A Multienzyme Metabolic Complex for Glucose Metabolism

项目摘要/摘要 这一建议的长期目标是了解代谢流的分子水平机制。 在人类细胞中的调节。在这个方案中，我们将研究亚细胞定位-功能关系 人类多酶代谢复合体，调节人类细胞中的葡萄糖代谢。尽管 在我们对糖酵解酶及其复合体的了解方面取得了长足的进步，但仍然具有挑战性 解释葡萄糖流量的方向如何在空间和/或时间上在代谢节点处被调节 能量代谢和合成代谢的生物合成途径。现在，我们提供了令人信服的证据，证明 葡萄糖代谢中的胞质、速率决定酶在空间上被组织成多酶复合体， 即人类细胞质中大小不一的“葡萄糖体”。我们假设空间 葡萄糖体的组装在亚细胞中以大小依赖的方式调节葡萄糖通量的方向 级别。在Aim1中，我们将确定每种大小的葡萄糖体簇的精确代谢功能 亚细胞水平，从而为它们在整体上的集体代谢结果提供了一个量化原则 级别。将进行定量二次离子质谱学成像和13C代谢通量分析 确定糖体簇的特定大小分配系数作为其代谢功能。在目标2中，我们 将提供关于多个代谢途径如何作为网络相互调节的机械性见解 在亚细胞水平管理代谢分流。细胞内荧光共振能量转移显微镜 体外免疫沉淀将被用来绘制蛋白质-蛋白质相互作用的网络和它的 不同大小的星团中的变化。在目标3中，我们将讨论葡萄糖体簇是如何在空间上改变的 在功能上对细胞周期进程做出贡献。流式细胞术、延时荧光活细胞 成像、细胞同步和质谱学将被用来在功能上关联 葡萄糖体随着细胞周期的变化而聚集。我们设想，在人体内，决定速率的酶的代谢活性 葡萄糖代谢在葡萄糖体簇内被空间调节，以控制葡萄糖通量的方向 细胞。拟议中的研究将极大地促进我们对葡萄糖流量调节的理解 亚细胞水平，从而在人类代谢性疾病，如癌症和糖尿病中的调节失调。 总而言之，这种新的理解水平将揭示迄今未被认识到的新陈代谢的重要性。 隔室作为治疗干预的新靶点。

Project Summary/Abstract The long-term objective of this proposal is to understand the molecular-level mechanism of metabolic flux regulation in human cells. In this proposal, we will investigate the subcellular localization-function relationship of human multienyzme metabolic complex that regulate glucose metabolism in human cells. Despite considerable advances in our knowledge of glycolytic enzymes and their complexes, it is still challenging to explain how the direction of glucose flux is spatially and/or temporally regulated at metabolic nodes between energy metabolism and anabolic biosynthetic pathways. Now, we provide compelling evidence that all the cytosolic, rate-determining enzymes in glucose metabolism are spatially organized into a multienzyme complex, namely the “glucosome,” in various sizes in the cytoplasm of human cells. We hypothesize that the spatial assembly of glucosomes regulates the direction of glucose flux in a size-dependent manner at subcellular levels. In Aim1, we will determine a precise metabolic function of each size of glucosome clusters at subcellular levels, thus providing a quantitative principle for their collective metabolic outcomes at ensemble levels. Quantitative secondary ion mass spectrometric imaging and 13C-metabolic flux assays will be performed to determine size-specific partition coefficients of glucosome clusters as their metabolic functions. In Aim 2, we will provide mechanistic insights of how multiple metabolic pathways are reciprocally regulated as a network to govern metabolic shunts at subcellular levels. Intracellular fluorescence resonance energy transfer microscopy and in vitro immunoprecipitation will be used to map the network of protein-protein interactions and its alterations in differently sized clusters. In Aim 3, we will address how glucosome clusters are spatially altered to functionally contribute to the cell cycle progression. Flow cytometry, time-lapse fluorescence live-cell imaging, cell synchronization and mass spectrometry will be employed to functionally correlate the sizes of glucosome clusters with the cell cycle. We envision that metabolic activities of the rate-determining enzymes in glucose metabolism are spatially regulated inside glucosome clusters to govern the direction of glucose flux in cells. The proposed research will significantly advance our understanding of glucose flux regulation at subcellular levels and thus its dysregulation in human metabolic diseases, like cancer and diabetes. Collectively, this new level of understanding will divulge the importance of a heretofore unrecognized metabolic compartment as a novel target for therapeutic intervention.