Abstract Ever since the first characterization of peroxisomes, a central theme has been their involvement in cellular hydrogen peroxide (H 2 O 2 ) metabolism. While the reputation of H 2 O 2 drastically changed from an exclusively toxic molecule to a signaling messenger, the regulatory role of peroxisomes in these signaling events is still largely underappreciated. This is mainly because the number of known protein targets of peroxisome-derived H 2 O 2 is rather limited and testing of specific targets is predominantly based on knowledge previously gathered in related fields of research. To gain a broader and more systematic insight into the role of peroxisomes in redox signaling, an unbiased approach is urgently needed. To accomplish this goal, we have combined a previously developed cell system in which peroxisomal H 2 O 2 production can be modulated with a yeast AP-1-like-based sulfenome mining strategy to inventory protein thiol targets of peroxisome-derived H 2 O 2 in different subcellular compartments. Using this unbiased approach, we were able to identify specific and common targets of peroxisome-derived and exogenous H 2 O 2 in peroxisomes, the cytosol, and mitochondria. We also observed that the sulfenylation kinetics profiles of key targets belonging to different protein families can vary considerably. In addition, we obtained compelling but indirect evidence that peroxisome-derived H 2 O 2 may oxidize at least some of its targets through a redox relay mechanism. In conclusion, given that sulfenic acids function as key intermediates in H 2 O 2 signaling, the findings presented in this study provide initial but critical insight into how peroxisomes may be integrated in the cellular H 2 O 2 signaling network. Highlights YAP1C-trapping is a robust tool to assess the peroxisomal H 2 O 2 -dependent sulfenome Exogenous and peroxisome-derived H 2 O 2 have both common and distinct targets ANXA2, PRDX1, and SKP1 are major targets of peroxisome-derived H 2 O 2 The sulfenylation kinetics profiles of key redox-active proteins vary considerably Production of H 2 O 2 inside peroxisomes directly impacts the mitochondrial sulfenome

Peroxisome-derived hydrogen peroxide can modulate the sulfenylation profiles of key redox signaling proteins