Role of Hsp27 in regulation of Neutrophil Apoptosis

描述（由申请人提供）：活化中性粒细胞（pmn）在脓毒症、缺血再灌注损伤和免疫复合物介导的疾病中起关键作用。我们实验室的长期目标是阐明Akt在调节PMN功能中的作用。这一提议将验证Hsp27作为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。本研究的具体目的是：1 .确定与Akt信号复合物相关的蛋白受Hsp27调控。特异性目的2：确定Akt- hsp27复合物破坏后蛋白变化对Akt活化和中性粒细胞凋亡调控的影响。特异性目的3 -确定Akt-Hsp27破坏诱导的中性粒细胞凋亡的信号通路。特异性目的4-鉴定Akt自磷酸化位点，并确定其对Akt- hsp27信号复合物组装、Akt活化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的相互作用。A)通过转导TAT-scrambled或TAT- Akt117-128肽，该肽包含Akt上的Hsp27结合位点。B)在PMNs中引入对照抗体或抗hsp27抗体。C)用重组或Hsp27 siRNA转染HK-11细胞。蛋白裂解物将进行Akt和Hsp27免疫沉淀、2D SDS-PAGE、MALDI-MS和LC-ESI-MS/MS分析，以确定Akt-Hsp27复合物中存在或不存在Akt-Hsp27相互作用时蛋白的变化。特异性靶2实验将确定与Akt- hsp27复合物相关/解离的蛋白（在特异性靶1中鉴定）是否影响Akt活性和中性粒细胞凋亡。在特定的目的3中，我们将确定在没有Akt-Hsp27相互作用的情况下诱导中性粒细胞凋亡的信号级联。在特定的目标中，4位点定向诱变和质谱技术将用于鉴定体外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被商业化生产，以鉴定中性粒细胞体内磷酸化位点，并确定其对Akt信号模块组装、Akt活化和中性粒细胞凋亡的影响。这些研究将引导我们找到在炎症性疾病中破坏中性粒细胞介导的组织损伤的新靶点。

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.