LIPOPROTEIN - MEMBRANE INSERTION/MODIFICATION/PROCESSING

本项目的总体目标是了解 生物合成和出口的分子细节 前脂蛋白。此过程需要事先使用 甘油和脂肪酸在加工前的脂质改性 前脂蛋白由特异的脂蛋白信号肽酶组成。这个 了解蛋白质分泌的机制很重要。 不仅是因为许多酶、激素、毒素、抗体 但也是因为最近重组DNA的出现 制造医学上重要蛋白质的技术。许多 脂蛋白在人体新陈代谢中也很重要，例如低密度脂蛋白。 密度脂蛋白和高密度脂蛋白。 将研究前脂蛋白转运的整个过程。 就其组成步骤而言：插入、修改和 正在处理。每一步的要求和具体情况将 通过基因操作和基因操作的组合进行检查 体外易位系统中的生化研究 大肠埃希氏菌倒置胞膜囊泡。这个 插入前脂蛋白Will的结构要求 使用信号序列和成熟区域发生变化的突变体，以及 该插入物的脂类专一性将用 由合成脂类形成的脂质体。三磷酸腺苷的作用 水解性、细胞质可溶性因子和膜蛋白 改性和加工中，以及前体的重要性 易位能力将通过纯化后进行评估 每个不同步骤的组件。用化学交联剂 双功能试剂，随后是免疫沉淀和 用膜泡和脂质体部分重建，将 用于识别所涉及的组件。最后，非- 具有最小结构变化的脂蛋白前体 相应的前脂蛋白将被比较，以测试 假设自发插入膜脂是 蛋白质转运的第一步。

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.