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

项目摘要（Lin项目） 细胞凋亡是一种细胞死亡程序，通常会消除功能失调的细胞，因此对人类健康至关重要。 健康细胞凋亡不足会导致癌症，而细胞凋亡过多会导致中风后的脑损伤。 为了开发有效的治疗这些疾病的方法，我们需要全面了解细胞凋亡是如何 是受管制的。线粒体膜穿孔是细胞凋亡的承诺步骤，其由 Bcl-2家族蛋白质（例如促凋亡Bax和抗凋亡Bcl-2）之间的相互作用。而 选择性靶向和抑制抗凋亡蛋白的抗癌药物已经在临床上显示出前景， 试验中，有限的促凋亡蛋白的结构信息已成为发展的瓶颈， 神经保护剂，可以减少中风的损害。特别是，活性Bax低聚物的结构， 可以在线粒体膜上形成巨大的孔，释放致命的线粒体因子是未知的。 由于天然Bax蛋白具有多种相互作用方式，并且可以产生不同类型的异质性。 低聚物不服从结构测定，一种替代和创新的实验方法， 需要一种方法来确定这些不同的低聚物的结构和它们可以形成的巨大孔。 我们假设：（A）适用于结构测定的均匀小寡聚体可以通过以下方法产生： 使用保留完整Bax寡聚体中可见的关键相互作用区域的截短或迷你Bax蛋白;（B）这些 小的寡聚体将是剖析完整的Bax蛋白在细胞中表达的机制的理想工具。 线粒体膜，并通过其抗凋亡化合物靶向Bax，以防止这种线粒体 渗漏和神经元细胞死亡。我们的项目为俄克拉荷马州眼镜蛇在结构生物学第二阶段 应用程序将测试我们的新假设在以下具体目标：（1）什么是迷你的结构， Bax寡聚体和它们如何通过完整的Bax促进巨孔组装？(2)如何抗凋亡 化合物阻断线粒体膜中Bax孔的形成？在目标1中，我们将使用分子生物学 技术来构建mini-Bax蛋白。我们将进行二硫化物交联实验和尺寸 排阻色谱法，以选择微型寡聚体形成蛋白和线粒体蛋白或脂质体 将使用染料释放测定来鉴定微孔形成低聚物。我们将进行结晶试验 与成孔，大小选择均匀的微型低聚物，并解决他们的结构，使用X射线 衍射法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 溶液中的蛋白质、去污剂胶束或脂质纳米盘，以揭示微型寡聚体结构。而且我们 将使用电子低温显微镜来揭示膜中的微孔结构，并使用诱变来评估 这些微结构如何在体外和体内通过完整Bax促进巨孔组装。我们的合作伙伴 博士大卫安德鲁斯发现了几种化合物，可以阻止Bax在一个不确定的二聚体上的寡聚化 在中风损伤体外模型中分期和保护原代神经元免受谷氨酸兴奋性毒性。在Aim中 2，我们将探索这些化合物是否抑制我们的mini-Bax蛋白的寡聚化而不是二聚化 如果是，这种抑制是否足以消除微孔形成。我们还将探讨这些 化合物迫使完整的Bax成为不能诱导线粒体膜穿孔的死端二聚体， 如果是这样，我们将解决死端二聚体结构。项目的预期成果是： 线粒体膜中对促凋亡Bax孔组装至关重要的分子相互作用， 以及抗凋亡化合物对该过程的调节。这些重大成果对 凋亡领域将是至关重要的，因为它们将揭示发展中国家的相关目标和机制。 更好的神经保护药物，以有效地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.