Structural Biology of Intramembrane Proteolysis

描述（由申请人提供）：调节膜内蛋白水解（RIP）是一种普遍保守的信号机制，从细菌到人类。RIP的一个重要步骤是通过脂质双分子层内的特定膜内蛋白酶对信号蛋白的跨膜片段进行位点特异性切割。这些膜内蛋白酶可分为四个家族：丝氨酸蛋白酶菱形、金属蛋白酶Site- 2蛋白酶（S2P）、天冬氨酸蛋白酶、早老素和信号肽肽酶。尽管研究深入，但这些膜内蛋白酶的结构和机制在很大程度上仍然未知。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射线晶体学和生化分析。取得了显著的初步成果；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)大肠杆菌菱形蛋白酶GlpG与抑制剂和底物复合物跨膜核心结构域的晶体结构测定。(2)原核生物和真核生物全长菱形蛋白酶晶体结构的测定。(3) jannaschii S2P膜内蛋白酶跨膜核心结构域晶体结构的测定。(4) jannaschii和大肠杆菌全长S2P膜内蛋白酶晶体结构的测定。(5)与底物肽复合物中S2P膜内蛋白酶晶体结构的测定。这些研究完成后，将揭示膜内蛋白酶菱形和S2P家族的结构、功能和机制。公共卫生相关性：在从细菌到人类的生物体中，调节膜内蛋白水解（RIP）是一种普遍保守的信号机制。RIP的一个重要步骤是通过脂质双分子层内的特定膜内蛋白酶对信号蛋白的跨膜片段进行位点特异性切割。本提案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.