Structural Biology of Intramembrane Proteolysis

描述（由申请人提供）：受调节的膜内蛋白水解（RIP）是从细菌到人类的物种中普遍存在的保守信号传导机制。RIP的一个重要步骤是通过脂质双层内的特异性膜包埋蛋白酶对信号蛋白中的跨膜片段进行位点特异性切割。这些膜内蛋白酶分为四个家族：丝氨酸蛋白酶菱形，金属蛋白酶位点-2蛋白酶（S2 P），和乙酰蛋白酶早老素和信号肽肽酶。尽管深入研究，这些膜内蛋白酶的结构和机制仍然在很大程度上未知。我们已经开始对丝氨酸蛋白酶菱形和金属蛋白酶S2 P进行系统的X射线晶体学和生化分析。已经取得了重要的初步成果;这里提议的工作将以我们的初步成果为基础，具体目标如下。(1)确定了E.大肠杆菌菱形蛋白酶GlpG与抑制剂和底物的复合物。(2)原核和真核生物全长菱形蛋白酶晶体结构的测定。(3)来自M.的S2 P膜内蛋白酶的跨膜核心结构域的晶体结构的确定。jannaschii。(4)M.全长S2 P膜内蛋白酶晶体结构的测定jannaschii和E.杆菌(5)与底物肽复合的S2 P膜内蛋白酶的晶体结构的测定。这些研究，完成后，将揭示重要的见解的菱形和S2 P家族的膜内蛋白酶的结构，功能和机制。公共卫生相关性：受调节的膜内蛋白水解（RIP）是一种普遍存在于从细菌到人类的生物体中的保守信号传导机制。RIP的一个重要步骤是通过脂质双层内的特异性膜包埋蛋白酶对信号蛋白中的跨膜片段进行位点特异性切割。该建议旨在了解这些膜内蛋白酶的结构和机制。

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.