Modulation of Neutrophil Apoptosis by Akt-Hsp27 Signalosome

点击翻译按钮获取中文摘要

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 JNK or p38 MAPKmediated protein phosphorylation, ubiquitination or subcellular redistribution of Akt-candidate proteins. In specific aim 1 we will generate TAT-fusion proteins to 6 Akt-associating and 6 Akt-dissociating proteins, identified by our proteomic studies and determine effects of transducing these proteins on Akt activation and neutrophil apoptosis. cDNA subclonning, 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 MAPK 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 JNK and p38 MAPK kinase assays, proteasome activity assays, transduction of relevant TATpeptides/ TAT-proteins 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, sitedirected mutagenesis studies, and confocal microscopic 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.