The prostanoids, a family of oxygenated metabolites of polyunsaturated fatty acids, have been ascribed important roles in a wide variety of physiological and pathological processes including inflammation, hemostasis, atherogenesis, thrombosis, and stroke. The first committed step in the biosynthesis of each of these prostanoids, a key point for control of the levels of these powerful mediators, is catalyzed by the cyclooxygenase activity of prostaglandin H synthase (PGHS). Two PGHS isoforms have been found. Protein and mRNA levels of the recently discovered second PGHS isoform, PGHS-2, are almost undetectable in quiescent cells, but are strongly and transiently induced in a few cell types by inflammatory mediators. In contrast, PGHS-1 is found in a wide variety of quiescent cells, bud its levels are modulated to only limited degree. It is clear that the two isoforms have distinct pathophysiological functions. The emerging concept has PGHS-2 responsible for prostanoid synthesis crucial to inflammation, whereas PGHS-l produces prostanoids for housekeeping functions. The long term goal is to understand the role that the biochemical characteristics of PGHS-2 play in its distinct functions in vivo.
The specific aims are: l) Identify the biochemical basis for differential cellular control of prostanoid synthesis by PGHS-2. Cultured cells expressing the human PGHs isoforms and the purified human enzymes themselves will be used to evaluate several aspects of catalytic control, including requirements for hydroperoxide activator, specificity for fatty acid substrate structure, and substrate compartmentation. 2) Characterize the reaction kinetics of human PGHS-2, and relate kinetic differences with human PGHS-l to specific protein structural differences. Purified human PGHS isoforms will be used to analyze individual cyclooxygenase and peroxidase reaction steps, to quantitate the kinetics of interactions with inhibitors, and to examine the role of tyrosyl free radicals in catalysis. Structural models of the human PGHS isoforms will be used to identify structural features of PGHS-2 for evaluation by site- directed mutagenesis. 3) Characterize the endoplasmic reticulum membrane attachment of PGHS-2 and compare it with that of PGHS-1. The nature of membrane attachment will be evaluated from the accessibility of particular polypeptide segrnents to the cytoplasm and the ER lumen, and from the effects of specific mutations on membrane association.
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