Proteomics and tracer metabolomics link GAPDH ISGylation to glycolytic control

BACKGROUND: Ubiquitin-like protein ISG15 (interferon-stimulated gene 15) is implicated in the regulation of central carbon metabolism, but conflicting findings across experimental systems limit mechanistic insight. Here, we apply 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. RESULTS: ISGylation preferentially targets metabolic enzymes, with marked enrichment among glycolytic proteins, suppressing the energy-yielding phase of glycolysis. Tracer metabolomics reveals a bottleneck at glyceraldehyde-3-phosphate dehydrogenase (GAPDH), reflected by accumulation of upstream intermediates and depletion of downstream metabolites. This arises from multisite ISGylation of lysines near its catalytic and regulatory regions, which reduces enzymatic activity without disrupting tetramer assembly. CONCLUSIONS: 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.