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持续缺氧和间歇性缺氧在神经元PC- 12和RN46A神经元细胞中诱导的缺氧诱导的akt相关通路，并确定这些信号通路如何影响细胞存活。我们将验证缺氧诱导的Akt信号模块内不同信号分子之间的相互作用，调节神经元细胞对持续和间歇性缺氧的耐受性或易感性的假设。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，并鉴定持续缺氧和间歇性缺氧时Akt相关蛋白的差异；(2)表征Akt信号模块内蛋白-蛋白相互作用；(3)确定信号模块中Akt -蛋白的选择性相互作用，这些相互作用是神经元在持续和间歇性缺氧条件下存活的基础；(4)。研究Akt信号模块中蛋白-蛋白相互作用的中断对参与缺氧诱导的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信号小体各组分的相互作用将通过TnT偶联转录、翻译、共免疫沉淀和GST下拉方法确定。将使用Transformer kit制作Akt及其结合蛋白的框架内和序列缺失突变体，以确定它们的Akt对接位点。最后，我们将引入靶向蛋白与Akt信号模块结合的特定对接位点对应的tat融合肽，并破坏其与Akt信号复合物的相互作用，以评估蛋白质与Akt信号模块结合/解离对持续和间歇性缺氧细胞存活的影响。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）的神经元细胞损失。

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.