Proteomics and tracer metabolomics link GAPDH ISGylation to glycolytic control

Ubiquitin-like protein ISG15 (interferon-stimulated gene 15) has been implicated in the regulation of central carbon metabolism, but conflicting findings across experimental systems have limited mechanistic insight. Here, we applied a multi-omics approach in cells ectopically expressing the ISGylation machinery independent of immune stimuli, to generate a systematic view of ISGylation in metabolic control. We found that ISGylation predominantly modifies metabolic proteins, including key glycolytic enzymes, and is associated with suppression of the energy-producing phase of glycolysis. Tracer metabolomics indicated a bottleneck at glyceraldehyde-3-phosphate dehydrogenase (GAPDH), marked by accumulation of its substrate and depletion of downstream metabolites. Functional assays demonstrated that ISGylation directly impairs GAPDH activity, and structural analysis revealed that ISG15 modifications cluster near the active site and dimerization interfaces, critical for enzymatic function. These findings identify GAPDH as a central metabolic checkpoint regulated by ISGylation and uncover a direct post-translational mechanism by which ISG15 controls energy metabolism.