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严格调控的细胞过程，在细胞的生死决定、组织的动态平衡、发育和癌症生物学中发挥着至关重要的作用。第一个发现的抗凋亡蛋白是Bcl2，我们的实验室发现了第一个哺乳动物自噬蛋白Beclin 1。在前一个项目期，我们证明了Bcl2作为一个抗自噬蛋白的功能，并确定了调节Bcl2和Beclin 1相互作用的关键生化机制(即应激诱导的Bcl2多位点磷酸化)。在下一个项目期，我们计划对调节Bcl2/Beclin 1相互作用的分子机制以及Bcl2调节Beclin 1依赖的自噬的体内生理意义有更深入的了解。在第一个具体目标中，我们将使用结构生物学、生化和细胞生物学的方法来定义新的分子决定因素来调节Bclin-2和Beclin 1之间的相互作用。我们将使用核磁共振(核磁共振)来确定细胞中被预测与Beclin 1(但不是其他含有BH3结构域的蛋白质)结合的残基，在功能分析中检验这些预测，并进行生化和细胞生物学研究，以研究我们在初步研究中发现的Beclin 1的饥饿调节的翻译后修饰(例如丝氨酸磷酸化、精氨酸甲基化)是否：(1)受Bcl2调节；(2)修改Bcl2/Beclin 1的相互作用；(3)修改Beclin 1的自噬活性。在第二个特定目标中，我们将使用表达突变形式的Bcl-2或Beclin 1的靶向突变小鼠来评估Bcl-2对Beclin 1自噬功能的体内生理意义，这些突变形式改变了Bcl-2/Beclin 1相互作用的正常调节。我们将在自噬和自噬依赖的生物过程的体内调节方面，表征先前产生的、被预测为结构性抑制Beclin 1功能的Bcl-2非磷酸化突变小鼠(以及根据我们的研究结果在第一个特定目标上新产生的小鼠)。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、组织内稳态、发育和癌症生物学的生死决策方面的意义。 与公共卫生相关：我们正在研究两种相互作用的蛋白质，即Bcl-2和Beclin 1，它们都已知在调节细胞的生存或死亡、多细胞生物体如何发展和适应不同形式的压力以及人类癌症如何发生和对治疗做出反应方面发挥作用。我们研究的目标是了解这些蛋白质之间的相互作用是如何调节的，以及这种相互作用如何有助于哺乳动物成功适应压力、正常发育和避免癌症的能力。

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.