LIPOPROTEIN--MEMBRANE INSERTION/MODIFICATION/PROCESSING

脂蛋白--膜插入/修饰/加工

• 批准号: 3300302
• 负责人: PHANG C. TAI
• 金额: $ 14.1万
• 依托单位: GEORGIA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-01 至 1994-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3300302
• 关键词: Escherichia coli; bacterial genetics; bacterial proteins; blood lipoprotein transport; lipid bilayer membrane; liposomes; membrane lipids; membrane proteins; membrane reconstitution /synthesis; mutant; protein biosynthesis; protein signal sequence; protein transport; recombinant DNA

The overall objective of this project is to understand the molecular details of the biosynthesis and export of prolipoproteins. This process involves prior modification with glycerol and fatty acids before processing of the lipid-modified prolipoprotein by specific lipoprotein signal peptidase. The understanding of the mechanism of protein secretion is important not only because many enzymes, hormones, toxins, antibodies are secreted but also because of the recent advent of recombinant DNA technology of making medically important proteins. Many lipoproteins are also important in human metabolism, e.g. low- density and high-density lipoproteins. The overall process of prolipoprotein translocation will be studied in terms of its component steps: insertion, modification, and processing. The requirements and the specificity of each step will be examined by a combination of genetic manipulation and biochemical studies in an in vitro translocation system with Escherichia coli inverted cytoplasmic membrane vesicles. The structural requirements for the insertion of prolipoprotein will employ mutants with altered signal sequence and mature region, and the lipid specificity for the insertion will be examined with liposomes formed with synthetic lipids. The roles of ATP hydrolysis, cytoplasmic soluble factors, and membrane proteins in modification and in processing, and the importance of precursor competency for translocation will be assessed with purified components for each distinct step. Chemical crosslinking with bifunctional reagents, followed by immunoprecipitation and partial reconstitution with membrane vesicles and liposomes, will be used to identify the components involved. Finally, non- lipoprotein precursors with minimal structural change from corresponding prolipoproteins will be compared to test the hypothesis that spontaneous insertion into membrane lipids is the first step of protein translocation.

该项目的总体目的是了解 生物合成和出口的分子细节 催化蛋白。 此过程涉及事先修改 甘油和脂肪酸在脂质改性之前加工之前 特异性脂蛋白信号肽酶的催化蛋白。 这 了解蛋白质分泌机制很重要 不仅因为许多酶，激素，毒素，抗体是 分泌，但也是由于重组DNA最近出现 制造医学重要蛋白质的技术。 许多 脂蛋白在人类代谢中也很重要，例如低的- 密度和高密度脂蛋白。 将研究流传蛋白易位的总体过程 就其组件步骤而言：插入，修改和 加工。 每个步骤的要求和特异性将 通过遗传操纵和 在体外易位系统中的生化研究 大肠杆菌倒的细胞质膜囊泡。 这 插入蛋白插入的结构要求将 使用具有改变信号序列和成熟区域的突变体，以及 将检查插入的脂质特异性 由合成脂质形成的脂质体。 ATP的角色 水解，细胞质可溶性因子和膜蛋白 修改和处理以及前体的重要性 易位能力将通过纯化评估 每个不同步骤的组件。 化学交联 双功能试剂，然后进行免疫沉淀和 与膜囊泡和脂质体的部分重构，将 用于识别所涉及的组件。 最后，非 - 脂蛋白前体具有最小的结构变化 将比较相应的催化蛋白，以测试 假设自发插入膜脂质是 蛋白质易位的第一步。