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

心磷脂-蛋白质相互作用的分子基础涉及内在细胞凋亡

• 批准号: 9342976
• 负责人: Valerian E Kagan
• 金额: $ 36.58万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9342976
• 关键词: Address; Animal Testing; Animals; Apoptosis; Apoptotic; Binding; Biological; Biological Assay; Biophysics; Cardiolipins; Cardiovascular Diseases; Cessation of life; Chemicals; Collaborations; Complex; Cytochrome c Group; Data; Disease; Drug Design; Drug Targeting; Electron Microscopy; Equus caballus; Event; Feedback; Fluorescence; Future; Generations; Heart; Huntington Disease; Inner mitochondrial membrane; Interdisciplinary Study; Intervention; Label; Link; Lipids; Magic; Malignant Neoplasms; Measurement; Mediating; Medical; Membrane; Membrane Fusion; Mitochondria; Mitochondrial Proteins; Modeling; Modification; Molecular; Molecular Structure; NMR Spectroscopy; Nerve Degeneration; Neurodegenerative Disorders; Optics; Outcome Study; Outer Mitochondrial Membrane; Oxides; Parkinson Disease; Peroxidases; Peroxides; Play; Process; Property; Protein Conformation; Proteins; Publications; Publishing; Reactive Oxygen Species; Recruitment Activity; Research Personnel; Resolution; Role; Sampling; Signal Transduction; Site; Solvents; Specificity; Spectrum Analysis; Stimulus; Structure; System; Techniques; Testing; Ursidae Family; Variant; Vesicle; Work; cytochrome c; design; experience; experimental study; flexibility; fluidity; inhibitor/antagonist; insight; mitochondrial membrane; molecular dynamics; mutant; neuron loss; oxidation; oxidized lipid; peroxidation; polyunsaturated fat; premature; protein structure; public health relevance; response; solid state nuclear magnetic resonance; stable isotope; stem; synergism; tool; uncontrolled cell growth

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.

项目摘要 线粒体细胞凋亡的故障在一系列神经退行性疾病和癌症中隐含 它导致过早的神经元死亡或不受控制的细胞生长。神经退行性的动物研究 疾病显示了针对线粒体早期凋亡事件的药物的好处，但许多关键 基础分子过程的各个方面细节仍然神秘。很明显线粒体 膜经历了广泛的重塑，脂质的氧化起着至关重要的作用，重新分布脂质 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。后者特别包括线粒体细胞色素C（CYT-C），因为它执行了两个基本功能： 它通过活性氧催化线粒体脂质心脂蛋白的氧化，而Cyt-C释放是一个 最终不可撤销的“死亡信号”。在广泛的初步研究中，我们定义了一个新的，整合的 将这些事件联系在一起的模型，并确定一个潜在的临界正反馈回路，该回路是基础的 线粒体凋亡的最早阶段。我们提出了将测试此模型的实验，该模型在 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 固态NMR光谱和完整的技术，以阐明Cyt-C和Cardiolipin如何结合 并通过诱导结构变化而相互作用以触发Cyt-C的凋亡性过氧化物酶活性 在蛋白质中。最近的出版物和广泛的初步结果表明我们如何使用高级 固态NMR提供居住和特定地点的分辨率，这是真正了解的必要条件 这种心磷脂/Cyt-C复合物的分子特征。其他结构和功能测量将 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。我们还将详细介绍多不饱和的心磷脂，脂质氧化，CYT-C和其他凋亡 玩家会影响线粒体膜的结构，流动性和稳定性。同样，固态NMR将是 强大的工具，可为复杂生物学的分子结构和动力学提供独特的见解 膜。拟议的综合实验方法，特别包括使用最新的实验方法 对于该系统，魔术角旋转固态NMR将是必不可少的，也是前所未有的。阵列 上述完整性将启用结构，生物物理和功能测量 实验技术由经验丰富的跨学科研究团队执行。我们的团队有一个 已发表的协作工作记录，并带来深入的专家 技术，膜生物物理学以及对Cyt-C和Cardiolipin在凋亡中的作用的研究。那， 该项目肯定会提供急需的和前所未有的分子洞察力。 内在凋亡的阶段，并有助于为针对这些事件以药物设计而持续不断的努力， 从神经退行性疾病到癌症的应用。