Quantification of compartmentalized metabolism in eukaryotic systems

2.项目总结摘要 酰基辅酶A硫代酯(acyl-COAs)是进化上保守的关键代谢中间体 在真核细胞的不同部分制造和使用。在不同的亚细胞隔间中，酰基-COA可以 被用来满足能量需求，作为信号分子，并作为翻译后的酰基供体 蛋白质的修饰(PTM)。生物分析化学领域尚未解决的主要挑战 是对这些关键代谢物的亚细胞局部池的定量，特别是在 线粒体、细胞核和胞浆，其中相同的代谢物可以具有不同的功能。我们会克服的 这项挑战有两个目标，利用稳定的同位素标记策略，定量质谱学， 药理学、毒理学和独特的细胞生物学模型。首先，我们将量化主要的动力和命运 用于在细胞和心脏组织中划分酰基-COA的碳底物。第二，我们将量化影响 用组蛋白脱乙酰酶抑制剂、电子传输链扰乱隔室特异性过程 关于酰基-COAs、下游代谢物和隔室特异性PTM的抑制剂和遗传模型。这 该项目将允许严格量化不同的新陈代谢区间，从而使它们的数量 在新陈代谢出现问题的正常和病理过程中进行研究和操作。

2. PROJECT SUMMARY ABSTRACT Acyl-coenzyme A thioesters (acyl-CoAs) are evolutionarily conserved critical metabolic intermediates that are made and used in distinct parts of the eukaryotic cell. In different sub-cellular compartments, acyl-CoAs can be used to meet energy needs, act as signaling molecules, and serve as acyl-donors for post-translational modifications (PTMs) of proteins. A major challenge remaining unaddressed in the field of bioanalytical chemistry is the quantitation of the subcellular localized pools of these critical metabolites, especially between the mitochondria, nucleus, and cytosol where the same metabolite can have distinct functions. We will overcome this challenge in two aims, leveraging stable isotope labeling strategies, quantitative mass spectrometry, pharmacology, toxicology, and unique cell biology models. First, we will quantify the kinetics and fate of major carbon substrates for compartmentalized acyl-CoAs in cells and heart tissue. Second, we will quantify the effect of perturbing compartment specific processes with histone deacetylase inhibitors, electron transport chain inhibitors, and genetic models on acyl-CoAs, downstream metabolites, and compartment specific PTMs. This project will allow the rigorous quantification of distinct compartments of metabolism, and thus, their quantitative study and manipulation in normal and pathological processes where metabolism goes awry.