Role of Hsp27 in regulation of Neutrophil Apoptosis

Hsp27 在中性粒细胞凋亡调节中的作用

基本信息

批准号：
7241372
负责人：
MADHAVI J RANE
金额：
$ 29.6万
依托单位：
UNIVERSITY OF LOUISVILLE
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-07-01 至 2008-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7241372
关键词：
apoptosis clinical research neutrophil phosphorylation proteins role

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 that Hsp27 modulates neutrophil apoptosis by functioning as a scaffolding protein for Akt activation and induction of neutrophil survival pathways. The specific aims of this proposal are: Specific Aim 1-To identify proteins whose association with the Akt signaling complex is regulated by Hsp27.Specific Aim 2-To determine the effect of protein changes, following disruption of Akt-Hsp27complex, on the regulation of Akt activation and neutrophil apoptosis. Specific Aim 3 -To identify signaling pathways that underlie Akt-Hsp27 disruption-induced neutrophil apoptosis. Specific Aim 4- To identify Akt auto-phosphorylation sites and determine their effect on assembly of Akt-Hsp27 signal complex, Akt activation, and neutrophil apoptosis. In specific aim 1 we will disrupt Akt/Hsp27 interaction by 3 separate approaches. A) By transducing TAT-scrambled or TAT- Akt117-128 peptide, which encompasses Hsp27 binding site on Akt. B) We will introduce control or anti-Hsp27 antibody into PMNs. C) Transfecting HK-11 cells with scrambled or Hsp27 siRNA. Protein lysates will be subjected to Akt and Hsp27 immunoprecipitation, 2D SDS-PAGE, MALDI-MS, and LC-ESI-MS/MS analysis to identify proteins changes within Akt-Hsp27 complex in the presence or absence of Akt-Hsp27 interaction. Specific aim 2 experiments will determine if proteins (identified in specific aim 1) that associate/ dissociate from Akt-Hsp27 complex, affect Akt activity and neutrophil apoptosis. In specific aim 3 we will identify signaling cascades that induce neutrophil apoptosis in the absence of Akt-Hsp27 interaction. In specific aim 4 site-directed mutagenesis and mass spectroscopic techniques will be used to identify in vitro Akt auto-phosphorylation sites. Phospho-specific antibodies to Akt auto-phosphorylation sites will be generated commercially to identify in vivo phosphorylation sites in neutrophils and to determine its effect on assembly of Akt signal module, Akt activation, and neutrophil apoptosis. 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 Hsp27 通过充当 Akt 激活和诱导中性粒细胞存活途径的支架蛋白来调节中性粒细胞凋亡的假设。该提案的具体目标是：具体目标 1 - 鉴定与 Akt 信号传导复合物关联受 Hsp27 调节的蛋白质。具体目标 2 - 确定 Akt-Hsp27 复合物破坏后蛋白质变化对调节的影响Akt 激活和中性粒细胞凋亡。具体目标 3 - 确定 Akt-Hsp27 破坏诱导的中性粒细胞凋亡的信号传导途径。具体目标 4 - 识别 Akt 自磷酸化位点并确定其对 Akt-Hsp27 信号复合物组装、Akt 激活和中性粒细胞凋亡的影响。在具体目标 1 中，我们将通过 3 种不同的方法破坏 Akt/Hsp27 相互作用。 A) 通过转导 TAT-乱序或 TAT-Akt117-128 肽，其包含 Akt 上的 Hsp27 结合位点。 B) 我们将向 PMN 中引入对照或抗 Hsp27 抗体。 C) 用乱序或 Hsp27 siRNA 转染 HK-11 细胞。对蛋白质裂解物进行 Akt 和 Hsp27 免疫沉淀、2D SDS-PAGE、MALDI-MS 和 LC-ESI-MS/MS 分析，以鉴定在存在或不存在 Akt-Hsp27 相互作用的情况下 Akt-Hsp27 复合物内的蛋白质变化。具体目标 2 实验将确定与 Akt-Hsp27 复合物结合/解离的蛋白质（在具体目标 1 中确定）是否影响 Akt 活性和中性粒细胞凋亡。在具体目标 3 中，我们将鉴定在没有 Akt-Hsp27 相互作用的情况下诱导中性粒细胞凋亡的信号级联。具体目标 4 将使用定点诱变和质谱技术来鉴定体外 Akt 自磷酸化位点。针对 Akt 自磷酸化位点的磷酸化特异性抗体将在商业上产生，以鉴定中性粒细胞中的体内磷酸化位点，并确定其对 Akt 信号模块组装、Akt 激活和中性粒细胞凋亡的影响。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。