We propose to thoroughly define the structural components of human P450c17 (17alpha-hydroxylase/17,20 lyase) that contribute to its two activities and to identify the physiologic factors that regulate the ratio of these two activities. In early childhood, adrenal P450c17 activity is limited almost exclusively to 17-hydroxylation. During adrenarche, adrenal synthesis of the weak androgen DHEA commences, resultant from acquisition of 17,20 lyase activity. The molecular basis for this shift in P450c17 activities, however, remains enigmatic. Based on data from site-directed mutagenesis experiments, we hypothesize that post-translational modification generates the physiologically relevant change in the three- dimensional structure of P450c17 that leads to the rise in adrenal 17,20 lyase activity during adrenarche. Preliminary data from the mentor's laboratory suggest that P450c17 undergoes hormone-responsive phosphorylation. We propose to characterize post-translational modifications of P450c17, regulation of this modification, and its effect on P450c17 activities. We will study how structure relates to function by building a computer model of P450c17 based on X-ray crystal structures of the 3 soluble P450's. We will substitute core structural units (alpha- helicies and beta-sheets) of homologous regions in P450c17 for those in P450's -cam, -terp, and -BM3; energy minimize; and add connecting loops. We will use the model to identify residues surrounding the substrate binding pocket likely to participate in substrate binding, reduction by P450-oxidoreductase, and catalysis for each of the 2 separate activities. Finally, we will test the model using site-directed mutagenesis to introduce structural changes predicted to alter one of the two activities. We will thoroughly characterize the activities of each mutant and post- translationally modified protein, including inhibition by alternate substrates and oxygenation at adjacent carbon atoms. We will incorporate the data from mutagenesis and modification experiments back into the model to refine our structure of P450c17. This proposal teams a PI with graduate training in steroid biochemistry, enzymology, and spectroscopic methods, a mentor who is a leader in the molecular biology of human steroidogenesis, and a collaborator with expertise in crystallography and computer modeling P450 enzymes. This project will complete the PI's training in molecular biology and preparation for independent investigation; improve the technology of building structural protein models based on known structures of related proteins; and advance our understanding of and ability to modulate androgen (and consequently estrogen) biosynthesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08DK002387-03
Application #
2770279
Study Section
Special Emphasis Panel (SRC)
Program Officer
Hyde, James F
Project Start
1996-09-01
Project End
2001-08-31
Budget Start
1998-09-10
Budget End
1999-08-31
Support Year
3
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Pediatrics
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Costa-Santos, Marivania; Kater, Claudio E; Dias, Eduardo P et al. (2004) Two intronic mutations cause 17-hydroxylase deficiency by disrupting splice acceptor sites: direct demonstration of aberrant splicing and absent enzyme activity by expression of the entire CYP17 gene in HEK-293 cells. J Clin Endocrinol Metab 89:43-8
Costa-Santos, Marivania; Kater, Claudio E; Auchus, Richard J et al. (2004) Two prevalent CYP17 mutations and genotype-phenotype correlations in 24 Brazilian patients with 17-hydroxylase deficiency. J Clin Endocrinol Metab 89:49-60
Khan, Naveed; Sharma, Kamalesh K; Andersson, Stefan et al. (2004) Human 17beta-hydroxysteroid dehydrogenases types 1, 2, and 3 catalyze bi-directional equilibrium reactions, rather than unidirectional metabolism, in HEK-293 cells. Arch Biochem Biophys 429:50-9
Fluck, Christa E; Miller, Walter L; Auchus, Richard J (2003) The 17, 20-lyase activity of cytochrome p450c17 from human fetal testis favors the delta5 steroidogenic pathway. J Clin Endocrinol Metab 88:3762-6
Gupta, Manisha K; Guryev, Oleg L; Auchus, Richard J (2003) 5alpha-reduced C21 steroids are substrates for human cytochrome P450c17. Arch Biochem Biophys 418:151-60
Sherbet, Daniel P; Tiosano, Dov; Kwist, Kerri M et al. (2003) CYP17 mutation E305G causes isolated 17,20-lyase deficiency by selectively altering substrate binding. J Biol Chem 278:48563-9
Wilson, Jean D; Auchus, Richard J; Leihy, Michael W et al. (2003) 5alpha-androstane-3alpha,17beta-diol is formed in tammar wallaby pouch young testes by a pathway involving 5alpha-pregnane-3alpha,17alpha-diol-20-one as a key intermediate. Endocrinology 144:575-80
Mathieu, Axel P; LeHoux, Jean Guy; Auchus, Richard J (2003) Molecular dynamics of substrate complexes with hamster cytochrome P450c17 (CYP17): mechanistic approach to understanding substrate binding and activities. Biochim Biophys Acta 1619:291-300
Auchus, Richard J; Sampath Kumar, A; Andrew Boswell, C et al. (2003) The enantiomer of progesterone (ent-progesterone) is a competitive inhibitor of human cytochromes P450c17 and P450c21. Arch Biochem Biophys 409:134-44
Arlt, Wiebke; Martens, John W M; Song, Maengseok et al. (2002) Molecular evolution of adrenarche: structural and functional analysis of p450c17 from four primate species. Endocrinology 143:4665-72

Showing the most recent 10 out of 20 publications