The secretory granules of human cells contain high levels of three serine proteinases: tryptase, chymase, and an enzyme similar to neutrophil cathepsin G. Understanding the structure and inhibitory mechanisms of proteinases from inflammatory cells, including the mast cell, is important for establishing the roles of proteinases and their inhibitors in inflammation. This proposal focuses on the continued characterization of mast cell proteinases and their mechanisms of inhibition. Using computer modeling techniques, the three-dimensional structure of chymase and its interactions with substrates, inhibitors, and proteoglycans will be investigated at the molecular level. Expression of analysis of mutant chymase. Mast cell cathepsin G and neutrophil cathepsin G differ in molecular weight, a dissimilarity that is likely related to differences in cellular processing. The biochemical and functional significance of the structural difference will be examined using mast cell cathepsin G isolated from skin. The plasma inhibitors alpha1-antichymotrypsin (ACT) and alpha1-proteinase inhibitor (alpha1-PI) inhibit human chymase. Kinetic and cleavage site analyses indicate that ACT and alpha1-PI may interact with chymase via distinct mechanisms. ACT and alpha1-PI variants will be synthesized by site directed mutagenesis and analyzed for their ability to function as substrates and/or inhibitors. These studies will provide insights into the mechanisms of serpin inhibition and thereby aid in the design of potent inflammatory proteinase inhibitors by genetic engineering. Tryptase has no apparent physiological inhibitor, but is regulated by an auto-activation process. This form of regulation is unique among serine proteinases. Investigations to establish the precise mechanism of tryptase auto-inactivation will involve defining conformational and structural changes as well as determining the factors that affect activity loss and reactivation. Definition of the properties of mast cell proteinases and the mechanisms of their inhibition by serpins will be useful for predicting behavior of proteinases and inhibitors in inflammatory processes and in the design of products which can interfere with these processes.
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