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: 8789382
• 负责人: Steven Michael Claypool
• 金额: $ 39.89万
• 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 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8789382
• 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; Dependency; Disease; Equipment and supply inventories; Family; Goals; Health; Heart; Heart Diseases; Human; Hypertrophic Cardiomyopathy; Individual; Inherited; Link; Mammalian Cell; Mediating; Metabolism; Mitochondria; Mitochondrial Diseases; Mitochondrial Matrix; Modeling; Molecular; Mus; Mutation; Myopathy; Oxidative Phosphorylation; Pathology; Phospholipid Metabolism; Phospholipids; Physiological; Physiology; Production; Protein Isoforms; Proteins; Relative (related person); 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; 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/腺嘌呤核苷酸转位酶（酵母AAC =哺乳动物ANT）介导ADP进入和ATP排出线粒体基质的1:1交换，因此是氧化磷酸化（OXPHOS）所必需的。最近，我们证明了酵母中主要的ADP/ATP载体Aac2p与呼吸超复合体物理相关，但仅在含有独特磷脂心磷脂（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，还有许多由CL代谢改变引起的病理，包括遗传性和糖尿病性心肌病。考虑到CL对Aac2p相互作用组的关键重要性，我们假设CL依赖的ADP/ATP载体相互作用组代表了线粒体在由CL代谢改变引起的多种疾病状态中的“阿喀琉斯之脚跟”。第一个具体目标是确定哺乳动物的ANT结合伙伴。一旦确定了相互作用蛋白的清单，将使用表达ANT1转运无效疾病等位基因的稳定细胞系来探测每种相互作用的功能重要性；以及基于cl的心肌病Barth综合征的小鼠模型和细胞系。这一目标的结果将为OXPHOS的这一关键成分对不同线粒体功能的生理重要性提供新的见解。第二个具体目标将确定CL在建立扩展ADP/ATP载体家族相互作用中的作用。具体来说，内源性ANT2的复杂组装将在两种缺cl的哺乳动物细胞模型中确定。此外，将在CL-null酵母中确定所有四种人类ANT异构体和另外两种酵母AAC异构体的复杂组装。最终，AAC同工异构体之间的高序列同源性将有助于确定Aac2p是否含有特定的CL结合基序，如果是，则确定最低氨基酸需求。这些信息将反过来用于确定Aac2p-CL对OXPHOS总体效率的相对重要性。最后，第三个目标将从生物化学角度探究Aac2p与呼吸超复合体之间的相互作用，最终目标是确定Aac2p内部负责这种关联的基序。此外，这种关联如何促进最佳OXPHOS将被剖析，重点是使用transport-active， interaction-null和transport-null， interaction-active Aac2p变体的相互作用的双方。通过定义Aac2p-呼吸超复合体对最佳线粒体功能的贡献，该目标的结果将为多种OXPHOS疾病提供敏锐的洞察力。这项应用的结果将显著影响我们对由于ANT突变和/或CL代谢紊乱而可能发生的ANT相互作用组改变后果的理解。反过来，将获得对导致心血管疾病的基本机制的更深入了解，心血管疾病是美国的头号死亡原因。