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: 6651137
• 负责人: WILLIAM MARGOLIN
• 金额: $ 22.28万
• 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/6651137
• 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。 尽管对这种基本细胞过程涉及的主要蛋白质有广泛的了解，但几乎没有了解这些蛋白质的靶向，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的生长以综合分裂的隔膜。 该项目的长期目标是阐明使用大肠杆菌作为模型系统的细胞分裂蛋白靶向和组装背后的分子机制，并利用细菌的新型，强大的细胞学方法。 一个关键的通用细胞分裂蛋白FTSZ，将自组装成在分裂位点的内膜表面的聚合环（Z环）中，并募集其他蛋白质，包括FTSA，ZIPA和一组整体膜蛋白，所有这些蛋白质都有助于完成septum。 该提案的第一个目的是获得FTSZ对FTSA和ZIPA募集的分子理解。 解决此问题的一种方法是通过涉及FTSZ-FTSA和FTSZ-ZIPA相互作用的FTSZ区域的诱变，以使蛋白质在相互作用中有缺陷。 一旦分离出这种突变体，选择基因内和外部抑制剂可能会揭示残基特异性接触。 新型的荧光原位蛋白相互作用测定将用于筛选蛋白质 - 蛋白质相互作用，然后进行生化测试直接相互作用。 第二个目标将研究FTSA在细胞分裂中的作用。 将表征纯化FTSA的酶促和结构特性。 另外，将测试FTSA与FTSZ，ZIPA和下游细胞分裂蛋白相互作用的能力。该项目的最终目的是了解Z形环将如何针对其确切的蜂窝地址。 核苷似乎会对Z形组件产生负面影响，并且核苷隔离可能会释放出负信号以允许精确的Z形定位。 将对核苷结构和染色体复制缺陷的突变体中的Z形组件进行研究，以确定核苷与Z形环之间的关系。 最后，由Minc，D和E组成的最小系统是Z圈组件和定位的重要调节器。 最小系统与FTSZ之间的关系将在生化和遗传上详细解决。 预计这项拟议的研究的结果将对细胞分裂的分子机制有更完整的了解，并应促进分离新的，更好的抗菌药物靶标。