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 口腔。 这些蛋白酶影响蛋白质的结构和活性 唾液和唾液中的唾液和细菌成分的数量 获得的薄膜。 已知许多蛋白水解活性是 硫醇依赖性蛋白酶，因此其活性将受到调节 由唾液半胱氨酸蛋白酶抑制剂。 唾液中的半胱氨酸蛋白酶抑制剂已被证明 是薄膜成分，也可能与软组织相互作用 口腔表面。 唾液胱抑素与唾液中胱抑素的相互作用 口腔的硬组织和软组织表面可能会影响 唾液中各种系统抑素亚型的活性和特异性。 的 工作的主要目标是：1）建立结构-功能 使用动力学研究唾液胱抑素主要亚型的关系 和化学修饰方法来探索参与 胱抑素功能中的特定侧链。 结果将是 使用分子集成到结构的整体模型中 建模，将使用定点诱变进一步探索 基于鸡半胱氨酸蛋白酶抑制剂基因的方法（我们可以使用）。 2）到 检查半胱氨酸蛋白酶抑制剂亚型与羟基磷灰石的相互作用或 脂质囊泡，作为硬组织和软组织表面的模型 口腔。 实验将测量胱抑素和 这些表面，并将确定所涉及的分子区域。 我们还将确定胱抑素是否发生构象变化 与这些表面的相互作用，以及无论活动或 硫醇蛋白酶抑制的特异性受到影响； 3) 进行 初步实验建立可用于 确定不同的半胱氨酸蛋白酶抑制剂亚型可能会产生哪些硫醇蛋白酶 与体内相互作用。 这些实验将使用化学交联 用光活化试剂和反亲和色谱法 证明相互作用，并反向酶谱检测抑制 活性与某些蛋白酶的比较。 这些目标的实现将给 详细了解相互作用的影响和作用 胱抑素亚型与口腔组织的结合，最终导致 以便我们进一步了解这些分子保护口腔的方式 蛀牙免受细菌、病毒和物理的有害影响 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.