Hypoxia-induced Akt Signaling module in Neuronal Cells.

神经元细胞中缺氧诱导的 Akt 信号传导模块。

• 批准号: 6774693
• 负责人: EVELYNE GOZAL
• 金额: $ 36.75万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6774693
• 关键词: SDS polyacrylamide gel electrophoresis; apoptosis; binding proteins; biological signal transduction; chimeric proteins; enzyme complex; enzyme mechanism; gene deletion mutation; hypoxia; immunoprecipitation; matrix assisted laser desorption ionization; neurogenetics; neurons; phosphorylation; protein kinase; protein protein interaction; protein sequence; proteomics; tissue /cell culture; western blottings

DESCRIPTION (provided by applicant): The purpose of this proposal is to elucidate hypoxia-induced Akt-related pathways induced in neuronal PC- 12 and RN46A neuronal cells by sustained hypoxia and by intermittent hypoxia, and to determine how these signaling pathways affect cell survival. We will test the hypothesis that hypoxia-induced interactions between different signaling molecules within the Akt signaling module, modulate tolerance or vulnerability to sustained and intermittent hypoxia in neuronal cells. We propose: (1) To identify, using proteomic approaches, members of the Akt signaling module in normoxic and hypoxic neuronal cells, and to identify differences in Akt-associated proteins during sustained and intermittent hypoxia; (2) To characterize protein-protein interactions within the Akt signaling modules; (3) To identify selective Akt - protein interactions within the signaling module that underlie neuronal survival to sustained and intermittent hypoxia; (4). To examine the effect of disruption of protein-protein interactions within the Akt signaling module on downstream genes involved in regulation of hypoxia-induced cellular apoptosis. We will use proteomic techniques, SDS-PAGE and MALDI-MS to identify the Akt-binding proteins co-immunoprecipitating with Akt in normoxic cells or cells exposed to sustained or intermittent hypoxia. Interactions of the components of the Akt signalosomes will be determined by TnT coupled transcription, translation, co-immunoprecipitation, and GST pull-down methods. Transformer kits will be used to make in frame and serial deletion mutants of Akt and its binding proteins in order to identify their Akt docking sites. Finally we will introduce into the cells TAT-fusion peptides corresponding to specific docking sites of targeted proteins binding to Akt signaling module, and disrupt their interaction with the Akt signaling complex to assess the effect of protein association/dissociation with the Akt signaling module, on cell survival to sustained and intermittent hypoxia. These studies will provide the groundwork for future intervention strategies aiming to prevent neuronal cell loss in diseases associated with intermittent and sustained hypoxia, such as sleep apnea and lung disease.

描述(申请人提供)：本建议的目的是阐明低氧诱导神经元PC-12和RN46A神经元细胞持续低氧和间歇性低氧诱导Akt相关的通路，并确定这些信号通路如何影响细胞存活。我们将测试这一假设，即低氧诱导Akt信号模块中不同信号分子之间的相互作用，调节神经细胞对持续和间歇性低氧的耐受性或易感性。我们建议：(1)利用蛋白质组学方法，鉴定常氧和低氧神经细胞中Akt信号模块的成员，以及在持续和间歇低氧过程中Akt相关蛋白的差异；(2)表征Akt信号模块中的蛋白质-蛋白质相互作用；(3)鉴定持续和间歇低氧下神经元存活所依赖的信号模块中Akt-蛋白质的选择性相互作用；(4)目的：研究Akt信号模块中蛋白质-蛋白质相互作用的中断对参与低氧诱导细胞凋亡调控的下游基因的影响。我们将使用蛋白质组学技术、SDS-PAGE和MALDI-MS来鉴定在常氧或持续或间歇低氧条件下与Akt免疫共沉淀的Akt结合蛋白。Akt信号小体各组成部分之间的相互作用将通过TNT偶联转录、翻译、免疫共沉淀和GST下拉方法来确定。转换试剂盒将用于制造Akt及其结合蛋白的框内和序列缺失突变体，以确定其Akt对接位点。最后，我们将目标蛋白与Akt信号模块结合的特定对接位置对应的TAT融合多肽引入细胞，并破坏它们与Akt信号复合体的相互作用，以评估蛋白与Akt信号模块的结合/解离对持续和间歇性低氧下细胞存活的影响。这些研究将为未来旨在防止与间歇性和持续性低氧相关的疾病(如睡眠呼吸暂停和肺部疾病)中的神经细胞丢失的干预策略提供基础。