Transformation of mitochondrial VDAC1 between protective and lethal states

线粒体 VDAC1 在保护状态和致死状态之间的转变

• 批准号: 9453027
• 负责人: AMADOU K. CAMARA
• 金额: $ 70.56万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9453027
• 关键词: Affect; Alzheimer' s Disease; Animal Model; Apoptosis; Apoptotic; Applications Grants; BAX gene; Binding; Biochemical; Bioenergetics; Biology; CRISPR/Cas technology; Cardiac; Cell Death; Cell Line; Cell Survival; Cell physiology; Cells; Characteristics; Disease; Electrophysiology (science); Electrostatics; Equilibrium; Free Energy; Functional disorder; Genes; Goals; Heart; Hexokinase A; Huntington Disease; Hypoxia; Ions; Link; Measurement; Membrane Lipids; Mitochondria; Mitochondrial Proteins; Modeling; Molecular; Molecular Biology; Mutation; Myocardial Ischemia; Necrosis; Nerve Degeneration; Organ; Outer Mitochondrial Membrane; Pathogenesis; Peripheral; Phosphorylation; Phosphorylation Site; Physiological; Pilot Projects; Play; Post-Translational Protein Processing; Predisposition; Principal Investigator; Property; Protein Dephosphorylation; Proteins; Proteomics; Reperfusion Injury; Research; Resistance; Role; Seminal; Site; Stress; Structure; Testing; Transgenic Animals; Transgenic Organisms; Translating; Voltage-Dependent Anion Channel; base; cardioprotection; clinically significant; comparative; design; experimental study; hexokinase; in vivo; in vivo Model; molecular dynamics; mutant; neoplastic; novel; pro-apoptotic protein; receptor; stressor

ABSTRACT The aim of this proposal is to investigate the mechanisms underlying transformation of the mitochondrial voltage-dependent anion channel (VDAC1) from protective to lethal states. We postulate that site-specific phosphorylation of VDAC1 is a key determinant of this transformation. In particular, we will test the hypothesis that phosphorylation of specific sites differentially regulates the gating properties of the channel, and also its ability to associate with pro- and anti-apoptotic proteins. We propose a novel paradigm that this phosphorylation-dependent differential modulation of VDAC1 can shift the balance between cell death and survival. Our studies will establish a mechanistic link between site-specific phosphorylation of VDAC1 and the impact on mitochondrial and cellular functions. In preliminary experiments, we have obtained novel proteomics results that identified candidate sites that impact VDAC1 gating properties and those that regulate its ability to bind anti- and pro-apoptotic proteins. Based on these results, we will systematically target residues that 1) regulate VDAC1 binding with pro- and anti-apoptotic proteins, and 2) alter the channel’s gating characteristics. We will employ a highly orchestrated experimental-computational approach. In Aim 1, we will identify and determine the impact of site-specific phosphorylation on the functional and binding properties of VDAC1. The multifaceted experimental approach will include electrophysiology at the single channel level, mitochondrial bioenergetic measurements, and biochemical and molecular biology approaches. In Aim 2, we will determine the structural impact of site-specific phosphorylation of VDAC1. Molecular dynamics (MD) simulations will be utilized to simulate VDAC1 to characterize the structural changes triggered by site-specific phosphorylation. Free energy calculations of substrate permeation will determine the molecular mechanism that underlies changes in gating due to phosphorylation of specific sites. Comparative MD simulations will be also used to investigate how phosphorylation at more peripheral sites alters the structure, the electrostatics properties affecting membrane/lipid interaction, and dynamics of the putative binding region for hexokinase, a major anti- apoptotic protein that targets VDAC1. In Aim 3, we will test the cardioprotective capacity of VDAC1 mutants in ex vivo and in vivo models. Using ex vivo hearts and in vivo animal models, we will test the transgenic expression of mutant VDAC1 that harbors a specific phosphorylation-resistant site that regulates hexokinase binding or a specific phosphorylated site(s) (phosphomimetic) that promotes cell survival. The proposed research, ranging from the single channel to the in vivo model, will fill a significant void in our understanding of how functional and structural changes in VDAC1 tip the balance between cell survival and cell death. Our long term goal is to delineate how post-translational modifications (PTMs) of VDAC1 contribute to the pathogenesis of ischemic heart disease. These studies could also provide a unique platform for investigating the role of VDAC1 PTMs in other mitochondrial-related diseases, such as Huntington’s or Alzheimer’s diseases.

抽象的 该提案的目的是研究线粒体转化的机制 我们假设该位点特定的电压依赖性阴离子通道（VDAC1）从保护状态到致死状态。 VDAC1 的磷酸化是这种转变的关键决定因素，我们将特别检验这一假设。 特定位点的磷酸化差异调节通道的门控特性，以及它的 我们提出了一种与促凋亡蛋白和抗凋亡蛋白相关的新范例。 VDAC1 的磷酸化依赖性差异调节可以改变细胞死亡和细胞死亡之间的平衡 我们的研究将建立 VDAC1 位点特异性磷酸化与生存之间的机制联系。 在初步实验中，我们获得了新的蛋白质组学。 结果确定了影响 VDAC1 门控特性和调节其能力的候选位点 根据这些结果，我们将系统地靶向以下残基：1) 调节 VDAC1 与促凋亡蛋白和抗凋亡蛋白的结合，2) 改变通道的门控特性。 在目标 1 中，我们将采用高度精心策划的实验计算方法。 确定位点特异性磷酸化对 VDAC1 功能和结合特性的影响。 多方面的实验方法将包括单通道水平的电生理学、线粒体 在目标 2 中，我们将确定生物能量测量以及生化和分子生物学方法。 VDAC1 位点特异性磷酸化的结构影响将是分子动力学 (MD) 模拟。 用于模拟 VDAC1 以表征位点特异性磷酸化触发的结构变化。 基质渗透的自由能计算将确定其背后的分子机制 由于特定位点磷酸化导致的门控变化也将用于比较 MD 模拟。 研究更外围位点的磷酸化如何改变结构、静电特性 影响膜/脂质相互作用，以及己糖激酶（一种主要的抗- 在目标 3 中，我们将测试 VDAC1 突变体的心脏保护能力。 离体和体内模型，我们将使用离体心脏和体内动物模型来测试转基因。 具有调节己糖激酶的特定磷酸化抗性位点的突变体 VDAC1 的表达 结合或促进细胞存活的特定磷酸化位点（磷酸化）。 从单通道到体内模型的研究将填补我们理解的一个重大空白 VDAC1 的功能和结构变化如何决定细胞存活和细胞死亡之间的平衡。 术语目标是描述 VDAC1 的翻译后修饰 (PTM) 如何促进发病机制 这些研究还可以为研究缺血性心脏病的作用提供一个独特的平台。 其他线粒体相关疾病（例如亨廷顿病或阿尔茨海默病）中的 VDAC1 PTM。