CYSTATIN-PROTEASE INTERACTIONS--THE EFFECTS OF SURFACES

多种细菌和哺乳动物来源的蛋白酶存在于细胞中。 口腔 这些蛋白酶影响蛋白酶的结构和活性。 唾液和细菌成分的数量，无论是在唾液中， 获得性表膜 已知许多蛋白水解活性是 巯基依赖性蛋白酶，因此其活性将被调节 唾液中的胱抑素 唾液中的半胱氨酸蛋白酶抑制剂已被证明 并且还可以与软组织相互作用 口腔的表面。 唾液半胱氨酸蛋白酶抑制剂与 口腔的硬组织和软组织表面可影响 唾液中各种系统抑制素同种型的活性和特异性。 的 本研究的主要目标是：1）建立结构-功能模型 唾液半胱氨酸蛋白酶抑制剂主要亚型之间的关系 和化学改性方法，以探讨 cystatin功能中的特异性侧链。 结果将 使用分子技术将其整合到结构的整体模型中， 建模，这将进一步探讨使用定点诱变 基于鸡半胱氨酸蛋白酶抑制剂基因的方法（我们可获得）。2)到 检查胱抑素同种型与 羟基磷灰石或 脂质囊泡，作为硬组织和软组织表面的模型， 口腔 实验将测量半胱氨酸蛋白酶抑制剂和 这些表面，并将决定所涉及的分子的区域。 我们还将确定是否半胱氨酸蛋白酶抑制剂经历构象变化 在与这些表面相互作用时， 巯基蛋白酶抑制的特异性受到影响;和3）进行 初步实验，以建立可用于 确定不同的半胱氨酸蛋白酶抑制剂亚型可以 在体内相互作用。 这些实验将使用化学交联 使用光活化试剂和反向亲和层析， 证明相互作用，并反向酶谱法检测抑制 对某些蛋白酶的活性。 这些目标的实现将使 详细了解的影响和相互作用的作用， 半胱氨酸蛋白酶抑制剂亚型与口腔组织，这将最终导致 我们进一步了解这些分子保护口腔黏膜的方式， 空腔的有害影响的细菌，病毒和物理 组织损伤

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.