Modulation of Neutrophil Apoptosis by Akt-Hsp27 Signalosome

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。这一提议将检验这一假设：HSP27通过作为支架蛋白调节Akt信号体内的关键蛋白质-蛋白质相互作用来调节中性粒细胞的生存/死亡反应。这一建议的具体目的是：特定目的1-确定在没有Akt-Hsp27相互作用的情况下，Akt相关/解离蛋白是否调节Akt的激活和中性粒细胞的凋亡。特定目的2-确定Akt-Hsp27中断诱导中性粒细胞凋亡的信号通路。具体目的3-确定Akt-Hsp27相互作用中断诱导中性粒细胞凋亡是否源于Akt及其候选蛋白的磷酸化和亚细胞内重分布的变化。在特定的目标1，我们将产生4个Akt结合蛋白和4个Akt解离蛋白的TAT融合蛋白，通过蛋白质组学研究，并确定这些蛋白转导这些蛋白对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、定点突变、蛋白转导、cDNA转染、体外激酶检测和细胞凋亡检测。具体目的2实验将集中在确定在Akt-Hsp27相互作用中断后激活p38Mark和JNK通路的机制。我们还将研究Akt-Hsp27中断对蛋白酶体激活调节的影响。为了实现这一目标，将进行体外激酶分析、蛋白酶体活性分析以及将相关TAT多肽转导到PMN中。在特定目标3中，我们将重点研究那些在特定目标1中调节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翻译后修饰来调节Akt激活和中性粒细胞凋亡，如由于Akt-Hsp27相互作用中断而发生的磷酸化和/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改变和/或改变它们的功能。为了达到这一特定的目的，将进行TAT-肽转导、细胞分离研究、蛋白质印迹、定点突变研究和共聚焦显微镜研究。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: 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.