Modulation of Neutrophil Apoptosis by Akt-Hsp27 Signalosome

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。该提案将验证假设：Hsp27通过作为支架蛋白调节Akt信号体内关键蛋白-蛋白相互作用来调节中性粒细胞存活/死亡反应。本研究的具体目的是：1 -确定Akt相关/解离蛋白是否在Akt- Hsp27不相互作用的情况下调节Akt活化和中性粒细胞凋亡。特异性目的2-确定Akt-Hsp27破坏诱导中性粒细胞凋亡的信号通路。特异性目的3-确定破坏Akt- hsp27相互作用诱导中性粒细胞凋亡是否源于Akt及其候选蛋白磷酸化和亚细胞重分布的改变。在具体目标1中，我们将生成4个Akt相关蛋白和4个Akt解离蛋白的tat融合蛋白，通过我们的蛋白质组学研究鉴定，并确定转导这些蛋白对Akt激活和中性粒细胞凋亡的影响。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将实现这一目标。特异性目的2实验将集中于确定Akt-Hsp27相互作用中断后p38 MARK和JNK通路激活的机制。我们还将研究Akt-Hsp27破坏对蛋白酶体活化调节的影响。为了实现这一目标，将进行体外激酶测定、蛋白酶体活性测定、相关tat肽转化为pmn的转导。在特定目标3中，我们将重点研究Akt相关或Akt解离蛋白在特定目标1中调节Akt激活和中性粒细胞凋亡。然后，我们将确定这些相关蛋白是否通过翻译后修饰（如磷酸化和/或泛素化）调节Akt活化和中性粒细胞凋亡，这是Akt- hsp27相互作用中断的结果。我们还将确定这些蛋白质修饰是否会导致这些蛋白质定位的改变和/或改变它们的功能。tat肽转导、细胞分离研究、western blotting、定点诱变研究和共聚焦显微镜研究将完成这一特定目标。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: 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.