Structural Biology of Intramembrane Proteolysis

描述(由申请人提供)：调节的膜内蛋白分解(RIP)是从细菌到人类的各种物种中普遍存在的一种保守的信号机制。RIP的一个基本步骤是信号蛋白中的跨膜片段被脂类双层中特定的膜嵌入的蛋白酶定点切割。这些膜内蛋白水解酶分为四个家族：丝氨酸蛋白酶、菱形、金属蛋白酶S2P、天冬氨酸蛋白酶、早老素和信号肽酶。尽管进行了深入的研究，但这些膜内蛋白水解酶的结构和机制仍然很大程度上是未知的。我们对丝氨酸蛋白酶菱形和金属蛋白酶S2P进行了系统的X射线结晶学和生物化学分析。已经取得了重大的初步成果；这里提出的工作将以我们的初步成果为基础，具体目标如下。(1)抑制物底物复合体中大肠杆菌菱形蛋白酶GlpG跨膜核心区晶体结构的测定。(2)原核生物和真核生物的全长菱形蛋白水解酶的晶体结构测定。(3)Jannaschii S2P膜内蛋白水解酶跨膜核心区结构的测定。(4)Jannaschii和E.Coli全长S2P膜内酶的晶体结构测定。(5)S2P膜内酶与底物多肽形成的复合体的晶体结构的测定。这些研究完成后，将揭示菱形和S2P家族的膜内蛋白水解酶的结构、功能和机制。公共卫生相关性：调节的膜内蛋白分解(RIP)是从细菌到人类的生物体中普遍存在的一种保守的信号机制。RIP的一个基本步骤是信号蛋白中的跨膜片段被脂类双层中特定的膜嵌入的蛋白酶定点切割。这项提议试图了解这些膜内蛋白水解酶的结构和机制。

DESCRIPTION (provided by applicant): Regulated intramembrane proteolysis (RIP) is an ubiquitously conserved signaling mechanism in species ranging from bacteria to humans. An essential step of RIP is the site-specific cleavage of a transmembrane segment in a signaling protein by a specific membrane-embedded protease within the lipid bilayer. These intramembrane proteases are classified into four families: the serine protease rhomboid, metalloprotease Site- 2 protease (S2P), and aspartyl proteases presenilin and signal-peptide peptidase. Despite intense investigation, the structure and mechanisms of these intramembrane proteases remain largely unknown. We have initiated systematic X-ray crystallographic and biochemical analyses of the serine protease rhomboid and the metalloprotease S2P. Significant preliminary results have been achieved; the work proposed here will build on our preliminary results with the following specific aims. (1) Determination of the crystal structure of the transmembrane core domain of the E. coli rhomboid protease GlpG in complex with inhibitor and substrate. (2) Determination of the crystal structure of full-length rhomboid proteases from prokaryotic and eukaryotic species. (3) Determination of the crystal structure of the transmembrane core domain of a S2P intramembrane protease from M. jannaschii. (4) Determination of the crystal structure of the full-length S2P intramembrane protease from M. jannaschii and from E. coli. (5) Determination of the crystal structure of a S2P intramembrane protease in complex with a substrate peptide. These studies, when completed, will reveal significant insights into the structure, function, and mechanism of the rhomboid and S2P families of intramembrane proteases. PUBLIC HEALTH RELEVANCE: Regulated intramembrane proteolysis (RIP) is an ubiquitously conserved signaling mechanism in organisms ranging from bacteria to humans. An essential step of RIP is the site-specific cleavage of a transmembrane segment in a signaling protein by a specific membrane-embedded protease within the lipid bilayer. This proposal seeks to understand the structures and mechanisms of these intramembrane proteases.