UCSF DIABETES, ENDOCRINOLOGY & METABOLISM TRAINING PROGRAM FACULTY RESEARCH SUMMARIES

My laboratory studies the molecular biology of human steroid hormone biosynthesis. Initial work concerned the identification of steroidogenic enzymes and the cloning of their cDNAs and genes. This included cDNAs or genes for the cholesterol side-chain cleavage enzyme (P450scc), 17alpha hydroxylase/ 17,20 lyase (P450c17), 21 hydroxylase (P450c21), adrenodoxin reductase, adrenodoxin, steroidogenic acute regulatory protein (StAR), vitamin D 1alpha hydroxylase (P450c1alpha) and two novel transcription factors (LBP-1b and LBP-9). We found that the extracellular matrix protein Tenascin-X is one of several novel genes in the P450c21 gene locus. The regulation of each steroidogenic gene was studied at the levels of activity, mRNA expression and fine promoter analysis. Mutations were sought causing human disease. My laboratory provided key evidence showing that gene conversion was the principal mechanism causing 21-hydroxylase deficiency and was first to determine the genetic basis of several diseases, including lipoid adrenal hyperplasia (StAR), isolated 17,20 lyase deficiency (electron transfer to P450c17), recessive Ehlers-Danlos Syndrome (Tenascin-X), vitamin D-dependent rickets (P450c1alpha), and Antley-Bixler Syndrome (P450 oxidoreductase).

Present work focuses on three questions.

(1) What is the mechanism of action of StAR? Steroidogenic cells store little steroid, hence the acute regulation of steroidogenesis, such as the secretion of cortisol in response to ACTH, is mediated at the level of access of cholesterol to the first enzyme, P450scc, in the mitochondria. The influx of cholesterol is regulated by StAR, which exerts its actions on the outer mitochondrial membrane before being imported. We are determining what proteins StAR interacts with, what conformational changes it undergoes, and how these move cholesterol into mitochondria.

(2) How is 17,20 lyase activity regulated? The 17,20 lyase activity of P450c17, which is required for all sex steroid synthesis, is regulated post-translationally by factors that influence its association with P450 oxidoreductase. One of these is the phosphorylation of P450c17 itself. We are identifying the relevant kinase and its site of action, as this kinase may also play a significant role in the polycystic ovary syndrome.

(3) Does P450 oxidoreductase (POR) play a significant role in the genetic variations in drug responses (pharmacogenomics)? POR transfers electrons to all microsomal P450s, including some steroidogenic enzymes and the major hepatic drug-metabolizing enzymes. Although POR knockout mice are embryonic lethal, we found POR mutations causing a steroidogenic disorder. We are now identifying and characterizing the enzymatic activities of POR mutants and polymorphisms in ethnically diverse normal individuals.

Selected References

Flück CE, Tajima T, Pandey AV, Arlt W, Okuhara K, Verge CF, Jabs EW, Mendonca BB, Fujieda K, Miller WL. Mutant P450 oxidoreductase causes disordered steroidogenesis with and without Antley-Bixler syndrome. Nature Genetics 36:228-230, 2004.

Yaworsky DC, Baker BY, Bose HS, Best KB, Jensen LB, Bell JD, Baldwin MA, Miller WL. pH-dependent interaction of the carboxyl-terminal helix of steroidogenic acute regulatory protein with synthetic membranes. J Biol Chem 280: 2045-2054, 2005.

Pandey AV, Miller WL. Regulation of 17,20 lyase activity by cytochrome b 5 and by serine phosphorylation of P450c17. J Biol Chem 280: 13265-13271, 2005.

Huang N, Pandey AV, Agrawal V, Reardon W, Lapunzina PD, Mowat D, Jabs EW, Van Vliet G, Sack J, Flück CE, Miller WL. Diversity and function of mutations in P450 oxidoreductase in patients with Antley-Bixler syndrome and disordered steroidogenesis. Am J Hum Genet 76: 729-749, 2005.

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