Modulation of Neutrophil Apoptosis by Akt-Hsp27 Signalosome

Akt-Hsp27 信号体对中性粒细胞凋亡的调节

基本信息

批准号：
7890434
负责人：
MADHAVI J RANE
金额：
$ 56.77万
依托单位：
UNIVERSITY OF LOUISVILLE
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-08-08 至 2012-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7890434
关键词：
Address Antigen-Antibody Complex Apoptosis Apoptotic Bacteria Binding Biological Assay Cell Fractionation Cell Survival Cessation of life Chimeric Proteins Complementary DNA Complex Confocal Microscopy Data Disease Excision Goals Granulocyte-Macrophage Colony-Stimulating Factor Heat Shock Protein 27 IL8 gene Immune system In Vitro Infection Infection Control Infectious Agent Inflammatory Injury Laboratories Lead Leukotriene B4 Lipopolysaccharides MAP Kinase Gene MAPK14 gene MAPK8 gene Mediating Neutrophil Activation Organ failure Pathway interactions Peptides Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Play Post-Translational Protein Processing Protein Kinase Protein-Serine-Threonine Kinases Proteins Proteomics Regulation Reperfusion Injury Reporting Role Scaffolding Protein Sepsis Signal Pathway Signal Transduction Site Site-Directed Mutagenesis System Testing Time Tissues Transfection Ubiquitin Ubiquitination Western Blotting Work base chemokine cytokine fighting killings member multicatalytic endopeptidase complex neutrophil novel novel strategies novel therapeutics pathogen prevent protein protein interaction research study response

项目摘要

DESCRIPTION (provided by applicant): Activated neutrophils (PMNs) play a critical role in sepsis, ischemia-reperfusion injury, and immune complex-mediated diseases. The broad long-term objective of our laboratory is to elucidate the role of Akt in regulating PMN functions. This proposal will test the hypothesis: Hsp27 regulates neutrophil cell survival/ death responses by modulating critical protein-protein interactions within the Akt signalosome by acting as a scaffolding protein. The specific aims of this proposal are: Specific Aim 1 - To determine whether Akt associated/dissociated proteins regulate Akt activation and neutrophil apoptosis in the absence of Akt- Hsp27 interaction. Specific Aim 2- To determine signaling pathways that underlie Akt-Hsp27 disruption induced neutrophil apoptosis. Specific Aim 3- To determine whether induction of neutrophil apoptosis by disruption of Akt-Hsp27 interaction results from changes in phosphorylation and subcellular redistribution of Akt and its candidate proteins. In specific aim 1 we will generate TAT-fusion proteins to 4 Akt-associating and 4 Akt-dissociating proteins, identified by our proteomic studies and determine effects of transducing these proteins on Akt activation and neutrophil apoptosis. cDNA subcloning, site-directed mutagenesis, protein transductions, cDNA transfections, in vitro kinase assays, and apoptosis assays will be performed to accomplish this aim. Specific aim 2 experiments will be focused on determining mechanisms that underlie activation of p38 MARK and JNK pathways after disruption of Akt-Hsp27 interaction. We will also investigate the impact of Akt-Hsp27 disruption on the regulation of proteasomal activation. In vitro kinase assays, proteasome activity assays, transduction of relevant TAT-peptides into PMNs will be performed to accomplish this aim. In specific aim 3, we will focus our work on those Akt-associated or Akt-dissociated proteins shown to regulate Akt activation and neutrophil apoptosis in specific aim 1. We will then determine if these relevant proteins regulate Akt activation and neutrophil apoptosis by undergoing a post-translational modification such as phosphorylation and/or ubiquitination as a consequence of disruption of Akt-Hsp27 interaction. We will also determine if these protein modifications would result in change in localization of these proteins and/or alter their function. TAT-peptide transduction, cell fractionation studies, western blotting, site-directed mutagenesis studies, and confocal microscopy studies will be performed to accomplish this specific aim. These studies will lead us to new targets for disrupting neutrophil-mediated tissue damage in inflammatory diseases.

描述（由申请人提供）：活化的中性粒细胞（PMN）在败血症、缺血再灌注损伤和免疫复合物介导的疾病中发挥关键作用。我们实验室的长期目标是阐明 Akt 在调节 PMN 功能中的作用。该提案将检验以下假设：Hsp27 通过充当支架蛋白来调节 Akt 信号体内关键的蛋白质-蛋白质相互作用，从而调节中性粒细胞的生存/死亡反应。该提案的具体目标是：具体目标 1 - 确定 Akt 相关/解离蛋白是否在 Akt-Hsp27 相互作用缺失的情况下调节 Akt 激活和中性粒细胞凋亡。具体目标 2 - 确定 Akt-Hsp27 破坏诱导中性粒细胞凋亡的信号传导途径。具体目标 3-确定通过破坏 Akt-Hsp27 相互作用诱导中性粒细胞凋亡是否是由于 Akt 及其候选蛋白的磷酸化和亚细胞重新分布的变化所致。在具体目标 1 中，我们将生成 4 个 Akt 关联蛋白和 4 个 Akt 解离蛋白的 TAT 融合蛋白，这些蛋白是通过我们的蛋白质组学研究鉴定的，并确定转导这些蛋白对 Akt 激活和中性粒细胞凋亡的影响。为了实现这一目标，将进行 cDNA 亚克隆、定点诱变、蛋白质转导、cDNA 转染、体外激酶测定和细胞凋亡测定。具体目标 2 实验将侧重于确定 Akt-Hsp27 相互作用破坏后 p38 MARK 和 JNK 通路激活的机制。我们还将研究 Akt-Hsp27 破坏对蛋白酶体激活调节的影响。将进行体外激酶测定、蛋白酶体活性测定、将相关 TAT 肽转导至 PMN 中以实现这一目标。在具体目标 3 中，我们将重点研究那些在具体目标 1 中显示可调节 Akt 激活和中性粒细胞凋亡的 Akt 相关或 Akt 解离蛋白。然后，我们将确定这些相关蛋白是否通过经历一个过程来调节 Akt 激活和中性粒细胞凋亡。由于 Akt-Hsp27 相互作用破坏而导致的翻译后修饰，例如磷酸化和/或泛素化。我们还将确定这些蛋白质修饰是否会导致这些蛋白质的定位变化和/或改变其功能。将进行 TAT 肽转导、细胞分级分离研究、蛋白质印迹、定点诱变研究和共聚焦显微镜研究来实现这一特定目标。这些研究将为我们找到破坏炎症性疾病中中性粒细胞介导的组织损伤的新目标。