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：
8090809
负责人：
Frederick s Southwick
金额：
$ 24.04万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-08-01 至 2011-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8090809
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 Inflammation Mediators Interferon Type II Interleukin-12 Interleukin-2 Invaded Investigation Killer Cells Knock-out Knockout Mice Length Link Listeria Listeria monocytogenes Lymphocyte Measures Mediating Microarray Analysis Microfilaments Molecular Profiling Movement Mus Mutagenesis Mutate Phagocytes Phagocytosis 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 Wild Type Mouse Yeasts ameboid movement antigen processing arm cell motility cell type design gain of function genetic regulatory protein immortalized cell immune function insight link protein macrophage mutant neutrophil new therapeutic target pathogen protein expression receptor research study response

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爬行至感染部位并摄入入侵的病原体。目前的资助重点是巨噬细胞中最丰富的肌动蛋白调节蛋白 CapG。目标 1 将探索敲除小鼠 CapG 的功能后果。 CapG缺失小鼠的巨噬细胞、中性粒细胞和树突状细胞移动和吸收异物的能力存在严重缺陷。为了确定 CapG 在功能上如何与细胞中的其他蛋白质相关，将通过 2-D 凝胶电泳和微测序以及基因微阵列分析来评估其他巨噬细胞蛋白质的补偿性变化。这些适应性蛋白质变化的功能意义将通过过度表达已识别的蛋白质并通过 RNA 干扰降低其浓度来评估。它们与 CapG 结合的能力将通过 Pull-down 和酵母-两种杂交测定进行评估。 CapG 的缺失导致细胞内细菌李斯特菌感染的易感性增加，表明细胞介导的免疫存在缺陷。将测量炎症介质 IL-2 和干扰素-γ 的水平。将检查 CD4 和 CDS 淋巴细胞反应、巨噬细胞和树突细胞抗原处理以及与 CD4 淋巴细胞的通讯、靶细胞的杀伤细胞裂解以及 B 细胞抗体的产生。 CapG 在吞噬细胞中的表达浓度远高于调节肌动蛋白组装所需的浓度，这增加了 CapG 可能发挥其他功能的可能性。将检查过度表达 CapG 对不同细胞系存活的影响。将 CapG 缺失的中性粒细胞和巨噬细胞的凋亡与野生型细胞进行比较。目标 2 将分析 CapG 功能获得性突变蛋白的结构-功能关系。肌动蛋白丝的切断和加帽是巨噬细胞运动的关键步骤。 PCR 诱变产生了一系列功能获得性 CapG 切割蛋白，晶体学研究揭示了它们的结构。芘基肌动蛋白和分析离心被用来定义这些重要过程的结构-功能关系。 CapG 的研究有望为细胞运动、先天性和细胞介导的免疫以及细胞存活的机制提供新的见解。这些研究可能为防御感染、控制自身免疫性疾病和调节细胞死亡时间提供新策略。

项目成果

期刊论文数量（56）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Isolation of an inhibitor of actin polymerization from human polymorphonuclear leukocytes.

从人多形核白细胞中分离肌动蛋白聚合抑制剂。

DOI：
发表时间：
1981-03-25
期刊：
影响因子：
0
作者：
Southwick, F S;Stossel, T P
通讯作者：
Stossel, T P

Platelet-activating factor both stimulates and "primes" human polymorphonuclear leukocyte actin filament assembly.

血小板激活因子既刺激又“启动”人类多形核白细胞肌动蛋白丝的组装。

DOI：
发表时间：
1987-12
期刊：
影响因子：
20.3
作者：
Shalit, M;Dabiri, G A;Southwick, F S
通讯作者：
Southwick, F S

Neutrophil actin dysfunction is a genetic disorder associated with partial impairment of neutrophil actin assembly in three family members.

中性粒细胞肌动蛋白功能障碍是一种遗传性疾病，与三个家庭成员的中性粒细胞肌动蛋白组装部分受损有关。

DOI：
发表时间：
1988-11
期刊：
影响因子：
0
作者：
Southwick, F S;Dabiri, G A;Stossel, T P
通讯作者：
Stossel, T P

The relationship between CR3 deficiency and neutrophil actin assembly.

CR3缺乏与中性粒细胞肌动蛋白组装之间的关系。

DOI：
发表时间：
1989-05-15
期刊：
影响因子：
20.3
作者：
Southwick, F S;Howard, T H;Holbrook, T;Anderson, D C;Stossel, T P;Arnaout, M A
通讯作者：
Arnaout, M A

Gelsolin and ADF/cofilin enhance the actin dynamics of motile cells.

凝溶胶蛋白和 ADF/丝切蛋白增强运动细胞的肌动蛋白动力学。

DOI：
10.1073/pnas.97.13.6936
发表时间：
2000-06-20
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
F. Southwick
通讯作者：
F. Southwick