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.

项目概要 线粒体细胞凋亡的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不受控制的细胞生长。神经退行性的动物研究。 已经显示出针对线粒体早期凋亡事件的药物的益处，但许多关键疾病 很明显，线粒体的潜在分子过程的细节仍然是个谜。 膜经历广泛的重塑，脂质的氧化起着关键作用，脂质的重新分布 物种作为关键的促凋亡信号，并且线粒体蛋白获得新的凋亡特异性 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，并且细胞色素C释放是 根据广泛的初步研究，我们定义了一个新的、综合的“死亡信号”。 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 我们提出了测试该模型的实验，其特点是 其核心是蛋白质-脂质相互作用和膜结构变化的协同作用，我们将利用尖端技术。 固态核磁共振波谱和补充技术阐明 cyt-c 和心磷脂如何结合 并通过诱导结构变化相互作用，触发 cyt-c 的促凋亡过氧化物酶活性 最近的一篇出版物和一系列广泛的初步结果展示了我们如何使用先进的技术。 固态 NMR 提供残基和位点特异性分辨率，这是真正理解所必需的 这种心磷脂/cyt-c 复合物的分子特征将进行额外的结构和功能测量。 涉及过氧化物酶测定、荧光测量、电子显微镜和各种光学 我们还将详细介绍多不饱和心磷脂、脂质氧化、细胞色素 C 和其他细胞凋亡的情况。 固态核磁共振将再次影响线粒体膜的结构、流动性和稳定性。 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 所提出的综合实验方法，特别包括使用最先进的技术。 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。 结构、生物物理和功能测量将通过上述补充得以实现 实验技术并由经验丰富的跨学科研究团队执行。 发表了合作工作的记录，并在关键实验中发挥了深入的专业知识 技术、膜生物物理学以及细胞色素-c 和心磷脂在细胞凋亡中的作用的研究。 这个项目肯定会为关键的早期疾病提供急需的、前所未有的分子洞察力。 内在细胞凋亡的各个阶段，并有助于为正在进行和未来针对这些事件进行药物设计的努力提供信息， 涵盖从神经退行性疾病到癌症的应用。