case studies will be presented, highlighting the importance of cyt-b5 in regulating the 17,20-lyase activity of human CYP17A1. 6. Inactivating cyt-b5 mutations cause true 17,20-lyase deficiency One of the first reports of human 17a-hydroxylase deficiency was by Biglieri et al. in 1966 [89] while Geller et al. reported a form of 17,20-lyase deficiency in 1997 [38]. It was initially thought that true 17,20-lyase deficiencies could be characterised by CYP17A1 mutations, while apparent 17,20-lyase deficiencies were ascribed to mutations in POR [90]. Given the pivotal role cyt-b5 plays in the activation of the CYP17A1 catalysed 17,20-lyase reaction, the search for mutations in cyt-b5, as an additional cause for 17,20-lyase deficiency, was a logical consequence. Earlier reports on a patient with cyt-b5 deficiency were published in 1986 and 1994 [91,92]. These studies, however, focused on methaemoglobinemia and did not report on adrenal or gonadal steroidogenesis. Giordano et al. did, however, observe pseudohermaphroditism in the patient [92]. It was only in 2010 that the first case of isolated 17,20-lyase deficiency, resulting from a cyt-b5 mutation, was reported. Kok et al. identified a consanguineous 46,XY infant homozygous for the cyt-b5 mutation W27X, which led to the introduction of a premature stop codon. The 17a-hydroxylase activity of CYP17A1 was not affected, but impaired 17,20-lyase function resulted in hypergonadotropic hypogonadism and impaired adrenal and gonadal C19 steroid production. As a result the patient was diagnosed with a 46,XY disorder of sex development (DSD) [31]. Idkowiak et al. subsequently identified three siblings with isolated CYP17A1 17,20-lyase deficiency [32]. It was found that the 17a-hydroxylase activity in these patients was not affected and that they exhibited normal glucocorticoid production. However, low levels of urinary androgen metabolites were reported, pointing to a 17,20-lyase deficiency accounting for the

low circulating levels of androgens reported in these patients. No mutations were found in the CYP17A1 and POR genes of these individuals. A missense mutation at the start of exon 2 of the CYPB5A gene, resulting in a H44L substitution, was, however, indicated. It was concluded that this mutation resulted in defective heme binding and subsequent inactivation of the protein due to the introduction of significant conformational changes. A subsequent comparison of all previously reported cases, with apparent isolated 17,20-lyase deficiency due to CYP17A1 or POR mutations, led to the conclusion that true 17,20-lyase deficiency is only observed in the case of inactivating cyt-b5 mutations [32]. This finding underlines the importance of cyt-b5 in normal steroidogenesis and the important role this haemoprotein plays in the promiscuous catalytic activity of CYP17A1. 7. Conclusion From the preceding discussion it is apparent that cyt-b5 can alter not only the specificity of single cytochrome P450-dependent enzymes during steroidogenesis, but that it can also alter the activity of other important enzymes crucial for normal steroid hormone homeostasis. The modulation of multiple reactions within steroidogenesis is accomplished by multiple and diverse reaction mechanisms. The resulting changes in enzyme specificity and affinity are species and substrate specific, thereby increasing the versatility and regulatory complexity of this important group of bio-catalysts. References [1] D. Keilin, E.F. Hartree, Effect of low temperature on the absorption spectra of haemoproteins; with observations on the absorption spectrum of oxygen, Nature 164 (4163) (1949) 254–259. [2] A.M. Pappenheimer Jr., C.M. Williams, The effects of diphtheria toxin on the Cecropia silkworm, J. Gen. Physiol. 35 (5) (1952) 727–740. [3] D.E. Hultquist, P.G. Passon, Catalysis of methaemoglobin reduction by erythrocyte cytochrome b5 and cytochrome b5 reductase, Nature 229 (8) (1971) 252–254. [4] S.R. Keyes, D.L. Cinti, Biochemical properties of cytochrome b5-dependent microsomal fatty acid elongation and identification of products, J. Biol. Chem. 255 (23) (1980) 11357–11364. [5] F. Paltauf, R.A. Prough, B.S.S. Masters, J.M. Johnston, Evidence for the participation of cytochrome b5 in plasmalogen biosynthesis, J. Biol. Chem. 249 (8) (1974) 2661–2662. [6] H. Fukushima, G.F. Grinstead, J.L. Gaylor, Total enzymic synthesis of cholesterol from lanosterol cytochrome b5-dependence of 4-methyl sterol oxidase, J. Biol. Chem. 256 (10) (1981) 4822–4826. [7] T. Kawano, Y. Kozutsumi, T. Kawasaki, A. Suzuki, Biosynthesis of Nglycolylneuraminic acid-containing glycoconjugates. Purification and characterization of the key enzyme of the cytidine monophospho-Nacetylneuraminic acid hydroxylation system, J. Biol. Chem. 269 (12) (1994) 9024–9029. [8] J.B. Schenkman, I. Jansson, Interactions between cytochrome P450 and cytochrome b5, Drug Metab. Rev. 31 (2) (1999) 351–364. [9] J.B. Schenkman, I. Jansson, The many roles of cytochrome b5, Pharmacol. Ther. 97 (2) (2003) 139–152. [10] A. Ito, R. Sato, Purification by means of detergents and properties of cytochrome b5 from liver microsomes, J. Biol. Chem. 243 (18) (1968) 4922–4923. [11] A. Ito, Cytochrome b5-like hemoprotein of outer mitochondrial membrane; OM cytochrome b. I. Purification of OM cytochrome b from rat liver mitochondria and comparison of its molecular properties with those of cytochrome b5, J. Biochem. 87 (1) (1980) 63. [12] D. Garfinkel, A comparative study of electron transport in microsomes, Comp. Biochem. Physiol. 34 (1963) 367–379. [13] S.J. Giordano, A.W. Steggles, The human liver and reticulocyte cytochrome b5 mRNAs are products from a single gene, Biochem. Biophys. Res. Commun. 178 (1) (1991) 38–44. [14] S.J. Giordano, A.W. Steggles, Differential expression of the mRNAs for the soluble and membrane-bound forms of rabbit cytochrome b5, Biochim. Biophys. Acta 1172 (1–2) (1993) 95–100. [15] P. Goosen, K. Storbeck, A.C. Swart, R. Conradie, P. Swart, Cytochrome b5 augments 3b-hydroxysteroid dehydrogenase/D5-D4 isomerase activity, J. Steroid Biochem. Mol. Biol. 127 (3–5) (2011) 238–247. [16] P. Goosen, A.C. Swart, K. Storbeck, P. Swart, Allosteric interaction between 3bhydroxysteroid dehydrogenase/D5-D4 isomerase and cytochrome b5 influences cofactor binding, FASEB J. 27 (2013) 322–332.

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Please cite this article in press as: K.-H. Storbeck, et al., Cytochrome b5 modulates multiple reactions in steroidogenesis by diverse mechanisms, J. Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.11.024