Role of Hsp27 in regulation of Neutrophil Apoptosis

描述(由申请人提供)：激活的中性粒细胞(PMN)在脓毒症、缺血-再灌注损伤和免疫复合体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。这项提议将验证HSP27通过作为Akt激活和诱导中性粒细胞存活途径的支架蛋白来调控中性粒细胞凋亡的假设。这项建议的具体目的是：特定目标1-确定其与Akt信号复合体的关联受Hsp27调控的蛋白质。特定目标2-确定Akt-Hsp27复合体破坏后蛋白质变化对Akt激活和中性粒细胞凋亡调节的影响。具体目标3-确定Akt-Hsp27中断诱导中性粒细胞凋亡的信号通路。特定目的4-确定Akt自身磷酸化位点，并确定它们对Akt-Hsp27信号复合体组装、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)通过转导杂乱的TAT或TAT-Akt117-128肽，它包含Akt上的Hsp27结合位点。B)我们将在PMN中引入对照或抗Hsp27抗体。C)将干扰或Hsp27 siRNA导入HK-11细胞。蛋白质裂解产物将经过Akt和Hsp27免疫沉淀、2D SDS-PAGE、MALDI-MS和LC-ESI-MS/MS分析，以确定Akt-Hsp27复合体在存在或不存在Akt-Hsp27相互作用时蛋白质的变化。特定目的2实验将确定与Akt-Hsp27复合体结合/解离的蛋白质(在特定目的1中确定)是否影响Akt活性和中性粒细胞凋亡。在特定的目标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反应。在特定的目的中，将使用4个定点突变和质谱学技术来鉴定体外Akt自动磷酸化位点。商业上将产生针对Akt自身磷酸化位点的磷酸化特异性抗体，以识别体内中性粒细胞的磷酸化位点，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。这些研究将引导我们找到破坏炎症性疾病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.