A Multienzyme Metabolic Complex for Glucose Metabolism

项目总结/摘要 这项建议的长期目标是了解代谢通量的分子水平机制 在人体细胞中的调节。在这个提议中，我们将研究亚细胞定位-功能关系 人多酶代谢复合物，其调节人细胞中的葡萄糖代谢。尽管 尽管我们对糖酵解酶及其复合物的知识有了相当大的进步，但仍然具有挑战性， 解释葡萄糖通量的方向如何在代谢节点处被空间和/或时间调节， 能量代谢和合成代谢途径。我们提供了有力的证据证明 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物， 即“葡糖体”，在人细胞的细胞质中具有各种大小。我们假设， 葡萄糖体的组装以大小依赖的方式调节亚细胞内葡萄糖通量的方向。 程度.在Aim 1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，从而提供了一个定量的原则，为他们的集体代谢结果在合奏 程度.将进行定量二次离子质谱成像和13 C代谢通量测定 以确定糖体簇的大小特异性分配系数作为其代谢函数。在目标2中， 将提供多种代谢途径如何作为一个网络进行调节的机制见解， 在亚细胞水平上控制代谢分流。细胞内荧光共振能量转移显微术 体外免疫沉淀将用于绘制蛋白质-蛋白质相互作用的网络， 改变不同大小的集群。在目标3中，我们将讨论糖体簇在空间上如何改变 在功能上有助于细胞周期的进展。流式细胞术，延时荧光活细胞 成像、细胞同步化和质谱将被用于功能性地关联 糖体簇与细胞周期的关系我们设想，代谢活动的速率决定酶，在 葡萄糖代谢在葡萄糖体簇内受到空间调节，以控制葡萄糖在葡萄糖体簇中的流动方向。 细胞拟议的研究将大大提高我们对葡萄糖流量调节的理解， 亚细胞水平，因此它在人类代谢疾病中失调，如癌症和糖尿病。 总的来说，这种新的理解水平将揭示迄今未被认识的代谢的重要性。 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.