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

总结 细胞凋亡对组织中细胞群的正常发育和功能至关重要，其异常增殖和凋亡是细胞凋亡的重要组成部分。 调节对于变性疾病和癌症具有重要意义。触发的关键步骤 细胞凋亡是线粒体外膜（MOMP）的透化。这个过程是紧密的 由Bcl-2蛋白家族调节，其被细分为促凋亡（例如，Bax），抗凋亡 (e.g., Bcl-xL）和仅BH 3调节蛋白（例如，出价）。尽管最近在表征方面取得了进展， 由于Bcl-2蛋白质的结构，该领域缺乏对蛋白质-蛋白质和蛋白质-脂质的机制的理解， 介导MOMP的相互作用。这些知识对于为未来奠定基础至关重要 开发旨在抑制或激活细胞凋亡的治疗策略。拟议 该项目旨在破译Bax/Bid/Bcl-xL的膜插入和重折叠途径 调节三联体和表征其膜调节的相互作用的框架内， Bcl-2蛋白调控凋亡的嵌入式模型。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，其具有结构 与Bcl-2蛋白相似。位点特异性标记与一组荧光（包括 各种类型的稳态和寿命淬灭和FRET）和电子顺磁共振 方法（DEER，O2/NiEDDA可访问性），辅以分子动力学计算机 模拟，将用于获得必要的结构，动态和热力学信息， 破译生理功能的机制。我们的初步数据表明， 开关和激活Bid/Bax/Bcl-xL调节三联体由静电和机械调节 脂质双层的性质。我们将追求以下具体目标：（1）表征膜- 在抗凋亡Bcl-xL中依赖性构象转换，（3）表征脂质依赖性膜 Bid/Bax/Bcl-xL调节三联体的募集，（3）表征孔- 形成Bax并被Bcl-xL破坏。通过对蛋白质分子机制的新认识- Bax/Bcl-xL/Bid调节三联体中蛋白质和蛋白质-脂质相互作用，我们期望提供更清晰的 控制MOMP的分子途径图。此外，本研究还将揭示 膜上的蛋白质构象转换将告知生物医学的其他调节系统， 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.