Conformational Properties of Cytochrome c in Apoptosis

细胞色素c在细胞凋亡中的构象特性

• 批准号: 8665444
• 负责人: Ekaterina PLETNEVA
• 金额: $ 30.02万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8665444
• 关键词: Animal Model; Animals; Apoptosis; Apoptosis Regulator; Apoptotic; Autoimmune Diseases; Binding; Bioenergetics; Caenorhabditis elegans; Cardiolipins; Cardiovascular Diseases; Cardiovascular system; Caspase; Cell Death; Collection; Coupled; Cytochrome c Peroxidase; Cytosol; Defect; Development; Dissociation; Drug Targeting; Event; Fluorescence; Fluorescence Resonance Energy Transfer; Foundations; Heme Group; Hemeproteins; Image; In Vitro; Ionic Strengths; Kinetics; Label; Life; Light; Link; Liposomes; Malignant Neoplasms; Masks; Measurement; Membrane; Methods; Mitochondria; Modeling; Molecular Conformation; Mutation; Nature; Nematoda; Nerve Degeneration; Outer Mitochondrial Membrane; Pathway interactions; Peroxidases; Physiological; Population; Process; Property; Proteins; Regulation; Reporting; Research; Role; Rupture; Series; Site; Solvents; Staging; Structure; Testing; Transgenic Organisms; Triplet Multiple Birth; Variant; Work; analog; cytochrome c; design; fluorophore; gastrointestinal; human disease; in vivo; in vivo Model; mitochondrial membrane; mutant; neoplastic cell; peroxidation; polypeptide; research study; triplet state

DESCRIPTION (provided by applicant): Defects in apoptotic mechanisms contribute to cancer, neurodegeneration, cardiovascular and autoimmune diseases. The release of the heme protein cytochrome c (cyt c) from mitochondria to the cytosol is a key event in initiation of apoptosis. A sluggish peroxidase in its native state, when bound to cardiolipin (CL), cyt c catalyzes cardiolipin peroxidation, which, in turn, contributes to the outer membrane permeation and cyt c release. Modulation of the cyt c-CL interactions could be a valuable drug target for many human diseases but the rational design of such strategies depends first on understanding of the conformational properties of CL-bound cyt c. We hypothesize that CL-bound cyt c is a dynamic state consisting of diverse conformations. As such, the cyt c peroxidase activity may be more robust in certain conformations. Using a collection of site-specific photophysical probes, the proposed studies will elucidate conformational properties of the CL-bound cyt c, effects of physiological modulators of cyt c conformations and peroxidase activity, and also develop methods for assessing cyt c conformational dynamics in vivo. Measurements of fluorescence resonance energy transfer (FRET) kinetics in multiple fluorophore-labeled cyt c derivatives will yield structural information through estimates of the distributions of distances between a fluorescent donor (D) and acceptor (A). Analysis of the lifetime of the triplet state of Zn-substituted cyt c will probe the accessibility of the heme group. Coupled with computations, these experiments will provide a model of the heterogeneous CL-bound cyt c state. We will probe the effects of physiologically-relevant increase in ionic strength, ATP binding and CL peroxidation on the composition of the cyt c conformational ensemble and correlate populations changes with cyt c peroxidase activity. With a number of cyt c mutants, we will test and differentiate effects of protein stability and the strength of the Fe-Met80 bond on the nature of CL-bound cyt c conformational ensemble and associated peroxidase activity. These studies will determine which structural changes in cyt c are particularly important for its CL-induced peroxidase function. FRET experiments with fluorescently-labeled cyt c in live C. elegans will evaluate our structural and mechanistic model in vivo and illustrate how conformational dynamics influence cyt c apoptotic release. Not only will the proposed studies determine the conformational composition of the elusive CL-bound cyt c state but also shed light on the mechanism of the cyt c release and provide a foundation for the design of regulators of apoptosis.

描述（由申请人提供）：细胞凋亡机制的缺陷导致癌症、神经变性、心血管和自身免疫疾病。血红素蛋白细胞色素 c (cyt c) 从线粒体释放到胞质溶胶是细胞凋亡启动的关键事件。细胞色素 c 是一种在天然状态下反应迟缓的过氧化物酶，当与心磷脂 (CL) 结合时，细胞色素 c 会催化心磷脂过氧化反应，进而促进外膜渗透和细胞色素 c 释放。细胞色素 c-CL 相互作用的调节可能是许多人类疾病的有价值的药物靶点，但此类策略的合理设计首先取决于对 CL 结合细胞色素 c 的构象特性的理解。我们假设 CL 结合的细胞色素 c 是由不同构象组成的动态状态。因此，细胞色素c过氧化物酶活性在某些构象下可能更强大。使用一系列位点特异性光物理探针，拟议的研究将阐明 CL 结合的细胞色素 c 的构象特性、细胞色素 c 构象和过氧化物酶活性的生理调节剂的影响，并开发评估体内细胞色素 c 构象动力学的方法。多个荧光团标记的细胞色素 c 衍生物中荧光共振能量转移 (FRET) 动力学的测量将通过估计荧光供体 (D) 和受体 (A) 之间的距离分布来产生结构信息。对 Zn 取代的细胞色素 c 三重态寿命的分析将探讨血红素基团的可及性。结合计算，这些实验将提供异质 CL 结合细胞色素 c 状态的模型。我们将探讨生理相关的离子强度增加、ATP 结合和 CL 过氧化对 cyt c 构象整体组成的影响，并将群体变化与 cyt c 过氧化物酶活性相关联。通过许多 cyt c 突变体，我们将测试并区分蛋白质稳定性和 Fe-Met80 键强度对 CL 结合的 cyt c 构象整体和相关过氧化物酶活性的影响。这些研究将确定 cyt c 的哪些结构变化对其 CL 诱导的过氧化物酶功能特别重要。在活体线虫中使用荧光标记的细胞色素 c 进行的 FRET 实验将评估我们的体内结构和机制模型，并说明构象动力学如何影响细胞色素 c 凋亡释放。所提出的研究不仅将确定难以捉摸的 CL 结合的细胞色素 c 状态的构象组成，而且还将阐明细胞色素 c 释放的机制，并为细胞凋亡调节剂的设计提供基础。