The function and organization of MreB filaments

MreB 丝的功能和组织

• 批准号: 8593613
• 负责人: RANDY M MORGENSTEIN
• 金额: $ 5.22万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8593613
• 关键词: Actins; Address; Affect; Alanine; Amino Acids; Anabolism; Antibiotics; Bacteria; Bacterial Proteins; Beryllium; Binding; Biochemical; Biological Assay; Biological Models; Cell Growth Processes; Cell Shape; Cell Wall; Cell physiology; Cells; Cellular biology; Complex; Coupled; Cytoskeleton; Data; Dependence; Electron Microscopy; Elements; Enzymes; Escherichia coli; Eukaryotic Cell; Filament; Fluorescence; Genetic; Goals; Gram-Negative Bacteria; Growth; Homologous Gene; Human; Image; In Vitro; Knowledge; Learning; Length; Libraries; Life; Link; Maintenance; Maps; Metabolic; Molecular; Morphogenesis; Mutagenesis; Play; Polymers; Process; Property; Protein Binding; Proteins; Regulation of Cell Shape; Research; Role; Scanning; Structure; Surface; System; Testing; Work; antimicrobial; cell growth; cell growth regulation; clinically relevant; in vitro Assay; in vivo; interest; mutant; novel; phenomics; polymerization; protein protein interaction; public health relevance; research study; scaffold

DESCRIPTION (provided by applicant): Like eukaryotic cells, bacteria have multiple cytoskeletal elements. One of these bacterial cytoskeleton elements, MreB, a homolog of eukaryotic actin, has been shown to be involved in maintaining cell shape and protein localization. The mechanism by which MreB controls cell wall synthesis to maintain cell shape or direct protein localization remains unclear. MreB resembles actin structurally, and both proteins form filaments within cells. However, MreB does not form a helical structure when purified as it does in vivo. Hundreds of interacting proteins are known for eukaryotic actin, but only one interacting partner (RodZ) has been confirmed for MreB, indicating there are probably unknown accessory proteins. In order to better understand MreB function, I propose to focus on identifying and characterizing its interacting partners. In particular, I have developed a bimolecular fluorescent complementation (BiFC) assay in Escherichia coli, which will enable the study of MreB-protein interactions in vivo. I will initially use the assay to perform a functional analysis of MreB filaments, though the expression of MreB point mutants with enhanced or reduced binding to itself, followed by in vitro polymerization assays and electron microscopy to confirm these results. The role of RodZ, a known binding partner, will also be looked at for its role in filament stabilization. Secondly, I will look at the role of MreB for proper cell growth. Iwill test the hypothesis that MreB acts as a scaffold for a cell wall synthesis complex. I will also being to explore the hypothesis that MreB interacts with cellar metabolic proteins to properly localize them. An alanine scanning mutagenesis will be performed on surface exposed residues of MreB to determine if there is a single interaction domain on MreB that other proteins bind. The results of this proposal will create new knowledge of the functionality of MreB, which can then be applied by the field to the creation of novel antimicrobial compounds.

描述（由申请人提供）：与真核细胞一样，细菌具有多种细胞骨架元件。 MreB 是这些细菌细胞骨架元件之一，是真核肌动蛋白的同源物，已被证明参与维持细胞形状和蛋白质定位。 MreB 控制细胞壁合成以维持细胞形状或直接蛋白质定位的机制仍不清楚。 MreB 在结构上类似于肌动蛋白，两种蛋白质在细胞内形成丝状结构。然而，MreB 在纯化时不会像在体内那样形成螺旋结构。真核肌动蛋白已知有数百种相互作用蛋白，但 MreB 只确认了一种相互作用伴侣 (RodZ)，表明可能存在未知的辅助蛋白。为了更好地理解 MreB 功能，我建议重点关注识别和表征其相互作用的伙伴。特别是，我在大肠杆菌中开发了一种双分子荧光互补 (BiFC) 测定法，这将使 MreB 蛋白相互作用的体内研究成为可能。我将首先使用该测定对 MreB 丝进行功能分析，通过增强或减少与其自身结合的 MreB 点突变体的表达，然后进行体外聚合测定和电子显微镜来确认这些结果。 RodZ 是一种已知的结合伴侣，其在细丝稳定方面的作用也将受到关注。其次，我将研究 MreB 对细胞正常生长的作用。我将检验 MreB 作为细胞壁合成复合物支架的假设。我还将探索 MreB 与细胞代谢蛋白相互作用以正确定位它们的假设。将在 MreB 表面暴露的残基上进行丙氨酸扫描诱变，以确定 MreB 上是否存在与其他蛋白质结合的单一相互作用结构域。该提案的结果将创造关于 MreB 功能的新知识，然后该领域可以将其应用于创建新型抗菌化合物。