Peroxisomal interactome mapping enables network-based modelling of function and disease.

Peroxisomal dysfunction contributes to a broad spectrum of multisystem disorders, yet mechanistic understanding and therapeutic options remain limited, posing significant challenges for clinical management. Network-based computational strategies support hypothesis generation, biomarker discovery, and drug repurposing, but their usage is constrained by incomplete human interactome coverage-especially by scarcity of high-confidence protein-protein interaction (PPI) data for peroxisomal proteins. We present the first comprehensive map of the peroxisomal interactome, generated using an automated, informatics-guided bioluminescence resonance energy transfer strategy. We profiled PPIs for 92 peroxisomal proteins and six isoforms, validating 68% of known interactions and identifying 333 novel ones. Integration with curated PPIs yielded an expanded peroxisomal interactome, enriched for drug targets and disease-associated proteins. A disease-linked subnetwork enabled prioritization of drug repurposing candidates. Tissue-specific expanded peroxisomal interactome variants, derived from transcriptomic data, revealed distinct functional submodules across nine tissues. Gene ontology analysis of 1,272 non-peroxisomal interactors suggested pathways contributing to tissue-specific vulnerability. Our approach provides a systems-level framework for mechanistic insight in peroxisomal disease, the identification of treatment targets, and application to other organelle systems.