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: 8471123
• 负责人: Ekaterina PLETNEVA
• 金额: $ 28.97万
• 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/8471123
• 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）：凋亡机制的缺陷有助于癌症，神经退行性，心血管和自身免疫性疾病。血红素蛋白细胞色素C（Cyt C）从线粒体到细胞质的释放是启动凋亡的关键事件。 Cyt C抑制心脏脂蛋白（CL）时，其本地状态的缓慢的过氧化物酶会催化心磷脂过氧化，这反过来又有助于外膜渗透和Cyt c释放。 CYT 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标，但是此类策略的理性设计首先取决于对Cl-Bound-Boun-Bound Cyt Cyt c的构象性质的理解。我们假设CL结合的Cyt C是由不同构象组成的动态状态。因此，在某些构象中，Cyt C过氧化物酶活性可能更强大。使用位点特异性的光物理探针的集合，提出的研究将阐明CL结合Cyt C的构象性质，细胞构构和过氧化物酶活性的生理调节剂的影响，还开发用于评估体内细胞构象动态的方法。多个荧光团标记的Cyt 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）之间距离的分布来产生结构信息。分析Zn取代的Cyt C的三胞胎状态的寿命将探测血红素组的可及性。加上计算，这些实验将提供异质CL结合Cyt C状态的模型。我们将探测与生理强度，ATP结合和CL过氧化对Cyt C构象合奏组成的离子强度，ATP结合和CL过氧化的影响，并将种群与Cyt C过氧化物酶活性的变化相关。在许多Cyt C突变体的情况下，我们将测试和区分蛋白质稳定性的影响以及Fe-Met80键对Cl结合Cyt C构象合奏和相关过氧化物酶活性的性质的强度。这些研究将确定Cyt C中的哪些结构变化对于其CL诱导的过氧化物酶功能尤为重要。在活体秀中使用荧光标记的Cyt C进行的FRET实验将在体内评估我们的结构和机械模型，并说明构象动力学如何影响Cyt C凋亡释放。拟议的研究不仅会确定难以捉摸的Cl结合Cyt C状态的构象组成，而且还阐明了Cyt C释放的机理，并为凋亡调节剂设计提供了基础。