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

项目摘要 线粒体凋亡的功能障碍与一系列神经退行性疾病和癌症有关， 导致神经元过早死亡或细胞生长失控。神经退行性疾病的动物研究 已经显示出靶向线粒体中早期凋亡事件的药物的益处，但许多关键的 潜在的分子过程的细节方面仍然是个谜。很明显，线粒体 膜经历广泛的重塑，脂质的氧化起着关键作用， 物种充当关键的促凋亡信号，线粒体蛋白获得新的细胞凋亡特异性 功能协调发展的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： 它通过活性氧催化线粒体脂质心磷脂的氧化，而细胞色素C的释放是一种 最后不可撤销的“死亡信号”。通过广泛的初步研究，我们确定了一个新的， 模型将这些事件联系在一起，并确定了一个潜在的关键正反馈循环， 线粒体凋亡的最早阶段。我们提出的实验，将测试这个模型，其功能在 其核心是蛋白质-脂质相互作用和膜结构变化的协同作用。我们将利用最先进的 固态核磁共振波谱学和补充技术用于阐明细胞色素C和心磷脂如何结合联合收割机 并通过诱导结构变化相互作用以触发cyt-c的促凋亡过氧化物酶活性 在蛋白质中。最近的出版物和广泛的初步结果表明，我们可以如何使用先进的 固态NMR提供残留物和位点特异性分辨率，这对于真正了解 这种心磷脂/cyt-c复合物的分子特征。额外的结构和功能测量将 涉及过氧化物酶测定、荧光测量、电子显微镜和各种光学 光谱学我们还将详细说明多不饱和心磷脂、脂质氧化、cyt-c和其他凋亡因子 运动员影响线粒体膜的结构、流动性和稳定性。固态核磁共振将再次成为 一个强大的工具，提供了独特的见解的分子结构和复杂的生物动力学 膜。建议的综合实验方法，特别是包括使用最先进的 魔角旋转固态核磁共振对于该系统来说既是必不可少的，也是前所未有的。阵列 结构，生物物理和功能测量将通过上述补充实现 实验技术，并由经验丰富的跨学科研究团队执行。我们的团队拥有 出版的合作工作的跟踪记录，并带来了承担在关键的实验深入的专业知识， 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，在本发明中， 这个项目肯定会提供急需的和前所未有的分子洞察到关键的早期 内在凋亡的阶段，并帮助告知正在进行和未来的努力，以针对这些事件的药物设计， 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.