Membrane-Mediated Interactions of the Bcl-xL/Bid/Bax Triad of Apoptotic Regulators

摘要 细胞凋亡对于组织中细胞群体的正常发育和功能是至关重要的，而细胞凋亡的动态变化与细胞的功能密切相关。 监管对退行性疾病和癌症具有重大意义。触发的关键步骤 细胞凋亡是指线粒体外膜的通透性。这一过程是紧密的 受Bcl2蛋白家族的调控，该家族又细分为促凋亡蛋白(如Bax)、抗凋亡蛋白 (例如，Bl-XL)和仅BH3调节蛋白(例如，Bid)。尽管最近在表征方面取得了进展 在Bcl2蛋白中，该领域缺乏对蛋白质-蛋白质和蛋白质-脂肪的机械理解 调节MOMP的相互作用。这些知识对于为未来奠定基础是必不可少的。 旨在抑制或激活细胞凋亡的治疗策略的发展。建议数 该项目旨在破译Bax/Bid/Bl-xL的膜插入和复性途径 调控三联体及其膜调控相互作用的特征 Bcl2蛋白调控细胞凋亡的嵌合模型。我们将借鉴我们的经验 其他膜插入蛋白，包括白喉毒素易位结构域，它具有结构 与Bcl2蛋白有相似之处。结合荧光电池的特定部位标记(包括 各种类型的稳态和寿命猝灭和FRET)和电子顺磁共振 方法(鹿，O2/NiEDDA可及性)，辅之以分子动力学计算机 将利用模拟来获得所需的结构、动力学和热力学信息 破译生理机能的机制。我们的初步数据表明构象 Bid/Bax/Bcl-xL三联体的开关激活受静电和机械调节 脂双分子层的性质。我们将追求以下具体目标：(1)表征膜- 抗细胞凋亡的Bcl-xL中的依赖构象转换，(3)脂依赖膜的特征 Bid/Bax/Bclxl调节三联体的募集，(3)膜插入途径的特征。 Bax的形成及BclxL对其的破坏作用通过对蛋白质分子机制的新见解- 在Bax/Bcl-xl/Bid调控三联体中的蛋白质和蛋白质-脂质相互作用，我们希望提供一个更清楚的 控制MOMP的分子途径图。此外，这项研究还将揭示 膜上的蛋白质构象转换将通知生物医学的其他调控系统 重要性。

Summary Apoptosis is crucial for proper development and function of cell populations in tissues, and its dys- regulation is of major relevance for degenerative diseases and cancer. The critical step in triggering apoptosis is the permeabilization of the mitochondrial outer membrane (MOMP). This process is tightly regulated by the Bcl-2 family of proteins, which is subdivided into pro-apoptotic (e.g., Bax), anti-apoptotic (e.g., Bcl-xL), and BH3-only regulator proteins (e.g., Bid). Despite recent advances in the characterization of Bcl-2 proteins, the field lacks a mechanistic understanding of the protein–protein and protein–lipid interactions that mediate MOMP. Such knowledge would be essential for setting the stage for the future development of therapeutic strategies aimed at either suppressing or activating apoptosis. The proposed project is aimed at deciphering the pathways of membrane insertion and refolding of the Bax/Bid/Bcl-xL regulatory triad and characterizing their membrane-modulated interactions within the framework of the Embedded Together model of apoptotic regulation by Bcl-2 proteins. We will draw from our experience with other membrane-inserting proteins, including the diphtheria toxin translocation domain, which has structural similarities to Bcl-2 proteins. Site-specific labeling in combination with a battery of fluorescence (including various types of steady-state and lifetime quenching and FRET) and electron paramagnetic resonance approaches (DEER, O2/NiEDDA accessibility), complemented by Molecular Dynamics computer simulations, will be utilized to obtain structural, dynamic and thermodynamic information necessary for deciphering the mechanism of physiological function. Our preliminary data indicate that conformational switching and activation Bid/Bax/Bcl-xL regulatory triad is modulated by the electrostatic and mechanical properties of the lipid bilayer. We will pursue the following specific aims: (1) Characterize membrane- dependent conformational switching in anti-apoptotic Bcl-xL, (3) Characterize lipid-dependent membrane recruitment of the Bid/Bax/Bcl-xL regulatory triad, (3) Characterize the membrane insertion pathway of pore- forming Bax and its disruption by Bcl-xL. By gaining new insights into molecular mechanisms of protein– protein and protein–lipid interactions in the Bax/Bcl-xL/Bid regulatory triad, we expect to provide a clearer map of the molecular pathways controlling MOMP. In addition, this study will reveal general principles of protein conformational switching on membranes that will inform other regulatory systems of biomedical importance.