Beclin 1-Bcl-2 Interactions: Effects on Autophagy

描述（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、组织的稳态、发育和癌症生物学中起着至关重要的作用。第一个发现的抗凋亡蛋白是Bcl-2，我们实验室发现了第一个哺乳动物自噬蛋白Beclin 1。在之前的项目期间，我们证明了Bcl-2作为抗自噬蛋白的功能，并确定了Bcl-2与Beclin 1相互作用调控的关键生化机制（即应激诱导的Bcl-2多点磷酸化）。在接下来的项目期间，我们计划深入了解Bcl-2/Beclin 1相互作用的分子调控机制，以及Bcl-2调控Beclin 1依赖性自噬的体内生理意义。在第一个特定目标中，我们将使用结构生物学，生化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因素。我们将使用核磁共振（NMR）识别细胞Bcl-2中预测选择性参与Beclin 1结合的残基（但不包括其他BH3结构域蛋白），在功能分析中测试这些预测，并进行生化和细胞生物学研究，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（例如丝氨酸磷酸化，精氨酸甲基化）是否：(1)受Bcl-2调控；(2)改变Bcl-2/Beclin - 1的相互作用；(3)改变Beclin - 1自噬活性。在第二个特定目标中，我们将评估Bcl-2调节Beclin 1自噬功能的体内生理意义，使用靶向突变小鼠表达Bcl-2或Beclin 1的突变形式，改变Bcl-2/Beclin 1相互作用的正常调节。我们将描述先前生成的Bcl-2非磷酸化突变小鼠，这些小鼠被预测会构成性地抑制Beclin 1的功能（以及基于我们的研究结果的新生成的小鼠，在第一个特定目标中），涉及自噬和自噬依赖性生物过程的体内调节。总之，这些研究有望帮助阐明Bcl-2/Beclin 1复合体控制自噬的分子机制，以及该复合体在调节细胞生死决定、组织稳态、发育和癌症生物学中的意义。

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. PUBLIC HEALTH RELEVANCE: We are studying two proteins, Bcl-2 and Beclin 1, that interact with each other and are each known to play a role in regulating whether cells live or die, how multicellular organisms develop and adapt to different forms of stress, and how human cancers occur and respond to treatment. The goal of our studies is to understand how the interaction between these proteins is regulated and how this interaction contributes to the ability of mammals to successfully adapt to stress, to develop normally, and to avoid cancer.