The molecular basis of cardiolipin-protein interactions implicated in intrinsic apoptosis

项目总结 线粒体凋亡功能障碍与一系列神经退行性疾病和癌症有关， 它会导致神经元过早死亡或细胞生长失控。神经退行性变的动物研究 疾病已经显示出针对线粒体早期凋亡事件的药物的好处，但许多关键的 潜在的分子过程的细节仍然是个谜。很明显，线粒体 膜经历了广泛的重塑，脂类的氧化起着关键作用，脂类重新分布 物种作为关键的促凋亡信号，线粒体蛋白获得新的凋亡特异性 功能。后者特别包括线粒体细胞色素c(Cyt-c)，因为它执行两个基本功能： 它催化活性氧物种氧化线粒体脂质心磷脂，细胞色素c的释放是一种 最后不可撤销的“死亡信号”。根据广泛的初步研究，我们已经定义了一个新的、综合的 将这些事件联系在一起的模型，并确定潜在的关键正反馈循环，该循环是 线粒体凋亡的早期阶段。我们提出了测试该模型的实验，该模型的特点是 它的核心是蛋白质-脂质相互作用和膜结构变化的协同作用。我们将利用尖端技术 固体核磁共振波谱及辅助技术研究Cyt-c与心磷脂的结合 并相互作用，通过诱导结构变化来触发cyt-c的促凋亡过氧化物酶活性。 在蛋白质中。最近的一份出版物和大量的初步结果表明，我们可以使用高级 固态核磁共振提供残基和位点的特定分辨率，这将是真正了解 这种心磷脂/Cyt-c复合体的分子特征。其他结构和功能测量将 包括过氧化物酶分析、荧光测量、电子显微镜和各种光学方法 分光镜。我们还将详细介绍多不饱和心磷脂、脂质氧化、Cyt-c和其他细胞凋亡 玩家影响线粒体膜的结构、流动性和稳定性。同样，固态核磁共振将是一种 强大的工具，提供对复杂生物的分子结构和动力学的独特见解 膜。拟议的综合实验方法，具体包括使用最先进的 魔角旋转固态核磁共振，将是这一系统的关键，也是史无前例的。一个数组 上述互补性将使结构、生物物理和功能测量成为可能 实验技术，并由经验丰富的跨学科研究团队执行。我们队有一支 发布了协作工作的跟踪记录，并带来了关键实验的深入专业知识 技术、膜生物物理学以及Cyt-c和心磷脂在细胞凋亡中的作用的研究。因此， 这个项目肯定会提供急需的和前所未有的对枢纽早期的分子洞察力 固有的细胞凋亡阶段，并有助于为药物设计中针对这些事件的正在进行和未来的努力提供信息， 应用范围从神经退行性疾病到癌症。

PROJECT SUMMARY Malfunctioning of mitochondrial apoptosis is implicated in a range of neurodegenerative diseases and cancers, where it leads to premature neuronal death or uncontrolled cell growth. Animal studies of neurodegenerative disorders have shown the benefits of drugs that target early apoptotic events in mitochondria, but many critical aspects specifics of the underlying molecular processes remain enigmatic. It is clear that mitochondrial membranes undergo extensive remodeling, that oxidation of lipids plays a critical role, that redistributed lipid species act as critical pro-apoptotic signals, and that mitochondrial proteins attain new apoptosis-specific functions. The latter notably includes mitochondrial cytochrome c (cyt-c), as it performs two essential functions: it catalyzes the oxidation of the mitochondrial lipid cardiolipin by reactive oxygen species, and cyt-c release is a final irrevocable “death signal”. Informed by extensive preliminary studies, we have defined a new, integrated model that ties together these events, and identifies a potential critical positive feedback loop that underlies the earliest stages of mitochondrial apoptosis. We propose experiments that will test this model, which features at its core a synergy of protein-lipid interactions and membrane structural changes. We will leverage cutting-edge solid-state NMR spectroscopy and complementary techniques to elucidate how cyt-c and cardiolipin combine and interact to trigger the pro-apoptotic peroxidase activity of cyt-c, through the induction of structural changes in the protein. A recent publication and a broad array of preliminary results show how we can use advanced solid-state NMR to provide the residue- and site-specific resolution that will be necessary to truly understand the molecular features of this cardiolipin/cyt-c complex. Additional structural and functional measurements will involve peroxidase assays, fluorescence measurements, electron microscopy, and various optical spectroscopies. We will also detail how polyunsaturated cardiolipin, lipid oxidation, cyt-c, and other apoptotic players impact the structure, fluidity, and stability of mitochondrial membranes. Again solid-state NMR will be a powerful tool that provides unique insights into the molecular structure and dynamics of complex biological membranes. The proposed integrated experimental approach, specifically including the use of state-of-the-art magic-angle-spinning solid-state NMR, will be both essential and also unprecedented for this system. An array of structural, biophysical, and functional measurements will be enabled by the abovementioned complementary experimental techniques and executed by an experienced interdisciplinary research team. Our team has a published track record of collaborative work and brings to bear an in-depth expertise in the key experimental techniques, membrane biophysics, as well as the study of the roles of cyt-c and cardiolipin in apoptosis. Thus, this project is certain to provide much-needed and unprecedented molecular insight into the pivotal early stages of intrinsic apoptosis, and help inform ongoing and future efforts to target these events for drug design, spanning applications from neurodegenerative disease to cancer.