Biochemical and Physiological Phenotypes of CV Dysfunction In Human Cell Models

抽象的。 电子传递链的复合体V(CV，或ATP合成酶)是细胞能量的中心酶 抓捕。CV由电子传输链产生的质子梯度驱动合成三磷酸腺苷。这个 临床基因测序的出现突显了这种关键酶缺乏的破坏性影响。 CV亚基的致病变异可引起多系统疾病，包括中风、神经病变、共济失调、 视网膜病和心肌病。CV的遗传变异频繁，无法区分致病因素 来自未知意义的变异的突变是临床上的一个挑战，阻碍了对 预后和合理的处理方法。然而，目前尚不存在心血管功能的临床试验。这阻碍了 我们对基因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心血管功能 并最终预测CV变异的临床意义。我们之前证明了基础三磷酸腺苷 在CV缺乏的情况下，水平是正常的，而ATP合成率可能很低，这表明临床上 症状是由于CV不能适应不断增加的代谢需求。直接酶促反应 用循环伏安法测试ATP合成是不可能的，因为循环伏安法的底物是质子动力，这是 当酶被提纯后就会消散。因此，我们建议测定人类细胞(成纤维细胞， 包括新的候选基因在内的不同CV遗传变异的模型。我们有 观察到CV变异的生化效应会导致不同的生化后遗症；因此，我们将 还测定了氧耗、线粒体膜电位、基质pH、CV组装和 线粒体脊结构及其与临床表现的相关性。我们预计这将是 方法将提供生化和形态特征，告知变异体的致病性。我们 假设在CV缺陷细胞系中观察到的稳态ATP水平是正常的，尽管低 酶的通量意味着循环功能是对细胞代谢状态的反应。我们将推出一系列 刺激性(刺激-反应)测试程序，涉及调节营养水平(葡萄糖、αKG) 以及暴露于细胞应激(半乳糖、脂多糖)。通过严格研究生物化学 CV不足的后果包括在细胞应力的动态模型中，我们将奠定基础 在此基础上开发临床诊断方法，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亚单位基因的致病变异会引起CV的变化 生物能功能导致多种下游生化缺陷，从而预测临床表现。 此外，我们认为复杂的V缺乏严重程度的临床表现受 发现遗传缺陷的生化和营养环境。对此进行实验性的操作 环境可以确定营养治疗方法。

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.