CYSTATIN-PROTEASE INTERACTIONS--THE EFFECTS OF SURFACES

胱抑素-蛋白酶相互作用——表面的影响

• 批准号: 3223299
• 负责人: JOHN D FEATHERSTONE
• 金额: $ 16.33万
• 依托单位: EASTMAN DENTAL CENTER
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-02-01 至 1996-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3223299
• 关键词: X ray crystallography; affinity chromatography; cell membrane; chemical association; chemical models; chickens; computer simulation; conformation; crosslink; gel filtration chromatography; high performance liquid chromatography; hydroxyapatites; intermolecular interaction; ion exchange chromatography; papain; posttranslational modifications; protease inhibitor; protein denaturation; protein isoforms; protein structure function; saliva; site directed mutagenesis; surface property

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.

多种细菌和哺乳动物来源的蛋白酶存在于 口腔。这些蛋白水解酶会影响一种 唾液和细菌成分的数量，包括唾液和 后天得来的膜。已知的许多蛋白质分解活性是 硫醇依赖的蛋白水解酶，因此它们的活性将受到调节 唾液中的半胱氨酸氨基转移酶。唾液中的半胱氨酸氨基已被证明 是薄膜成分，也可以与软组织相互作用 口腔的表面。唾液半胱氨酸氨基转移酶与细胞外基质的相互作用 口腔的硬组织和软组织表面可能会影响 唾液中各种收缩素异构体的活性和特异性。这个 工作的主要目标是：1)建立结构-功能 应用动力学方法研究唾液胱抑素主要异构体之间的相互关系 和化学修饰的方法，以探索参与 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，这将进一步探索使用定点突变 基于鸡半胱氨酸氨基转移酶基因的方法(我们可以获得)。2)至 观察胱抑素亚型与羟基磷灰石的相互作用 脂泡，作为人体硬组织和软组织表面的模型 口腔。实验将测量半胱氨酸氨基转移酶和 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 在与这些表面相互作用时，无论活动或 硫醇蛋白水解酶抑制的特异性受到影响；3)传导 初步实验以建立可用于 确定不同的胱抑素亚型可能存在的硫醇蛋白 在活体内相互作用。这些实验将使用化学交联剂 使用光活化试剂和反向亲和层析来 演示相互作用，并通过反向酶谱检测抑制作用 活性与某些蛋白水解酶。这些目标的实现将给 详细了解相互作用的影响和作用 半胱氨酸氨基转移酶与口腔组织的异构体，最终导致 对我们进一步了解这些分子保护口腔的方式 空洞免受细菌、病毒和身体的有害影响 组织损伤。