Regulation of Actin Filament Formation in Phagocytes

吞噬细胞中肌动蛋白丝形成的调节

基本信息

批准号：
7266208
负责人：
Frederick s Southwick
金额：
$ 35.32万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
1985
资助国家：
美国
起止时间：
1985-08-01 至 2010-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7266208
关键词：
A Mouse Actin-Binding Protein Actins Affect Affinity Analytical Centrifugation Antibody Formation Apoptosis Applications Grants B-Lymphocytes Bacteria Binding Biological Assay CD4 Positive T Lymphocytes Cell Death Cell Line Cell Nucleus Cell Survival Cells Cellular Immunity Chemotaxis Communication Complex Contractile Proteins Cytolysis Cytoplasm Defect Dendritic Cells Embryo Fibroblasts Filament Gelsolin Gene Expression Gene Proteins Genes Grant HL-60 Cells Hela Cells Immune System Diseases Infection Infection Control Inflammation Inflammatory Interferon Type II Interleukin-12 Interleukin-2 Invaded Investigation Killer Cells Knock-out Knockout Mice Length Link Listeria Listeria monocytogenes Lymphocyte Measures Mediating Mediator of activation protein Microarray Analysis Microfilaments Molecular Profiling Movement Mus Mutagenesis Mutate Phagocytes Phagocytosis Polymerase Chain Reaction Predisposition Process Proteins RNA Interference Recombinants Regulation Regulation of Cell Shape Role Salmonella typhimurium Series Signal Transduction Site Structure Structure-Activity Relationship Time Two-Dimensional Gel Electrophoresis Two-Hybrid System Techniques Undifferentiated Upper arm Wild Type Mouse Yeasts ameboid movement antigen processing cell motility cell type design gain of function genetic regulatory protein immortalized cell immune function insight link protein macrophage mutant neutrophil novel therapeutics pathogen protein expression receptor research study response therapeutic target

项目摘要

DESCRIPTION (provided by applicant): The regulation of actin filament dynamics in neutrophils and macrophages allows cells to crawl to sites of infection and ingest invading pathogens. The present grant focuses on the most abundant actin regulatory protein in macrophages, CapG. Aim 1 will explore the functional consequences of knocking out CapG in mice. CapG-null mice have profound defects in the ability of their macrophages, neutrophils and dendritic cells to move and take in foreign material. In order to determine how CapG functionally relates to other proteins in the cell, the compensatory changes in other macrophage proteins will be assessed by 2-D gel electrophoresis and microsequencing, as well as by gene microarray analysis. The functional significance of these adaptive protein changes will be assessed by over-expressing the identified proteins and by lowering their concentrations by RNA interference. Their ability to bind to CapG will be assessed by pull-down and yeast-two hybrid assays. Loss of CapG results in increased susceptibility to infection by the intracellular bacterium Listeria, indicating a defect in cell-mediated immunity. Levels of the inflammatory mediators IL-2 and interferon-gamma will be measured. CD4 and CDS lymphocyte responses, macrophage and dendritic cell antigen processing and communication with CD4 lymphocytes, killer cell lysis of target cells, and B cell antibody production will be examined. CapG is expressed in phagocytes at far higher concentrations than required to regulate actin assembly, raising the possibility that CapG may serve other functions. The effects of over-expressing CapG on the survival of different cell lines will be examined. Apoptosis of CapG-null neutrophils and macrophages will be compared to wild-type cells. Aim 2 will analyze structure-function relationships in CapG gain-of-function mutant proteins. Actin filament severing and capping are critical steps for macrophage movement. PCR mutagenesis has created a series of gain-of-function CapG severing proteins and crystallographic studies have revealed their structure. Pyrenyl actin and analytical centrifugation are being used to define the structure-function relationship of these vital processes. Investigations of CapG promise to provide new insights into cell motility, innate and cell-mediated immunity, and the mechanisms underlying cell survival. These studies may provide new strategies for defending against infections, controlling auto-immune diseases and regulating the timing of cell death.

描述（由申请人提供）：中性粒细胞和巨噬细胞中肌动蛋白丝动力学的调节使细胞可以爬向感染部位并摄取侵入病原体。目前的赠款重点是巨噬细胞中最丰富的肌动蛋白调节蛋白，CAPG。 AIM 1将探索在小鼠中淘汰CAPG的功能后果。 CAPG-NULL小鼠在其巨噬细胞，中性粒细胞和树突状细胞移动和吸收异物的能力方面具有深远的缺陷。为了确定CAPG功能与细胞中的其他蛋白质的关系，其他巨噬细胞蛋白的代偿性变化将通过2-D凝胶电泳和微钉测序以及基因微阵列分析来评估。这些自适应蛋白变化的功能意义将通过过度表达鉴定蛋白并通过RNA干扰降低其浓度来评估。它们与CAPG结合的能力将通过下拉和酵母 - 两种混合动力分析来评估。 CAPG的丧失导致细胞内细菌对感染的敏感性增加，表明细胞介导的免疫力缺陷。将测量炎症介质IL-2和干扰素伽马的水平。 CD4和CD淋巴细胞反应，巨噬细胞和树突状细胞抗原加工以及与CD4淋巴细胞，靶细胞的杀伤细胞裂解以及B细胞抗体产生的相关性。 CAPG在吞噬细胞中的浓度远高于调节肌动蛋白组装所需的浓度，从而提高了CAPG可以提供其他功能的可能性。将检查过度表达CAPG对不同细胞系生存的影响。将CAPG无嗜中性粒细胞和巨噬细胞的凋亡与野生型细胞进行比较。 AIM 2将分析CAPG功能获得突变蛋白中的结构功能关系。肌动蛋白细丝切断和封盖是巨噬细胞运动的关键步骤。 PCR诱变创造了一系列功能获得的CAPG切断蛋白质，晶体学研究揭示了它们的结构。拟南芥肌动蛋白和分析离心被用于定义这些重要过程的结构功能关系。 CAPG的调查有望提供有关细胞运动，先天和细胞介导的免疫力以及细胞存活的机制的新见解。这些研究可能会为防御感染，控制自身免疫性疾病和调节细胞死亡的时间提供新的策略。