An intimate and multifaceted partnership: cardiolipin and the mitochondrial ADP/ATP carrier

亲密且多方面的伙伴关系：心磷脂和线粒体 ADP/ATP 载体

• 批准号: 10604895
• 负责人: Steven Michael Claypool
• 金额: $ 56万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-10 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10604895
• 关键词: 3-Methylglutaconic aciduria type 2; Adenine Nucleotide Translocase; Binding; Binding Sites; Biochemical; Bioenergetics; Biology; Biophysics; Cardiolipins; Cardiomyopathies; Cardiovascular Diseases; Cause of Death; Cell model; Child; Co-Immunoprecipitations; Complement; Complex; Computer Analysis; Dependence; Detergents; Disease; Electron Transport Complex III; Engineering; Enzymes; Family; Genes; Goals; Health; Heart Diseases; Human; Individual; Knowledge; Link; Lipids; Mammals; Mediating; Membrane; Metabolism; Mitochondria; Mitochondrial Diseases; Mitochondrial Matrix; Modeling; Molecular; Molecular Conformation; Multiprotein Complexes; Mutation; Myopathy; Oxidative Phosphorylation; Pathogenicity; Pediatric Cardiomyopathy; Phospholipid Metabolism; Phospholipids; Positioning Attribute; Production; Protein Isoforms; Proteins; Proteomics; Role; SLC25A4 gene; SLC25A5 gene; Saccharomyces cerevisiae; Series; Structure; Testing; United States; Yeasts; clinically relevant; complex IV; crosslink; dimer; functional disability; inhibitor; insight; mitochondrial fitness; mitochondrial membrane; molecular modeling; mutant; novel; protein complex; protein function; protein protein interaction; rational design; respiratory; thermostability; unnatural amino acids

Mitochondrial ADP/ATP carriers (Aac) mediate the 1:1 exchange of ADP into and ATP out of the mitochondrial matrix, an activity that is required for oxidative phosphorylation. Previously, we made the exciting discovery that the major yeast ADP/ATP carrier, Aac2, associates with the respiratory supercomplex (RSC; higher order assemblies of individual respiratory complexes) but only in the context of mitochondrial membranes that contain the unique phospholipid cardiolipin. Subsequently, we established that there is substantial overlap between the interactomes of yeast Aac2 and two human Aac isoforms. When combined, our results demonstrate that cardiolipin is of general importance to the extended and clinically relevant Aac family which participate in numerous evolutionarily conserved and cardiolipin-dependent protein-protein interactions that are therefore presumed to be functionally important. These collective findings strongly support our central hypothesis that the cardiolipin-dependent Aac interactome represents the mitochondrion’s “Achilles’ heel” in the multiple disease states that result from altered cardiolipin metabolism. In our ongoing efforts to drill into the cardiolipin- dependency of Aac2 we determined that cardiolipin promotes both the tertiary and quaternary assembly of Aac2, and excitingly, it does so via distinct mechanisms. We hypothesize that these two separable structural roles of cardiolipin with respect to Aac2 assembly reflect specific Aac2-cardiolipin interactions occurring within the folded carrier or on its periphery. From within, we speculate that three conserved cardiolipin-binding sites support the carriers folded structure and potentially enable its transport-related conformational dynamics. Armed with a series of rationally designed cardiolipin-binding Aac2 mutants, we will test our hypothesis using a suite of structural, biochemical, biophysical, and functional analyses. On the periphery, we hypothesize that the defining role of cardiolipin for the association of Aac2 with respiratory supercomplexes, composed in yeast of a complex III dimer and 1-2 copies of complex IV, involves individually weak interactions between Aac2-cardiolipin, Aac2- cardiolipin-RSC, and Aac2-RSC that when combined stabilize these multi-protein complexes. In Aim 2, mutations will be engineered into both Aac2 and specific complex III and IV subunits to disrupt this conserved interaction and then test our hypothesis that the cardiolipin-dependent association between Aac2 and the respiratory supercomplex is functionally and reciprocally beneficial. In testing a novel sixth model as to the functional relevance of RSCs, in this case those RSCs physically associated with Aac, results from this aim may help provide a contextual framework for the other proposed RSC-related models which are currently debated. Overall, results from this proposal will significantly impact our understanding of the consequences of alterations in the Aac interactome that may occur due to mutations in Aac and/or perturbations in cardiolipin 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.

线粒体ADP/ATP载体（Aac）介导ADP与ATP的1：1交换 基质，氧化磷酸化所需的活性。此前，我们有一个令人兴奋的发现， 主要的酵母ADP/ATP载体Aac 2与呼吸超复合物（RSC;高阶 个体呼吸复合物的组装），但仅在含有 独特的磷脂心磷脂随后，我们确定， 酵母Aac 2和两种人Aac同种型的相互作用组。结合起来，我们的结果表明， 心磷脂对于广泛的和临床相关的Aac家族具有普遍重要性， 因此，许多进化上保守的和心磷脂依赖的蛋白质-蛋白质相互作用， 被认为是功能重要的。这些集体发现有力地支持了我们的中心假设，即 心磷脂依赖性Aac相互作用组是多发性硬化症的致命弱点， 心磷脂代谢改变引起的疾病。在我们不断深入研究心磷脂的过程中- Aac 2的依赖性我们确定心磷脂促进Aac 2的三级和四级组装， 令人兴奋的是，它通过不同的机制来实现。我们假设，这两个可分离的结构作用， 心磷脂相对于Aac 2组装的相互作用反映了在折叠的心磷脂内发生的特异性Aac 2-心磷脂相互作用。 载体或其外围。从内部来看，我们推测三个保守的心磷脂结合位点支持了 载体折叠结构，并可能使其运输相关的构象动力学。手持 一系列合理设计的心磷脂结合Aac 2突变体，我们将使用一套 结构、生物化学、生物物理和功能分析。在外围，我们假设， 心磷脂在Aac 2与呼吸超复合物结合中的作用 III二聚体和复合物IV的1-2个拷贝，涉及Aac 2-心磷脂、Aac 2-磷脂和Aac 2-磷脂之间的单独弱相互作用。 心磷脂-RSC和Aac 2-RSC，它们在组合时稳定这些多蛋白复合物。在目标2中， 将被工程化到Aac 2和特定的复合物III和IV亚基中，以破坏这种保守的相互作用 然后检验我们的假设，即Aac 2和呼吸系统之间的心磷脂依赖性联系， Supercomplex在功能上和生物学上是有益的。在测试一种新的第六种模型时， RSC的相关性，在这种情况下，那些与Aac物理相关的RSC，从这一目标产生的结果可能有助于 为目前正在讨论的其他拟议RSC相关模型提供一个背景框架。总的来说， 这一提议的结果将大大影响我们对改变的后果的理解。 Aac相互作用组可能由于Aac突变和/或心磷脂代谢紊乱而发生。反过来， 更好地了解导致心血管疾病的基本机制，心血管疾病是导致 在美国死亡，将获得。