A variety of bacterial and mammalian derived proteases are present in the oral cavity. These proteases influence the structure and activity of a number of salivary and bacterial components, both in saliva and in the acquired pellicle. Many of the proteolytic activities are known to be thiol dependent proteases, and as such will have their activity modulated by the salivary cystatins. The cystatins in saliva have been shown to be pellicle components, and may also interact with the soft tissue surfaces of the oral cavity. Interactions of salivary cystatins with the hard and soft tissue surfaces of the oral cavity may influence the activity and specificity of the various systatin isoforms in saliva. The principal goals of the work are: 1) to establish structure-function relationships in the major isoforms of salivary cystatin using kinetic and chemical modification approaches to explore the involvement of specific side chains in the functions of cystatin. Results will be integrated into an overall model of the structure using molecular modeling, which will be further explored using site directed mutagenesis approaches based on the chicken cystatin gene (available to us). 2) to examine the interaction of cystatin isoforms with hydroxyapatite or lipid vesicles, as models of the hard and soft tissue surfaces of the oral cavity. Experiments will measure interactions between cystatin and these surfaces, and will determine the regions of the molecule involved. We will also determine whether cystatin undergoes conformational changes upon interaction with these surfaces, and whether or not the activity or specificity of thiol protease inhibition is affected; and 3) to conduct preliminary experiments to establish protocols that can be used to determine which thiol proteases the different cystatin isoforms may interact with in vivo. These experiments will use chemical cross-linking with photoactivatable reagents and reverse affinity chromatography to demonstrate interaction, and reverse zymography to detect inhibitory activity vs certain proteases. The achievement of these goals will give a detailed understanding of the effects and role of interaction of cystatin isoforms with oral cavity tissues, which will eventually lead to our further understanding of the way these molecules protect the oral cavity from the deleterious effects of bacteria, viruses and physical tissue damage.
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