Biochemical and Physiological Phenotypes of CV Dysfunction In Human Cell Models

摘要。 电子传递链的复合物V（CV或ATP合酶）是细胞能量的中心酶 捕获. CV在电子传递链产生的质子梯度驱动下合成ATP。的 临床基因测序的出现突出了缺乏这种关键酶的破坏性影响。 CV亚单位中的致病性变体引起多系统疾病，包括中风、神经病、共济失调， 视网膜病和心肌病CV中的遗传变异是频繁的，并且无法区分致病性 从未知意义的变体中突变是一个临床挑战，阻碍了对 预测和合理的管理方法。然而，没有CV功能的临床试验。这阻碍了 我们对遗传变异进行分类的能力。我们的目标是开发一种生物化学方法来评估CV功能 并最终预测CV变异的临床意义。我们以前证明，基础ATP 水平是正常的CV缺乏，而ATP合成率可能很低，这表明临床 症状起因于CV不能适应增加的代谢需求。直接酶 通过CV测试ATP合成是不可能的，因为CV的底物是质子动力， 当酶被纯化时，它就消失了。因此，我们建议测定我们的人细胞（成纤维细胞， 跨线粒体胞质杂交体）模型，包括新的候选基因。我们有 观察到CV变体的生化效应导致不同的生化后遗症;因此，我们将 还测定氧消耗、线粒体膜电位、基质pH、CV组装， 线粒体嵴结构，并将结果与临床表现相关联。我们预计， 这种方法将提供一个生物化学和形态学的概况，告知变异的致病性。我们 假设在CV缺陷细胞系中观察到稳态ATP水平是正常的，尽管低ATP水平， 酶通量意味着CV功能对细胞代谢状态有反应。我们将推出一系列 涉及调节营养水平（葡萄糖，αKG）的刺激（刺激-反应）测试程序 和暴露于细胞应激（半乳糖、脂多糖）。通过严格调查 CV缺陷的后果，包括在细胞应激的动态模型，我们将建立基础 在此基础上开发临床诊断测定法，以确认CV突变致病性和治疗反应。 这一建议的中心假设是：CV亚基基因的致病性变异引起CV的变化， 生物能量功能导致预测临床表现的多种下游生化缺陷。 此外，我们认为复合物V缺乏症的临床表现的严重程度受 生物化学和营养环境中的遗传缺陷发现自己。实验性的操作 环境可以识别营养治疗方法。

ABSTRACT. Complex V (CV, or ATP synthase) of the electron transport chain is the central enzyme of cellular energy capture. CV synthesizes ATP driven by the proton gradient generated by the electron transport chain. The advent of clinical gene sequencing has highlighted the devastating effects of deficiency of this critical enzyme. Pathogenic variants in CV subunits give rise to multi-system disease, including strokes, neuropathy, ataxia, retinopathy, and cardiomyopathy. Genetic variation in CV is frequent, and the inability to distinguish pathogenic mutations from the variants of unknown significance is a clinical challenge preventing understanding of prognosis and rational approach to management. However, no clinical test for CV function exists. This thwarts our ability to classify genetic variants. Our Goal is to develop a biochemical approach to evaluating CV function and ultimately predicting the clinical significance of CV variants. We previously demonstrated that basal ATP levels are normal with CV deficiency while the rate of ATP synthesis can be low, suggesting that clinical symptoms result from an inability of CV to accommodate an increased metabolic demand. Direct enzymatic testing of ATP synthesis by CV is impossible, as the substrate for CV is the proton motive force, which is dissipated when the enzyme is purified. We therefore propose to assay ATP flux in our human cell (fibroblast, transmitochondrial cybrid) models of diverse CV genetic variants, including novel candidate genes. We have observed that the biochemical effects of CV variants result in diverse biochemical sequelae; therefore, we will also assay oxygen consumption, mitochondrial membrane potential, matrix pH, CV assembly, and mitochondrial cristae structure and correlate results with clinical manifestations. We anticipate that this approach will furnish a biochemical and morphologic profile that informs the pathogenicity of the variant. We hypothesize that the observation that steady-state ATP levels are normal in CV deficient cell-lines despite low enzymatic flux implies that CV function is responsive to cellular metabolic state. We will introduce a series of provocative (stimulus-response) testing procedures that involve modulation of nutrient levels (glucose, αKG) and exposure to cell stress (galactose, lipopolysaccharide). By rigorously investigating the biochemical consequences of CV deficiency including in dynamic models of cellular stress, we will establish the foundation on which to develop clinical diagnostic assays to confirm CV mutation pathogenicity and treatment response. The Central Hypothesis of this proposal is: pathogenic variants in CV subunit genes evoke changes in CV bioenergic function resulting in diverse downstream biochemical defects that predict clinical presentation. Further, we propose that the clinical manifestations of Complex V deficiency severity are influenced by the biochemical and nutritional milieu in which the genetic deficiency finds itself. Experimental manipulation of this milieu may identify nutritional therapeutic approaches.