Biochemical and Physiological Phenotypes of CV Dysfunction In Human Cell Models

人类细胞模型中CV功能障碍的生化和生理表型

• 批准号: 10714339
• 负责人: Rebecca Ganetzky
• 金额: $ 44.5万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-15 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10714339
• 关键词: ATP Synthesis Pathway; Affect; Ataxia; Biochemical; Biological Assay; Brain; Candidate Disease Gene; Cardiomyopathies; Cell Line; Cell model; Cell physiology; Cells; Cellular Stress; Classification; Clinical; Crista ampullaris; Defect; Diagnosis; Diagnostic; Disease; Electron Transport; Enzymes; Exposure to; Eye; Fibroblasts; Foundations; Functional disorder; Future; Galactose; Genes; Genetic; Genetic Variation; Glucose; Goals; Heart; Human; Interphase Cell; Lipopolysaccharides; Liver; Membrane Potentials; Metabolic; Metabolic stress; Mitochondria; Mitochondrial Respiratory Chain Deficiencies; Modeling; Morphology; Mutation; Neuropathy; Nutrient; Nutritional; Oxygen Consumption; Pathogenicity; Phenotype; Physiological; Procedures; Prognosis; Proton-Motive Force; Protons; Respiratory Chain; Response to stimulus physiology; Retinal Diseases; Series; Severities; Source; Stroke; Structure; Symptoms; System; Testing; Therapeutic; Variant; Work; alpha ketoglutarate; body system; clinical diagnostics; clinical predictors; clinically significant; diagnostic assay; genetic variant; mitochondrial membrane; novel; oligomycin sensitivity-conferring protein; prevent; research clinical testing; treatment response; variant of unknown significance

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.

抽象的。 电子传递链的复合体 V（CV 或 ATP 合酶）是细胞能量的中心酶 CV 通过电子传递链产生的质子梯度驱动合成 ATP。 临床基因测序的出现凸显了这种关键酶缺乏的破坏性影响。 CV亚基的致病性变异会引起多系统疾病，包括中风、神经病、共济失调、 视网膜病和心肌病的遗传变异很常见，并且无法区分致病因素。 意义不明的变体产生的突变是一个临床挑战，阻碍了对这一问题的理解 然而，目前还没有针对 CV 功能的临床测试。 我们对遗传变异进行分类的能力 我们的目标是开发一种评估 CV 功能的生化方法。 并最终预测 CV 变异的临床意义。我们之前证明了基础 ATP。 CV 缺乏时水平正常，而 ATP 合成率可能较低，这表明临床 症状是由于 CV 无法适应增加的直接酶促需求而导致的。 通过 CV 测试 ATP 合成是不可能的，因为 CV 的底物是质子动力，即 因此，我们建议对人类细胞（成纤维细胞、 我们拥有多种 CV 遗传变异的传输软骨细胞杂种 (transochondrial cybrid) 模型，包括新的候选基因。 观察到CV变异的生化效应会导致多种生化后遗症，因此，我们将 还可以测定耗氧量、线粒体膜电位、基质 pH 值、CV 组装和 我们预计线粒体嵴结构并将结果与​​临床表现相关联。 我们的方法将提供生化和形态学特征，以告知变异的致病性。 坚持认为，尽管 CV 缺陷细胞系的稳态 ATP 水平较低，但稳态 ATP 水平是正常的。 酶通量意味着 CV 功能对细胞代谢状态有反应，我们将介绍一系列。 涉及营养水平调节（葡萄糖、αKG）的激发（刺激-反应）测试程序 和暴露于细胞应激（半乳糖、脂多糖）通过严格研究生化。 CV 缺陷的后果，包括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方法来确认 CV 突变的致病性和治疗反应。 该提案的中心假设是：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 缺乏严重程度的临床表现受到以下因素的影响： 遗传缺陷在生化和营养环境中的实验操纵。 环境可以确定营养治疗方法。