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    • The consensus amino acid recognition sequence for p

    2023-09-19

    The consensus amino cyp450 inhibitors recognition sequence for p38α substrates is (Ser/Thr)Pro (Cuadrado and Nebreda, 2010), typically assisted by upstream docking motifs (Remenyi et al, 2005, Sharrocks et al, 2000). P450c17 has 32 Ser and 25 Thr residues, of which only Thr 341 and Ser 427 are immediately followed by Pro (Chung et al., 1987). However, when these residues were mutagenized to Ala, either singly (T341A; S472A) and in combination (T341A + S427A), the corresponding P450c17 proteins could still be phosphorylated in vitro by p38α to the same degree as wild-type P450c17, and all three mutants retained their p38α-induced acquisition of 17,20 lyase activity at a level indistinguishable from wild-type P450c17 (Tee and Miller, 2013). Thus p38α phosphorylates P450c17 at an as-yet unidentified, non-canonical site(s). Thus at least three mechanisms, all governing electron transfer from POR to P450c17, regulate the ratio of 17,20 lyase activity to 17α-hydroxylase activity: first, 17,20 lyase activity is favored by high molar ratios of POR to P450c17; second, cytochrome b5 acts as an allosteric factor to promote the interaction of POR and P450c17, favoring 17,20 lyase activity; third, 17,20 lyase activity is facilitated by the phosphorylation of serine and/or threonine residues on P450c17 by p38α. It must be noted, however, that while the roles of POR and cytochrome b5 identified in vitro have been confirmed by human loss-of-function mutations in vivo, no corresponding human experiment of nature has yet confirmed the role of P450c17 phosphorylation.
    Future directions Two human hyperandrogenic states that have resisted molecular characterization are premature exaggerated adrenarche and PCOS. Adrenarche is the pre-pubertal induction of the synthesis of adrenal C19 steroids (mainly DHEA and DHEAS), accompanied by growth of the adrenal zona reticularis and an induction of the transcription of the gene for cytochrome b5 (reviewed by Miller, 2009 and by Nakamura et al., 2009). PCOS is a common hyperandrogenic disorder affecting about 6% of women of reproductive age, principally characterized by hyperandrogenism and associated with insulin resistance and metabolic syndrome (reviewed by Diamanti-Kandarakis and Dunaif, 2012 and by Legro et al., 2013). Both of these conditions are characterized by an overproduction of DHEA and other C19 steroids (Goodarzi et al., 2014), and data with primary cultures of theca cells from normal and PCOS women indicate an overproduction of P450c17 (Nelson et al, 1999, Nelson et al, 2001). CYP17A1 gene transcription appears to be increased in PCOS theca cells (Wickenheisser et al, 2000, Wickenheisser et al, 2004), and may be responsive to MAP kinases such as p38 (Nelson-Degrave et al., 2005). Recent genome-wide association studies (GWAS) have implicated a gene called DENND1A in PCOS (Chen et al, 2011, Goodarzi et al, 2012, Louwers et al, 2013, Shi et al, 2012, Welt et al, 2012). DENND1A yields two principal transcripts: variant 1 (V1), encodes a 1009 AA protein (connecdenn 1) with a clathrin-binding domain thought to facilitate endocytosis, and C-terminal proline-rich domain; variant 2 (V2) encodes a 559 AA protein that contains the clathrin-binding domain, but lacks the proline-rich domain (Marat et al., 2011). V2 mRNA and protein are over-expressed in PCOS theca cells and V2 mRNA is abundant in urinary exosomes of women with PCOS but not normally-cycling women (McAllister et al., 2014). Over-expression of V2 in normal theca cells increases the abundance of P450c17 mRNA, augments DHEA production and increases the activity of transfected CYP11A1 and CYP17A1 promoter–reporter constructs and siRNA knockdown of V2 in PCOS theca cells reversed the PCOS phenotype (McAllister et al., 2014). Thus the DENND1A gene appears to be involved in PCOS and to affect P450c17 directly. We propose that DENND1A acts upstream from a MAP kinase signaling pathway, possibly involving ROCK1, that leads to activation of p38α, which then increases both 17,20 lyase activity by phosphorylating P450c17 and increases the amount of P450c17 by increasing CYP17A1 gene transcription (Fig. 6). These possibilities are under investigation.