Quantification of compartmentalized metabolism in eukaryotic systems

2.项目摘要 酰基-辅酶A硫酯（酰基-CoA）是进化上保守的关键代谢中间体， 是在真核细胞的不同部分制造和使用的。在不同的亚细胞区室中，酰基辅酶A可以 可用于满足能量需求，充当信号分子，并作为翻译后的酰基供体 蛋白质修饰（PTM）。生物分析化学领域中一个尚未解决的重大挑战 是这些关键代谢物的亚细胞定位池的定量，特别是在 线粒体、细胞核和胞质溶胶，其中相同的代谢物可以具有不同的功能。我们将克服 这个挑战有两个目标，利用稳定同位素标记策略，定量质谱， 药理学、毒理学和独特的细胞生物学模型。首先，我们将量化的动力学和命运的主要 在细胞和心脏组织中，碳底物用于区室化酰基辅酶A。第二，我们会量化效果 用组蛋白去乙酰化酶抑制剂干扰区室特异性过程，电子传递链 抑制剂和酰基辅酶A、下游代谢物和隔室特异性PTM的遗传模型。这 该项目将允许严格量化不同的代谢区室，因此，它们的定量 研究和操纵正常和病理过程中的新陈代谢出错。

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.