Bax Pore Formation in Apoptosis: Structures of Intermediates and Mechanism of Assembly (Lin)

项目总结(LIN项目) 细胞凋亡是一种细胞死亡程序，通常会消除功能障碍的细胞，因此对人类来说是必不可少的 健康。细胞凋亡不足会导致癌症，而细胞凋亡过多则会加重中风后的脑损伤。 为了开发这些疾病的有效治疗方法，我们需要全面了解细胞凋亡是如何 是受监管的。线粒体膜穿孔是细胞凋亡的承诺性步骤，受 Bcl2家族蛋白之间的相互作用，如促凋亡的Bax和抗凋亡的Bcl2。而当 选择性靶向和抑制抗细胞凋亡蛋白的抗癌药物在临床上显示出良好的前景 试验中，有限的促凋亡蛋白的结构信息已成为发展的瓶颈 可以减少中风损伤的神经保护剂。具体地，活性Bax低聚物的结构 能否在线粒体膜上形成巨大的孔来释放致命的线粒体因子尚不清楚。 由于天然Bax蛋白有多种相互作用方式，并可产生不同类型的异质 不服从结构测定的低聚物，一种替代和创新的实验 需要一种方法来确定这些不同低聚物的结构和它们可能形成的巨大孔隙。 我们假设：(A)适合于结构测定的均一小分子齐聚物可以通过以下方式产生 使用截短或迷你Bax蛋白，保留在完整Bax寡聚体中看到的关键相互作用区域；(B)这些 小分子寡聚体将是分析完整的Bax蛋白穿孔细胞的机制的理想工具。 线粒体膜，抗凋亡化合物通过该膜靶向Bax来阻止这种线粒体 渗漏和神经细胞死亡。我们的俄克拉荷马州眼镜蛇结构生物学第二阶段项目 应用程序将在以下具体目标上检验我们的新假设：(1)迷你- BAX寡聚体及其如何通过完整的BAX促进巨孔组装？(2)抗细胞凋亡 化合物能阻断线粒体膜上Bax孔的形成？在目标1中，我们将使用分子生物学 构建迷你Bax蛋白的技术。我们将进行二硫化物交联实验和尺寸 排除层析法选择小寡聚体形成蛋白和线粒体蛋白或脂质体 染料释放实验将用于鉴定微孔形成的低聚物。我们将进行结晶试验 与成孔、尺寸选择的均一小分子低聚物结合，并用X-射线解析其结构 绕射。如果没有得到合适的晶体，我们将测量mini-Bax的小角X射线散射 在溶液、洗涤剂胶束或脂质纳米盘中显示蛋白质，以揭示微小低聚结构。此外，我们 将使用电子冷冻显微镜来揭示膜中的微孔结构，并通过诱变来评估 这些微小结构如何通过完整的Bax在体外和体内对巨孔组装做出贡献。我们的合作者 大卫·安德鲁斯博士发现了几种化合物，可以阻止Bax在一种未定义的二聚体上进行齐聚 在中风损伤的体外模型中，分级和保护初级神经元免受谷氨酸兴奋毒性。在AIM 2，我们将探索这些化合物是否抑制了我们的mini-Bax蛋白的寡聚而不是二聚化 如果是这样的话，这种抑制是否足以消除微孔的形成。我们还将探索这些 化合物迫使完整的Bax进入末端二聚体，而不会导致线粒体膜穿孔和 如果是这样的话，我们将解决死胡同的二聚体结构。预期的项目成果是 对于线粒体膜上的促凋亡Bax孔组装至关重要的分子相互作用， 并通过抗细胞凋亡化合物对这一过程进行调控。这些重大成果对 细胞凋亡领域将是至关重要的，因为它们将揭示相关的靶点和发展机制。 更好的神经保护药物，有效对抗中风造成的脑损伤。

Project Summary (Lin Project) Apoptosis is a cell death program that normally eliminates dysfunctional cells, and hence is essential to human health. Insufficient apoptosis leads to cancer, while excessive apoptosis worsens brain damage after stroke. To develop effective treatments for these diseases we need a comprehensive understanding of how apoptosis is regulated. Mitochondrial membrane perforation is the commitment step of apoptosis that is regulated by interactions among the Bcl-2 family proteins, such as the pro-apoptotic Bax and the anti-apoptotic Bcl-2. While anti-cancer drugs that selectively target and inhibit the anti-apoptotic proteins have shown promise in clinical trials, limited structural information on the pro-apoptotic proteins has become a bottleneck for development of neural protective agents that can reduce stroke damage. In particular, structures of active Bax oligomers that can form giant pores in the mitochondrial membrane to release deadly mitochondrial factors are unknown. Since native Bax proteins have multiple ways to interact and can generate different types of heterogeneous oligomers that are not amenable to structure determination, an alternative and innovative experimental approach is required to determine the structures of these different oligomers and the giant pores they can form. We hypothesize that: (A) Homogeneous small oligomers suitable for structure determination can be created by using truncated or mini-Bax proteins that retain key interacting regions seen in intact Bax oligomers; (B) These small oligomers will be ideal tools to dissect the mechanism by which intact Bax proteins perforate the mitochondrial membrane, and by which anti-apoptotic compounds target Bax to prevent this mitochondrial leakage and neuronal cell death. Our project for the Oklahoma COBRE in Structural Biology Phase II application will test our novel hypotheses in the following Specific Aims: (1) What are the structures of the mini- Bax oligomers and how do they contribute to the giant pore assembly by intact Bax? (2) How do anti-apoptotic compounds block Bax pore formation in the mitochondrial membrane? In Aim 1, we will use molecular biology techniques to construct mini-Bax proteins. We will perform disulfide crosslinking experiments and size exclusion chromatography to select the mini-oligomer forming proteins and a mitochondrial protein or liposomal dye release assay will be used to identify the mini-pore forming oligomers. We will conduct crystallization trials with the pore-forming, size-selected homogeneous mini-oligomers and solve their structures using X-ray diffraction. If suitable crystals are not obtained, we will measure small-angle X-ray scattering of the mini-Bax proteins in solution, detergent micelle, or lipid nanodisc to reveal the mini-oligomeric structures. Moreover, we will use electron cryo-microscopy to reveal the mini-pore structures in membranes, and mutagenesis to assess how these mini-structures contribute to mega pore assembly by intact Bax in vitro and in vivo. Our collaborator Dr. David Andrews has discovered several compounds that block Bax oligomerization at an undefined dimer stage and protect primary neurons from glutamate excitotoxicity in an in vitro model for stroke damage. In Aim 2, we will explore if these compounds inhibit the oligomerization but not dimerization of our mini-Bax proteins and if so, whether this inhibition is sufficient to abolish mini-pore formation. We will also explore if these compounds force intact Bax into a dead-end dimer that cannot induce mitochondrial membrane perforation and if so, we will solve the dead-end dimer structure. The anticipated project outcomes are the structural details of the molecular interactions that are critical for pro-apoptotic Bax pore assembly in the mitochondrial membrane, and the regulation of this process by anti-apoptotic compounds. The impact of these significant outcomes to the apoptosis field will be vital, because they will reveal the relevant targets and mechanisms for developing better neural protective drugs to effectively combat brain damage from stroke.