Modulation of Neutrophil Apoptosis by Akt-Hsp27 Signalosome

Akt-Hsp27 信号体对中性粒细胞凋亡的调节

• 批准号: 7627843
• 负责人: MADHAVI J RANE
• 金额: $ 5.53万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-10 至 2008-08-07
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7627843
• 关键词: Address; Antigen-Antibody Complex; Apoptosis; Apoptotic; Bacteria; Binding; Biological Assay; Cell Fractionation; Cell Survival; Cessation of life; Chimeric Proteins; Complementary DNA; Complex; Data; Disease; Disruption; Excision; Goals; Granulocyte-Macrophage Colony-Stimulating Factor; Heat Shock Protein 27; IL8 gene; Immune system; In Vitro; Infection; Infection Control; Infectious Agent; Inflammatory; Injury; Laboratories; Lead; Leukotriene B4; Lipopolysaccharides; MAP Kinase Gene; MAPK14 gene; MAPK8 gene; Mediating; Microscopic; Mitogen-Activated Protein Kinase Kinases; Molecular; Mutagenesis; Neutrophil Activation; Organ failure; Pathway interactions; Peptides; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Play; Post-Translational Protein Processing; Protein Kinase; Protein-Serine-Threonine Kinases; Proteins; Proteomics; Rate; Regulation; Reperfusion Injury; Reporting; Role; Scaffolding Protein; Sepsis; Signal Pathway; Signal Transduction; Site; Site-Directed Mutagenesis; System; Testing; Time; Tissues; Transfection; Ubiquitination; Western Blotting; Work; base; chemokine; cytokine; fighting; intracellular protein transport; killings; member; multicatalytic endopeptidase complex; neutrophil; novel; novel strategies; novel therapeutics; pathogen; prevent; protein localization location; protein protein interaction; research study; response

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.

活化的中性粒细胞 (PMN) 在脓毒症、缺血再灌注损伤和免疫中发挥着关键作用 复杂介导的疾病。我们实验室的长期目标是阐明 Akt 调节 PMN 功能。该提案将检验以下假设：Hsp27 调节中性粒细胞 通过调节 Akt 信号体内关键的蛋白质-蛋白质相互作用来响应生存/死亡 通过充当支架蛋白。该提案的具体目标是： 具体目标 1- 确定 Akt 相关/解离蛋白是否调节 Akt 激活和中性粒细胞凋亡 缺乏 Akt-Hsp27 相互作用。具体目标 2- 确定 Akt 背后的信号传导途径- Hsp27 破坏诱导中性粒细胞凋亡。具体目标 3 - 确定是否诱导 JNK 或 p38 MAPK 介导的变化导致 Akt-Hsp27 相互作用破坏导致中性粒细胞凋亡 Akt 候选蛋白的蛋白磷酸化、泛素化或亚细胞重新分布。 在具体目标 1 中，我们将生成 6 个 Akt 关联蛋白和 6 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的蛋白质，并确定转导这些蛋白质对 Akt 的影响 激活和中性粒细胞凋亡。 cDNA 亚克隆、定点突变、蛋白质 将进行转导、cDNA 转染、体外激酶测定和细胞凋亡测定 实现这一目标。具体目标 2 实验将侧重于确定以下机制： 是 Akt-Hsp27 相互作用破坏后 p38 MAPK 和 JNK 通路激活的基础。我们将 还研究了 Akt-Hsp27 破坏对蛋白酶体激活调节的影响。体外 JNK 和 p38 MAPK 激酶测定、蛋白酶体活性测定、相关 TAT 肽的转导/ 将 TAT 蛋白转化为 PMN 来实现这一目标。在具体目标 3 中，我们将 我们的工作重点是那些显示可调节 Akt 激活的 Akt 相关或 Akt 解离蛋白 和中性粒细胞凋亡的具体目标1。然后我们将确定这些相关蛋白是否调节 通过进行翻译后修饰（例如 Akt-Hsp27 相互作用破坏导致的磷酸化和/或泛素化。我们将 还确定是否 这些蛋白质修饰将导致这些蛋白质的定位发生变化 和/或改变它们的功能。 TAT 肽转导、细胞分级分离研究、蛋白质印迹、定点 将进行诱变研究和共焦显微镜研究来实现这一目标 具体目标。这些研究将引导我们找到破坏中性粒细胞介导的组织的新目标 炎症性疾病的损害。