Biology subjects

Kyoung, M.

Publications and source records attributed to Kyoung, M..

5 recordsLinked to original sources

bioRxiv2026

Functional Contribution of Multienzyme Glucosome Condensates to Cellular Redox Homeostasis in Cancer Cells

Glucosomes are liquid-liquid phase-separated condensates observed in human cells, formed by phosphofructokinase and other rate-determining enzymes in glycolysis and gluconeogenesis. While glucosomes are spatially formed into small-, medium-, and large-sized assemblies in cancer cells, medium-sized glucosomes are functionally characterized to shunt glucose flux to the pentose phosphate pathway (PPP). As the PPP is the primary pathway responsible for maintaining cytosolic NADPH levels during oxidative stress, we hypothesize that medium-sized glucosomes regulate cellular redox homeostasis through the promotion of the PPP. In this work, we started treating Hs578T cells with hydrogen peroxide (H2O2) to evaluate how glucosomes respond to redox perturbation. High-content imaging demonstrated that H2O2 significantly promotes medium-sized glucosomes at both single-cell and population levels. The extracellular acidification rate by Seahorse extracellular flux analysis then corroborated that H2O2 effectively diverts glycolytic flux to the PPP through the upregulation of medium-sized glucosomes. We then investigated the glutathione redox cycle as a potential mechanistic link between medium-sized glucosomes and H2O2 detoxification. Treatment with oxidized glutathione (GSSG), but not reduced glutathione (GSH), markedly increased the population of cells showing medium-sized glucosomes. Moreover, shRNA-mediated knockdown of glutathione reductase, which converts GSSG to GSH at the expense of NADPH, attenuated H2O2-induced glucosome formation in Hs578T cells. Collectively, we demonstrate that glucosome-mediated metabolic reprogramming couples glucose metabolism to the glutathione redox cycle to facilitate H2O2 detoxification, thereby establishing the functional role of glucosomes in cellular redox homeostasis.

cell biology↗

bioRxiv2022

Mechanistic insights of glucosome condensate formation by stochastic modeling approaches

Human liver-type phosphofructokinase 1 (PFKL) has been shown to play a scaffolder role to recruit and organize glycolytic and gluconeogenic enzymes into a multienzyme metabolic condensate, the glucosome, that regulates glucose flux in living human cells. However, it has been challenging to characterize which factors control phase separation of PFKL and so glucosome condensates in a living cell, thus hampering to understand a mechanism of reversible glucosome formation and its functional contribution to human cells. In this work, we developed a stochastic model in silico using the principal of Langevin dynamics to investigate how biological properties of PFKL contribute to the formation of glucosome condensates. Molecular dynamics simulation using the Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS) revealed the importance of an intermolecular interaction between PFKLs, an effective concentration of PFKL at a region of interest, and a pre-organization of its own self-assembly in formation of PFKL condensates and control of their sizes. Such biological properties that define intracellular dynamics of PFKL appear to be essential for phase separation of PFKL and thus formation of glucosome condensates. Collectively, our computational study provides mechanistic insights of glucosome formation, particularly an initiation step through the formation of PFKL condensates in living human cells.

biochemistry↗

bioRxiv2022

High-content quantitative high-throughput screening identifies a cell cycle-associated signaling cascade that regulates a multienzyme metabolic assembly for glucose metabolism

We have previously demonstrated that human liver-type phosphofructokinase 1 (PFK1) recruits other rate-determining enzymes in glucose metabolism to organize multienzyme metabolic assemblies, the glucosomes, in human cells. However, it has remained largely elusive how glucosomes are reversibly assembled and disassembled to functionally regulate glucose metabolism in human cells. We developed a high-content quantitative high-throughput screening (qHTS) assay to evaluate the impact of small molecule libraries on the formation of PFK1-mediated glucosome assemblies from stably transfected HeLa Tet-On cells. Initial qHTS with a library of pharmacologically active compounds directed following efforts to kinase-inhibitor enriched collections. Consequently, three active compounds that were known to inhibit cyclin-dependent kinase 2, ribosomal protein S6 kinase and Aurora kinase A, respectively, were identified and further validated under high-resolution fluorescence single-cell microscopy. Subsequent knockdown studies using small-hairpin RNAs confirmed an active role of Aurora kinase A on the formation of PFK1 assemblies in HeLa cells. Importantly, all the identified protein kinases here have been investigated as key signaling nodes of one specific cascade that controls cell cycle progression in human cells. Collectively, our qHTS approaches unravel a cell cycle-associated signaling cascade that regulates the formation of PFK1-mediated glucosome assembly in human cells.

biochemistry↗

bioRxiv2022

Functional regulation of 4D metabolic network between multienzyme glucosome condensates and mitochondria

Glucose metabolism is biochemically intertwined between energy metabolism and building block biosynthesis in living cells. However, it has not been investigated yet how its metabolic network is orchestrated to govern glucose flux in space and time. Since we reported that human enzymes in glucose metabolism are spatially organized into metabolically active membraneless compartments (i.e., glucosomes), we have employed lattice light sheet microscopic imaging and other biophysical and biochemical techniques to understand their functional significance in cellular metabolism. Now, we demonstrated that glucosome assemblies behave like liquid droplets in human cells and thus reversibly respond to environmental changes. In addition, we characterized a molecular architecture of the glucosome, which appears to be constructed from higher-ordered oligomeric structures of its scaffolder enzyme along with transient enzyme-enzyme interactions. Importantly, we found that enzymatic compositions of glucosomes are altered when they are spatially in proximity to mitochondria to functionally couple glycolysis with mitochondrial metabolism in human cells. Collectively, we envision that the subcellular localization-function relationship between glucosomes and mitochondria may represent one of fundamental principles by which 4-dimensional metabolic networks are not only dynamically but also efficiently regulated in living human cells. One Sentence SummaryInvestigation of a 4D functional network of glucose metabolism uncovers a fundamental principal of subcellular metabolic regulation in human cells.

biochemistry↗

bioRxiv2022

Functional oscillation of a multienzyme glucosome assembly during cell cycle progression

Glucose metabolism has been studied extensively to understand functional interplays between metabolism and a cell cycle. However, our understanding of cell cycle-dependent metabolic adaptation particularly in human cells remains largely elusive. Meanwhile, human enzymes in glucose metabolism are shown to functionally organize into three different sizes of a multienzyme metabolic assembly, the glucosome, to regulate glucose flux in a size-dependent manner. Here, using fluorescence single-cell imaging techniques, we discover that glucosomes spatiotemporally oscillate during a cell cycle in an assembly size-dependent manner. Importantly, their oscillation at single-cell levels is in accordance with functional contributions of glucose metabolism to cell cycle progression at a population level. Collectively, we demonstrate functional oscillation of glucosomes during cell cycle progression and thus their biological significance to human cell biology.

cell biology↗