Prostaglandins and hydroxy fatty acids are a family of biologically potent eicosanoids derived from arachidonic acid. These substances are rapidly catabolized and either inactivated or activated further. Prostaglandins are catabolized first by NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) followed by delta13-15-ketoprostaglandin reductase (13-PGR) resulting in inactive metabolites. Cloning, characterization and structure and activity relationship at 15-PGDH have been extensively studied. However, regulation of the 15-PGDH gene expression has not been explored in any detail. We have cloned the genomic DNA of mouse 15-PGDH gene and have sequenced the 5?-flanking region. We plan to identify specific regulatory elements involved in gene expression, induced by PMA and dexamethosone and to elucidate the signal transduction pathway of androgen activated 15-PGDH expression. We have also cloned and expressed the cDNA of porcine lung 13-PGR in an active form. We propose to identify the binding domains of NADPH and 15-ketoprostaglandins of 13-PGR by photoaffinity labeling studies and to elucidate the structure and function of the enzyme by site-directed mutagenesis. Finally, we propose to purify and characterize a novel 5-hydroxyeicosanoid dehydrogenase (5-HEDH) which generates a powerful activator of leukocytes, 5-KETE, from 5-HETE. The results of this research program should provide a molecular basis of how the expression of 15-PGDH is regulated and a molecular basis of catalysis of 13-PGR. Furthermore, the program should uncover novel properties of 5-HEDH and shed some light on the role of this enzyme in the inflammatory and allergic reactions.
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