The goals of this project are to elucidate the structures, functions and regulation of enzymes related to 3alpha-hydroxysteroid dehydrogenase, as this group of closely related enzymes plays an important role in metabolism of endogenous compounds including androgens, corticosteroids, prostaglandins and bile acids, as well as drugs and xenobiotics such as benzo(a)pyrene. Thus far, we have identified and sequenced seven different rat cDNA clones from cDNA libraries derived from liver, lung and kidney, and two human liver cDNA clones encoding proteins with high degrees of structural homology. Several of the rat clones have been expressed in bacteria at high levels and one shown to have 3alpha-hydroxysteroid dehydrogenase activity. Furthermore, our preliminary Western blot study showed the presence of additional enzymes in brain, eye and testis. We, therefore, propose the following specific aims for the next three-year grant period.
In specific aim 1, we will search for new cDNA clones by screening cDNA libraries with a) monoclonal antibody probes, b) cDNA as probes under low stringency conditions, c) degenerate oligonucleotide probes, and d) degenerate oligonucleotides as primers for the polymerase chain reaction to amplify related sequences.
In specific aim 2, We will determine the substrate specificities of rat aldo/keto reductases after expressing the cDNA clones in bacterium E. coli.
Specific aim 3 will deal with the purification of the expressed proteins for X-ray crystallography evaluation.
In specific aim 4, we will determine the tissue distributions of various rat aldo/keto reductases by RNA blot hybridization, RNAse protection assays, immunocytochemical staining and in situ hybridization.
Specific aims 5 and 6 will focus on the delineation of the induction of enzymes by xenobiotics, and the regulation of their expression by various hormonal factors.
