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Aims: Peroxisomes are ubiquitous, single-membrane-bounded organelles that contain considerable amounts of enzymes involved in the production or breakdown of hydrogen peroxide (H 2 O 2 ), a key signaling molecule in multiple biological processes and disease states. Despite this, the role of this organelle in cross-compartmental H 2 O 2 signaling remains largely unclear, mainly because of the difficulty to modulate peroxisomal H 2 O 2 production in a selective manner. This study aimed at establishing and validating a cellular model suitable to decipher the complex signaling processes associated with peroxisomal H 2 O 2 release., Results: Here, we report the development of a human cell line that can be used to selectively generate H 2 O 2 inside peroxisomes in a time- and dose-controlled manner. In addition, we provide evidence that peroxisome-derived H 2 O 2 can oxidize redox-sensitive cysteine residues in multiple proteins within (e.g., peroxiredoxin-5 [PRDX5]) and outside (e.g., nuclear factor kappa B subunit 1 [NFKB1] and subunit RELA proto-oncogene [RELA], phosphatase and tensin homolog [PTEN], forkhead box O3 [FOXO3], and peroxin 5 [PEX5]) the peroxisomal compartment. Furthermore, we show that the extent of protein oxidation depends on the subcellular location of the target protein and is inversely correlated to catalase activity and cellular glutathione content. Finally, we demonstrate that excessive H 2 O 2 production inside peroxisomes does not induce their selective degradation, at least not under the conditions examined., Innovation: This study describes for the first time a powerful model system that can be used to examine the role of peroxisome-derived H 2 O 2 in redox-regulated (patho)physiological processes, a research area in need of further investigation and innovative approaches., Conclusion: Our results provide unambiguous evidence that peroxisomes can serve as regulatory hubs in thiol-based signaling networks.

Significance: Peroxisomes are organelles that are best known for their role in cellular lipid and hydrogen peroxide (H 2 O 2 ) metabolism. Emerging evidence suggests that these organelles serve as guardians and modulators of cellular redox balance, and that alterations in their redox metabolism may contribute to aging and the development of chronic diseases such as neurodegeneration, diabetes, and cancer. Recent Advances: H 2 O 2 is an important signaling messenger that controls many cellular processes by modulating protein activity through cysteine oxidation. Somewhat surprisingly, the potential involvement of peroxisomes in H 2 O 2 -mediated signaling processes has been overlooked for a long time. However, recent advances in the development of live-cell approaches to monitor and modulate spatiotemporal fluxes in redox species at the subcellular level have opened up new avenues for research in redox biology and boosted interest in the concept of peroxisomes as redox signaling platforms., Critical Issues: This review first introduces the reader to what is known about the role of peroxisomes in cellular H 2 O 2 production and clearance, with a focus on mammalian cells. Next, it briefly describes the benefits and drawbacks of current strategies used to investigate the complex interplay between peroxisome metabolism and cellular redox state. Furthermore, it integrates and critically evaluates literature dealing with the interrelationship between peroxisomal redox metabolism, cell signaling, and human disease., Future Directions: As the precise molecular mechanisms underlying many of these associations are still poorly understood, a key focus for future research should be the identification of primary targets for peroxisome-derived H 2 O 2 .