Hypoxia-induced Akt Signaling module in Neuronal Cells.

描述（由申请人提供）：本提案的目的是阐明通过持续缺氧和间歇缺氧在神经元PC- 12和RN 46 A神经元细胞中诱导的缺氧诱导的Akt相关途径，并确定这些信号传导途径如何影响细胞存活。我们将测试的假设，缺氧诱导的Akt信号模块内的不同信号分子之间的相互作用，调节神经元细胞的耐受性或脆弱性，持续和间歇性缺氧。我们建议：（1）利用蛋白质组学方法鉴定常氧和缺氧神经元细胞中Akt信号模块的成员，并鉴定持续和间歇缺氧过程中Akt相关蛋白的差异;（2）表征Akt信号模块中蛋白质-蛋白质相互作用;（3）鉴定信号传导模块内的选择性Akt -蛋白相互作用，其是神经元在持续和间歇性缺氧下存活的基础;（四）、研究Akt信号模块中蛋白质-蛋白质相互作用的破坏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来鉴定在常氧细胞或暴露于持续或间歇性缺氧的细胞中与Akt共免疫沉淀的Akt结合蛋白。Akt信号体组分的相互作用将通过TnT偶联转录、翻译、免疫共沉淀和GST下拉方法来确定。Transformer试剂盒将用于制备Akt及其结合蛋白的框内和连续缺失突变体，以鉴定它们的Akt对接位点。最后我们将靶蛋白与Akt信号模块结合的特定对接位点对应的TAT融合肽导入细胞中，并破坏其与Akt信号复合物的相互作用，以评估蛋白与Akt信号模块的结合/解离对细胞在持续和间歇性缺氧条件下存活的影响。这些研究将为未来的干预策略提供基础，旨在预防与间歇性和持续性缺氧相关的疾病（如睡眠呼吸暂停和肺部疾病）中的神经元细胞丢失。

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.