Medical Student Research Fellowship for Summer 2007

Mentor: Richard J. Auchus
Department: Internal Medicine/Endocrinology & Metabolism
Room number: Y9.308
Mail Code: 8857
Phone number: 8-6751
E-mail: richard.auchus@UTSouthwestern.edu
Project title: biochemistry, physiology, and genetics of human steroid biosynthesis

Human subjects IRB approved project number (where applicable): 0902-505, 1203-776, 1203-811

Animal subjects IRB approved project number (where applicable): N/A

Project Type (patient-based research, animal-based research, or basic research; this characterization is only to permit a general classification for grouping similar types of projects)

basic research and patient-based research

Brief Description of Project:

Basic:
We are interested in determining the biochemical principles and structural features that govern how hydroxysteroid dehydrogenases function in living cells as primarily oxidative or reductive enzymes. This project involves site-directed mutagenesis and modification of tissue culture conditions to alter the directional preference of these enzymes in vivo. We then perform detailed in vivo and in vitro enzymology studies of the altered enzymes to understand why these changes are observed. We are growing crystals of the mutant enzymes complexed with nicotinamide cofactors to determine how these structural changes influence cofactor affinities and thus alter the enzyme's physiology.

Clinical:
We are developing liquid chromatography/tandem mass spectrometry (LC-MS/MS) assays for serum mineralocorticoids to define the physiology and genetics of mineralocorticoid-dependent hypertension, in collaboration with Ronald Victor in the Hypertension Division and the Dallas Heart Study. This study also involves a drug challenge with eplerenone and aliskerin in a crossover design, followed by biochemical profiling of each patient's mineralocorticoid production pattern. We thus intend to determine biochemical signatures that predict response to a specific antihypertensive drug, and we will then work backwards to deduce the genetic basis for this pharmacogenetic effect.

We are also studying congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21OHD). Many subjects with severe or complete 21OHD who cannot make any cortisol or aldosterone as children somehow develop the capacity to produce nearly normal amounts of these steroids in adulthood. We believe that certain hepatic cytochromes P450 (CYP2C enzymes) with 21-hydroxylase activity can compensate in adults with 21OHD and that genetic variants in these enzymes modify the severity of 21OHD in adults. To test this hypothesis, we will bring adults with severe 21OHD into the GCRC and stop their therapy for 2 days. We will measure steroid hormones and precursors in blood and urine and correlate these values with their CYP2C genotypes.

Previous Research Activities or Publications with Medical Students:
Tim C. Lee worked with me for a year after graduating college and during the summer after his first year in medical school (1996-1998). He and I developed our yeast system for studying enzymology of steroidogenic cytochromes P450 (Auchus et al 1998 J Biol Chem 273:3158-3165; Lee et al 1999 J Clin Endocrinol Metab 84:2104-2110). Tim received the Academic Pediatric Society/Society for Pediatric Research (APS/SPR) Student Research Award in 1999 for this work. Tim was chief resident in pediatrics at UCLA and is now in private practice.
Kavita Vyas, worked in my lab the summer of 2000, characterizing the biochemistry of fusion proteins to probe the mechanism of action of cytochrome b5 on CYP17. Kavita won the award for best poster at the 39th Medical Student Research Forum, which was presented at the Endocrine Society meeting in June 2001, and she is now an MS4 at Columbia and applying for residency in Internal Medicine.
Daniel Sherbet worked in my lab for 2 summers (2001-2002, characterizing a mutation in CYP17 that causes isolated 17, 20-lyase deficiency by a novel mechanism. Daniel's oral presentation at the 40th Medical Student Research Forum was selected as the best talk of the day. The work was presented at the Endocrine Society meeting in June, 2002 and published (Sherbet et al J Biol Chem 278:48563-48569). Daniel spent 2004-05 as a medical student research Fellow of the Howard Hughes Medical Institute studying hydroxysteroid dehydrogenases (HSDs). He and I have a book chapter and review article (Mol Cell Endocrinol) in press and are writing a manuscript about his HSD work. He is an Internal Medicine intern at UTSW and is planning to do endocrinology or cardiology.
Kristen Bruce, studied (-)-progesterone (the enantiomer of progesterone) as an inhibitor of CYP17 and CYP21 in 2001 and started our work on HSD directionality. Her work on (-)-progesterone was published (Auchus et al Arch Biochem Biophys 409:134-144). She is now in dental school.
David Stidd worked here in 2003. He synthesized deuterium-labeled steroids to measure kinetic isotope effects for CYP17 and CYP21, and he won an award for best basic science poster at the 42nd Medical Student Research Forum for this work. He came back to the lab for 6 months in 2006 to finish off his work, and we are in the process of writing a manuscript. David is an MS4 who is applying for residency in neurosurgery.
Andrew Brandmeier was a SURF student in 2003. He engineered mutations to change the directional preference of AKR1C9 (rat liver 3?-hydroxysteroid dehydrogenase), which was published in 2006 (Papari-Zareei et al Endocrinology 147:1591-1597). He is now an MD-PhD student at Indiana University.
Siareyah Rambally identified a mutation in a patient with 17?-hydroxysteroid type 3 deficiency in 2003. She also demonstrated that the magnitude of the directional preference for human 17?-hydroxysteroid type 1 in transfected cells was not fixed but could be varied by changes in culture medium that alter intracellular nicotinamide cofactor concentrations. She is co-author with Dan Sherbet on the review article in press and the manuscript in preparation. She is currently an MS4 applying for residency in Internal Medicine.
Neema Chokshi and Sarita Singeetham helped to organize our adrenal vein sampling serum bank, leading to an Endocrine Society abstract and now a manuscript (resubmitted with revisions to J Clin Endocrinol Metab) about new approaches to the diagnosis and understanding the physiology of primary aldosteronism. On the side, they sequenced the CYP17 gene from a patient with 17-hydroxylase deficiency and won an award for top clinical science poster at the 2006 MSRF. They are currently MS3s.

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