This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cytochrome P450c17 catalyzes both 17-hydroxylation and 17,20 oxidative cleavage of C21 steroids, in both human adrenal glands and gonads. While adrenal 17-hydroxylase activity remains fairly constant after birth, adrenal lyase activity, reflected by serum dehydroepiandrosterone sulfate, rises during adrenarche at about age 8 and then declines progressively after age 50. We hypothesize that a post-translational modification of P450c17 regulates its lyase activity. Recently, our laboratory showed that P450c17 is phosphorylated in NCI-H295 cells and that treatment of human fetal adrenal microsomes with alkaline phosphatase abolishes lyase activity without affecting 17-hydroxylation. These data suggest that phosphorylation/dephosphorylation regulates the lyase activity of P450c17. To study the structural and functional consequences of this process in detail, we must first determine the sites and extent of P450c17 phosphorylation. We propose to use bacterially expressed, His-tagged, catalytically active (i.e. has 17-alpha-hydroxylase activity) human P450c17, before and after treatment with the enriched kinase fraction. The proteins will be treated with proteolytic enzymes and the peptides will be characterized by LC/MS or other mass spectrometric methods. Phosphopeptides will be identified by mass differences specific to phosphorylation that are present in the samples treated with the enriched kinase fraction and absent in the dephosphorylated samples. The extend of phosphorylation will be determined by comparing the peptide peak area ratios of the dephosphorylated/phosphorylated peptide of interest. Results will direct site-directed mutagenesis experiments to systematically abolish phosphorylation sites and to study the functional consequences of such mutations. We will conduct enzymatic experiments to determine the precise step(s) in the P450 catalytic cycle altered by the key phosphorylation events. Finally, we will incorporate results into a computer graphics model of P450c17, constructed by assembling consensus core structural units found in bacterial P450s, to understand the structural basis for the changes in activity regulated by phosphorylation. These data will enable us to better understand and treat clinical problems involving dysregulation and inhibition of androgen biosynthesis, such as the polycystic ovary syndrome and prostatic hyperplasia or cancer.
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