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/腺嘌呤核苷酸移位酶（酵母AAC =哺乳动物ANT）介导ADP与线粒体基质的1：1交换，因此是氧化磷酸化（OXPHOS）所需的。最近，我们证明了主要的ADP/ATP载体在酵母酿酒酵母，Aac2p，物理上与呼吸超复合物，但只有在线粒体膜的情况下，含有独特的磷脂心磷脂（CL）。ANT1缺陷和突变与包括肥厚型心肌病在内的许多疾病有关。此外，存在由CL代谢的改变引起的多种病理，包括遗传性和糖尿病性心肌病。鉴于CL对Aac2p相互作用组的至关重要性，我们假设CL依赖性ADP/ATP载体相互作用组代表了CL代谢改变导致的多种疾病状态中的神经元的"阿喀琉斯之踵"。第一个具体目标的目标是鉴定哺乳动物ANT结合配偶体。一旦确定了相互作用蛋白的库存，将使用表达ANT1的转运无效疾病等位基因的稳定细胞系以及基于CL的心肌病Barth综合征的鼠模型和细胞系来探测每种相互作用的功能重要性。从这一目标的结果将提供新的洞察生理的重要性，这一关键组成部分的OXPHOS不同的线粒体功能。第二个具体目标将确定CL在建立扩展的ADP/ATP载体家族的相互作用组中的作用。具体而言，内源性ANT 2的复杂组装将在两个CL缺陷的哺乳动物细胞模型中确定。此外，将在无CL酵母中测定所有四种人ANT同种型和两种其他酵母AAC同种型的复合物组装。最终，AAC同种型之间的高序列同源性将有助于确定Aac2p是否含有特异性CL结合基序，如果是，则定义最小氨基酸需求。该信息将进而用于定义Aac2p-CL对总体OXPHOS效率的相对重要性。最后，第三个目标将以生物化学方式询问Aac2p和呼吸超复合物之间的相互作用，最终目标是定义Aac2p内负责这种关联的基序。此外，这种关联如何促进最佳OXPHOS将被解剖，重点是使用运输活性，相互作用无效和运输无效，相互作用活性Aac2p变体的相互作用的两侧。通过定义Aac2 β-呼吸超复合物对最佳线粒体功能的贡献，来自该目的的结果将提供对多种OXPHOS病症的敏锐洞察。本申请的结果将显著影响我们对ANT相互作用组中可能由于ANT突变和/或CL代谢扰动而发生的改变的后果的理解。反过来，对心血管疾病的基本机制有了更深入的了解，心血管疾病是美国的头号死因。