TARGETING AND ASSEMBLY OF E COLI CELL DIVISION PROTEINS

大肠杆菌细胞分裂蛋白的靶向和组装

• 批准号: 6946697
• 负责人: WILLIAM MARGOLIN
• 金额: $ 4.67万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6946697
• 关键词: Escherichia coli; cell cycle; cell cycle proteins; gene mutation; protein protein interaction; protein structure function

Cell division is essential for proliferation of bacteria, and appears to rely on a molecular machine positioned at the site of division. Despite extensive knowledge of the major proteins involved in this fundamental cellular process, very little is understood about how these proteins are targeted, how they assemble into a complex, and how they radically alter normal cell wall growth in order to synthesize the division septum. The long term goals of this project are to elucidate the molecular mechanisms behind the targeting and assembly of cell division proteins, using Escherichia coli as a model system and taking advantage of new, powerful cytological methods for bacteria. A key universal cell division protein, FtsZ, self-assembles into a polymeric ring (the Z-ring) at the surface of the inner membrane at the division site and recruits other proteins, including FtsA, ZipA, and a group of integral membrane proteins, all of which help to complete the septum. The first aim of this proposal is to gain a molecular understanding of the recruitment of FtsA and ZipA by FtsZ. One way that this will be addressed is by mutagenesis of a region of FtsZ that is involved in FtsZ-FtsA and FtsZ-ZipA interactions in order to obtain proteins defective in interaction. Once such mutants are isolated, selection for intragenic and extragenic suppressors may reveal residue-specific contacts. Novel fluorescent in situ protein interaction assays will be used to screen for protein-protein interactions, followed by biochemical tests for direct interaction. The second aim will investigate the role of FtsA in cell division. Enzymatic and structural properties of purified FtsA will be characterized. In addition, the ability of FtsA to interact with FtsZ, ZipA, and downstream cell division proteins in the membrane will be tested. The final aims of the project are to understand how the Z-ring gets targeted to its exact cellular address. The nucleoid appears to negatively influence Z-ring assembly, and it is possible that nucleoid segregation releases a negative signal to allow precise Z-ring positioning. Studies of Z-ring assembly in mutants defective in nucleoid structure and chromosome replication will be carried out to determine the relationship between the nucleoid and the Z-ring. Finally, the Min system, which consists of MinC, D, and E, is an important regulator of Z- ring assembly and positioning. The relationship between the Min system and FtsZ will be addressed in detail both biochemically and genetically. The results of this proposed research are expected to provide a more complete understanding of the molecular mechanisms of cell division, and should facilitate the isolation of new and better antimicrobial drug targets.

细胞分裂对于细菌的增殖至关重要，并且似乎依赖于位于分裂部位的分子机器。 尽管人们对参与这一基本细胞过程的主要蛋白质有广泛的了解，但人们对这些蛋白质如何靶向、它们如何组装成复合物以及它们如何从根本上改变正常细胞壁生长以合成分裂隔膜了解甚少。 该项目的长期目标是利用大肠杆菌作为模型系统，并利用新的、强大的细菌细胞学方法，阐明细胞分裂蛋白的靶向和组装背后的分子机制。 关键的通用细胞分裂蛋白 FtsZ 在分裂位点的内膜表面自组装成聚合环（Z 环），并招募其他蛋白，包括 FtsA、ZipA 和一组整合膜蛋白，所有这些都有助于完成隔膜。 该提案的首要目的是从分子角度了解 FtsZ 招募 FtsA 和 ZipA 的情况。 解决这个问题的一种方法是对参与 FtsZ-FtsA 和 FtsZ-ZipA 相互作用的 FtsZ 区域进行诱变，以获得相互作用缺陷的蛋白质。 一旦分离出此类突变体，对基因内和基因外抑制子的选择可能会揭示残基特异性接触。 新型荧光原位蛋白质相互作用测定将用于筛选蛋白质-蛋白质相互作用，然后进行直接相互作用的生化测试。 第二个目标是研究 FtsA 在细胞分裂中的作用。 将表征纯化的 FtsA 的酶学和结构特性。 此外，还将测试 FtsA 与膜中 FtsZ、ZipA 和下游细胞分裂蛋白相互作用的能力。该项目的最终目标是了解 Z 形环如何定位到其确切的蜂窝地址。 类核似乎会对 Z 环组装产生负面影响，并且类核分离可能会释放负信号以允许精确的 Z 环定位。 将进行类核结构和染色体复制缺陷突变体中 Z 环组装的研究，以确定类核和 Z 环之间的关系。 最后，Min系统由MinC、D和E组成，是Z形环装配和定位的重要调节器。 Min 系统和 FtsZ 之间的关系将从生化和遗传学方面进行详细探讨。 这项研究的结果预计将提供对细胞分裂分子机制的更全面的了解，并有助于分离新的、更好的抗菌药物靶点。