DISCOVERING STRUCTURAL DETERMINANTS OF EXTENDED SPECIFICITY GRANZYMES

发现扩展特异性颗粒酶的结构决定因素

• 批准号: 6456820
• 负责人: SANDRA W RUGGLES
• 金额: $ 27.32万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6456820
• 关键词: Mammalia; animal tissue; biological products; biomedical resource; biotechnology; drug /agent; genetics; human tissue; respiratory system; technology /technique

The serine protease family termed the granzymes has been implicated in the cytotoxic lymphocyte mediated cell death mechanism. Each granzyme is proposed to have an individual primary and extended specificity, like a fingerprint, yet the family has above 50% sequence similarity. How is it that these enzymes have such unique cleavage sites and yet are so similar in their primary sequences? This leads us to hypothesize that the components of extended specificity in this scaffold must come from just a few amino acids, varied through evolution to provide differing sequence specificities. By finding these essential amino acids in granzyme B and defining their interactions with the substrate, the specificities of the granzyme B-like family can be both predicted and engineered. Experiments address these issues through three pathways: solving the structure of granzyme B, using alignments of the family to determine the residues directly involved in specificity, and using site directed mutagenesis to discover the specific differences at these sites. The Computer Graphics Laboratory is invaluable for all three aspects of this project. MidasPlus is used extensively to draw conclusions regarding this family of proteins.

丝氨酸蛋白酶家族称为颗粒酶 与细胞毒性淋巴细胞介导的细胞死亡机制有关。 建议每个颗粒酶具有一个单独的主要和扩展 特异性，例如指纹，但家庭的序列超过50％ 相似。 这些酶如何具有如此独特的乳沟 站点的主要序列如此相似？ 这导致 我们假设在此中扩展特异性的组成部分 脚手架必须仅来自几个氨基酸，不同 提供不同序列特异性的进化。 通过查找 这些在颗粒酶B中的必需氨基酸并定义了它们 与底物的相互作用，颗粒的特异性 可以预测和设计类似B的家庭。 实验 通过三个途径解决这些问题：解决 颗粒酶B，使用家族的比对来确定残基 直接参与特异性，并使用定向诱变 在这些站点发现具体差异。 计算机 图形实验室对于所有三个方面都是无价的 项目。 Midasplus广泛用于得出有关的结论 这个蛋白质家族。