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

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Abstract 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 (H 2 O 2 ) to evaluate how glucosomes respond to redox perturbation. High-content imaging demonstrated that H 2 O 2 significantly promotes medium-sized glucosomes at both single-cell and population levels. The extracellular acidification rate by Seahorse extracellular flux analysis then corroborated that H 2 O 2 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 H 2 O 2 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 H 2 O 2 -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.

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