Cardiolipin and the mitochondrial ADP/ATP carrier interactome

心磷脂和线粒体 ADP/ATP 载体相互作用组

• 批准号: 8992907
• 负责人: Steven Michael Claypool
• 金额: $ 40.5万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-15 至 2018-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8992907
• 关键词: 3-Methylglutaconic aciduria type 2; Active Biological Transport; Adenine Nucleotide Translocase; Affinity Chromatography; Alleles; Amino Acid Motifs; Amino Acids; Binding; Cardiolipins; Cardiomyopathies; Cardiovascular Diseases; Cause of Death; Cell Culture Techniques; Cell Line; Cell model; Child; Complex; Congenital cardiomyopathy; Dependency; Disease; Equipment and supply inventories; Family; Goals; Health; Heart; Heart Diseases; Human; Hypertrophic Cardiomyopathy; Individual; Link; Mammalian Cell; Mediating; Metabolism; Mitochondria; Mitochondrial Diseases; Mitochondrial Matrix; Modeling; Molecular; Mutation; Myopathy; Oxidative Phosphorylation; Pathology; Phospholipid Metabolism; Phospholipids; Physiological; Physiology; Production; Protein Isoforms; Proteins; Role; SLC25A4 gene; SLC25A5 gene; Saccharomyces cerevisiae; Sequence Homology; Side; Testing; United States; Variant; Work; Yeasts; base; defined contribution; design; diabetic cardiomyopathy; insight; mitochondrial dysfunction; mitochondrial membrane; mouse model; mutant; novel; respiratory; skeletal; stable cell line

DESCRIPTION (provided by applicant): The ADP/ATP carrier/Adenine nucleotide translocase (yeast AAC = mammalian ANT) mediates the 1:1 exchange of ADP into and ATP out of the mitochondrial matrix and is thus required for oxidative phosphorylation (OXPHOS). Recently, we demonstrated that the major ADP/ATP carrier in the yeast Saccharomyces cerevisiae, Aac2p, physically associates with the respiratory supercomplex but only in the context of mitochondrial membranes that contain the unique phospholipid cardiolipin (CL). ANT1 deficiencies and mutations have been linked to numerous diseases including hypertrophic cardiomyopathy. Moreover, there are a multitude of pathologies caused by alterations in CL metabolism including both inherited and diabetic cardiomyopathies. Given the critical importance of CL for the Aac2p interactome, we hypothesize that the CL-dependent ADP/ATP carrier interactome represents the mitochondrion's "Achilles' heel" in the multiple disease states that result from altered CL metabolism. The goal of the first specific aim is to identify mammalian ANT binding partners. Once an inventory of interacting proteins is determined, the functional importance of each interaction will be probed using stable cell lines expressing a transport-null disease allele of ANT1; and both a murine model and cell lines of the CL-based cardiomyopathy, Barth syndrome. Results from this aim will provide novel insight into the physiological importance of this critical component of OXPHOS for diverse mitochondrial functions. The second specific aim will determine the role of CL in establishing the interactome of the extended ADP/ATP carrier family. Specifically, the complex assembly of endogenous ANT2 will be ascertained in two CL-deficient mammalian cell models. In addition, the complex assembly of all four human ANT isoforms and two other yeast AAC isoforms will be determined in CL-null yeast. Ultimately, the high sequence homology between AAC isoforms will facilitate efforts to determine if Aac2p contains specific CL binding motifs and if so, define the minimal amino acid requirements. This information will in turn be used to define the relative importance of Aac2p-CL to overall OXPHOS efficiency. Finally, the third aim will biochemically interrogate the interaction between Aac2p and the respiratory supercomplex with the ultimate goal of defining the motifs within Aac2p responsible for this association. In addition, how this association promotes optimal OXPHOS will be dissected focusing on both sides of the interaction using transport-active, interaction-null and transport-null, interaction-active Aac2p variants. By defining the contributio of Aac2p- respiratory supercomplex to optimal mitochondrial function, results from this aim will provide keen insight into multiple OXPHOS disorders. Results from this application will significantly impact our understanding of the consequences of alterations in the ANT interactome that may occur due to mutations in ANT and/or perturbations in CL metabolism. In turn, a greater understanding of basic mechanisms contributing to cardiovascular disease, the number one cause of death in the United States, will be obtained.

描述（由申请人提供）：ADP/ATP载体/腺嘌呤核苷酸易位酶（酵母AAC =哺乳动物ANT）介导了ADP从线粒体基质中的1：1交换，因此氧化磷酸化（oxphos）是必需的。最近，我们证明了酿酒酵母AAC2P中的主要ADP/ATP载体物理与呼吸道超复合物物理缔合，但仅在包含独特的磷脂cardiolipin（CL）的线粒体膜的背景下。 ANT1缺陷和突变与包括肥厚性心肌病在内的许多疾病有关。此外，由于CL代谢的改变，包括遗传性和糖尿病心肌病（包括遗传性和糖尿病性心肌病）引起了多种病理。鉴于CL对于AAC2P相互作用的重要性至关重要，我们假设Cl依赖性ADP/ATP载体相互作用组在多种疾病状态下代表了因Cl代谢改变而导致的多种疾病状态。第一个特定目的的目标是识别哺乳动物的蚂蚁约束伙伴。一旦确定了相互作用蛋白的清单，将使用表达ANT1的转运无疾病等位基因的稳定细胞系进行探测每种相互作用的功能重要性；以及基于CL的心肌病Barth综合征的鼠模型和细胞系。该目标的结果将提供对OXPHOS对不同线粒体功能的这种关键组成部分的生理重要性的新见解。第二个特定目的将决定CL在建立扩展ADP/ATP载体家族的相互作用中的作用。具体而言，将在两个CL缺陷型哺乳动物细胞模型中确定内源性ANT2的复杂组装。此外，将在Cl-Null酵母中确定所有四个人类蚂蚁同工型和其他两个酵母AAC同工型的复杂组装。最终，AAC同工型之间的高序列同源性将有助于确定AAC2P是否包含特定的CL结合基序，如果是的，则定义最小的氨基酸要求。此信息将依次用于定义AAC2P-CL对整体OXPHOS效率的相对重要性。最后，第三个目标将在生物化学上询问AAC2P与呼吸系统超复合之间的相互作用，其最终目标是定义负责该关联的AAC2P中的主题。此外，将如何使用运输活性，相互作用无效和交通互动激活的AAC2P变体来阐述这种关联如何促进最佳的Oxphos。通过定义AAC2P-呼吸超复合物对最佳线粒体功能的贡献，该目标的结果将为多种OXPHOS疾病提供敏锐的见解。该应用程序的结果将显着影响我们对蚂蚁相互作用变化的后果的理解，而蚂蚁相互作用的结果可能是由于蚂蚁和/或CL代谢中的扰动引起的。反过来，将获得对导致心血管疾病的基本机制的更深入的了解，这是美国的第一名死亡原因。