Regulation of anti- & pro-apoptotic functions of BAD

描述(申请人提供)：由Bcl2家族蛋白调控的凋亡性细胞死亡在急性和慢性神经病理中发挥重要作用，如中风、创伤性脑损伤、阿尔茨海默病、帕金森病和肌萎缩侧索硬化症。在体外谷氨酸处理的海马神经元和体内损伤后的脊髓神经元中，已经阐明了BAD蛋白在诱导海马神经元凋亡中的特殊作用。BAD是多个水平的翻译后修饰的靶点，包括磷酸化、蛋白水解性切割和细胞内重定位。然而，这些修饰调节神经元内BAD功能的方式仍然没有完全确定。该实验室进行了一项新的观察，即BAD可以是抗凋亡的，也可以是促凋亡的，这取决于细胞环境。因此，确定决定不良功能的细胞调控机制对于神经保护和抗癌干预都具有相当大的希望。特定目标1将使用BAD敲入突变小鼠和细胞培养模型来测试BAD的磷酸化、切割和线粒体靶向调节BAD从抗凋亡信号分子向促凋亡信号分子转化的假设。特殊目标2将使用细胞培养模型和免疫荧光成像技术来识别细胞死亡途径中被BAD抑制的步骤。特定目标3将使用原代皮质培养和小鼠来识别不良翻译后修饰和结合相互作用的差异，当BAD经历功能逆转时，这些差异在成熟过程中会有所不同。亲和纯化和酵母双杂交筛选将被用来寻找在发育过程中差异表达的新的不良调节蛋白。

DESCRIPTION (provided by applicant): Apoptotic cell death that is regulated by Bcl-2 family proteins plays a significant role in acute and chronic neuropathologies such as stroke, traumatic brain injury, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. A specific role for the Bcl-2 family protein BAD in inducing apoptosis has been elucidated for hippocampal neurons treated with glutamate in vitro and for neurons of the spinal cord following traumatic injury in vivo. BAD is a target for multiple levels of post-translational modification, including phosphorylation, proteolytic cleavage, and intracellular relocalization. The manner in which these modifications regulate the function of BAD within neurons, however, remains incompletely defined. This laboratory has made the novel observation that BAD can be either anti-apoptotic or pro-apoptotic depending on the cellular context. Defining the cellular mechanisms of regulation that determine BAD function therefore holds considerable promise for both neuroprotective and anti-cancer interventions.Specific Aim 1 will use BAD knock-in mutant mice and cell culture models to test the hypothesis that phosphorylation, cleavage, and mitochondrial targeting of BAD regulate the conversion of BAD from an anti-apoptotic to a pro-apoptotic signaling molecule. Specific Aim 2 will use cell culture models and immunofluorescent imaging techniques to identify the step in the cell death pathway that is inhibited by BAD. Specific Aim 3 will use primary cortical cultures and mice to identify differences in BAD post-translational modification and binding interactions that differ during maturation when BAD undergoes reversal of function. Affinity-purification and yeast-two-hybrid screening will be used to search for novel BAD regulatory proteins that are differentially expressed during development.