Beclin 1-Bcl-2 Interactions: Effects on Autophagy

描述（由申请人提供）：细胞凋亡和自噬都是受到严格调控的细胞过程，在细胞的生死决定、组织稳态、发育和癌症生物学中发挥关键作用。第一个发现的抗凋亡蛋白是Bcl-2，我们的实验室鉴定了第一个哺乳动物自噬蛋白Beclin 1。在上一个项目期间，我们证明了Bcl-2作为一种抗自噬蛋白的功能，并确定了一个关键的生化机制（即应激诱导的Bcl-2多位点磷酸化）调节Bcl-2和Beclin 1之间的相互作用。在下一个项目期间，我们建议更深入地了解调节Bcl-2/Beclin 1相互作用的分子机制，以及Bcl-2调节Beclin 1依赖性自噬的体内生理意义。在第一个具体目标中，我们将使用结构生物学，生物化学和细胞生物学方法来定义调节Bcl-2和Beclin 1之间相互作用的新分子决定簇。我们将使用核磁共振（NMR）来鉴定细胞Bcl-2中预测选择性参与Beclin 1结合的残基（但不是其它含有BH 3结构域的蛋白），在功能测定中测试这些预测，并进行生物化学和细胞生物学研究，以调查我们在初步研究中发现的饥饿调节的Beclin 1翻译后修饰是否在一些实施方案中，调节细胞凋亡（例如丝氨酸磷酸化、精氨酸甲基化）的作用是：（1）受Bcl-2调节;（2）修饰Bcl-2/Beclin 1相互作用;和（3）修饰Beclin 1自噬活性。在第二个具体目标中，我们将评估Bcl-2调节Beclin 1自噬功能的体内生理意义，使用表达Bcl-2或Beclin 1突变形式的靶向突变小鼠，这些突变形式改变Bcl-2/Beclin 1相互作用的正常调节。我们将表征先前产生的Bcl-2 nonphosphorylatable突变小鼠，预测组成性抑制Beclin 1功能（以及新产生的小鼠，基于我们的研究结果，在第一个具体目标）在体内调节自噬和自噬依赖的生物过程。总之，这些研究有望有助于阐明Bcl-2/Beclin 1复合物控制自噬的分子机制，以及该复合物在调节细胞、组织稳态、发育和癌症生物学的生死决定中的重要性。

DESCRIPTION (provided by applicant): Apoptosis and autophagy are both tightly regulated cellular processes that play crucial roles in life and death decisions of the cell, tissue homeostasis, development, and cancer biology. The first discovered anti-apoptotic protein was Bcl-2, and our laboratory identified the first mammalian autophagy protein, Beclin 1. In the previous project period, we demonstrated that Bcl-2 functions as an anti-autophagy protein, and identified a key biochemical mechanism (i.e. stress-induced Bcl-2 multisite phosphorylation) underlying the regulation of interactions between Bcl-2 and Beclin 1. During the next project period, we propose to gain a deeper understanding of both the molecular mechanisms regulating the Bcl-2/Beclin 1 interaction, and the in vivo physiological significance of Bcl-2 regulation of Beclin 1-dependent autophagy. In the first specific aim, we will use structural biology, biochemical, and cell biology approaches to define novel molecular determinants that regulate the interaction between Bcl-2 and Beclin 1. We will use nuclear magnetic resonance (NMR) to identify residues in cellular Bcl-2 that are predicted to be selectively involved in binding to Beclin 1 (but not other BH3 domain containing proteins), test these predictions in functional assays, and perform biochemical and cell biology studies to investigate whether starvation-regulated post-translational modifications of Beclin 1 that we have identified in preliminary studies (e.g. serine phosphorylation, arginine methylation) are: (1) regulated by Bcl-2; (2) modify Bcl-2/Beclin 1 interactions; and (3) modify Beclin 1 autophagic activity. In the second specific aim, we will evaluate the in vivo physiological significance of Bcl-2 modulation of the autophagy function of Beclin 1, using targeted mutant mice that express mutant forms of either Bcl-2 or Beclin 1 that alter the normal regulation of the Bcl-2/Beclin 1 interaction. We will characterize previously generated Bcl-2 nonphosphorylatable mutant mice that are predicted to constitutively inhibit Beclin 1 function (as well as newly generated mice based on our results with studies in the first specific aim) with respect to in vivo regulation of autophagy and autophagy-dependent biological processes. Together, these studies are expected to help elucidate the molecular mechanisms underlying the control of autophagy by the Bcl-2/Beclin 1 complex and the significance of this complex in regulating life and death decisions of the cell, tissue homeostasis, development, and cancer biology.