The solute carrier SLC25A17 sustains peroxisomal redox homeostasis in diverse mammalian cell lines.

Despite the crucial role of peroxisomes in cellular redox maintenance, little is known about how these organelles transport redox metabolites across their membrane. In this study, we sought to assess potential associations between the cellular redox landscape and the human peroxisomal solute carrier SLC25A17, also known as PMP34. This carrier has been reported to function as a counter-exchanger of adenine-containing cofactors such as coenzyme A (CoA), dephospho-CoA, flavin adenine dinucleotide, nicotinamide adenine dinucleotide (NAD+), adenosine 3',5'-diphosphate, flavin mononucleotide, and adenosine monophosphate. We found that inactivation of SLC25A17 resulted in a shift toward a more reductive state in the glutathione redox couple (GSSG/GSH) across HEK-293 cells, HeLa cells, and SV40-transformed mouse embryonic fibroblasts, with variable impact on the NADPH levels and the NAD+/NADH redox couple. This phenotype could be rescued by the expression of Candida boidinii Pmp47, a putative SLC25A17 orthologue reported to be essential for the metabolism of medium-chain fatty acids in yeast peroxisomes. In addition, we provide evidence that the alterations in the redox state are not caused by changes in peroxisomal antioxidant enzyme expression, catalase activity, H2O2 membrane permeability, or mitochondrial fitness. Furthermore, treating control and ΔSLC25A17 cells with dehydroepiandrosterone, a commonly used glucose-6-phosphate dehydrogenase inhibitor affecting NADPH regeneration, revealed a kinetic disconnection between the peroxisomal and cytosolic glutathione pools. Additionally, these experiments underscored the impact of SLC25A17 loss on peroxisomal NADPH metabolism. The relevance of these findings is discussed in the context of the still ambiguous substrate specificity of SLC25A17 and the recent observation that the mammalian peroxisomal membrane is readily permeable to both GSH and GSSG.

Functional Analysis of GSTK1 in Peroxisomal Redox Homeostasis in HEK-293 Cells.

Peroxisomes serve as important centers for cellular redox metabolism and communication. However, fundamental gaps remain in our understanding of how the peroxisomal redox equilibrium is maintained. In particular, very little is known about the function of the nonenzymatic antioxidant glutathione in the peroxisome interior and how the glutathione antioxidant system balances with peroxisomal protein thiols. So far, only one human peroxisomal glutathione-consuming enzyme has been identified: glutathione S-transferase 1 kappa (GSTK1). To study the role of this enzyme in peroxisomal glutathione regulation and function, a GSTK1-deficient HEK-293 cell line was generated and fluorescent redox sensors were used to monitor the intraperoxisomal GSSG/GSH and NAD+/NADH redox couples and NADPH levels. We provide evidence that ablation of GSTK1 does not change the basal intraperoxisomal redox state but significantly extends the recovery period of the peroxisomal glutathione redox sensor po-roGFP2 upon treatment of the cells with thiol-specific oxidants. Given that this delay (i) can be rescued by reintroduction of GSTK1, but not its S16A active site mutant, and (ii) is not observed with a glutaredoxin-tagged version of po-roGFP2, our findings demonstrate that GSTK1 contains GSH-dependent disulfide bond oxidoreductase activity.

Human peroxisomal NAD+/NADH homeostasis is regulated by two independent NAD(H) shuttle systems.

Reduced (NADH) and oxidized (NAD+) nicotinamide adenine dinucleotides are ubiquitous hydride-donating/accepting cofactors that are essential for cellular bioenergetics. Peroxisomes are single-membrane-bounded organelles that are involved in multiple lipid metabolism pathways, including beta-oxidation of fatty acids, and which contain several NAD(H)-dependent enzymes. Although maintenance of NAD(H) homeostasis in peroxisomes is considered essential for peroxisomal beta-oxidation, little is known about the regulation thereof. To resolve this issue, we have developed methods to specifically measure intraperoxisomal NADH levels in human cells using peroxisome-targeted NADH biosensors. By targeted CRISPR-Cas9-mediated genome editing of human cells, we showed with these sensors that the NAD+/NADH ratio in cytosol and peroxisomes are closely connected and that this crosstalk is mediated by intraperoxisomal lactate and malate dehydrogenases, generated via translational stop codon readthrough of the LDHB and MDH1 mRNAs. Our study provides evidence for the existence of two independent redox shuttle systems in human peroxisomes that regulate peroxisomal NAD+/NADH homeostasis. This is the first study that shows a specific metabolic function of protein isoforms generated by translational stop codon readthrough in humans.

Enhanced Levels of Peroxisome-Derived H2O2 Do Not Induce Pexophagy but Impair Autophagic Flux in HEK-293 and HeLa Cells.

Peroxisomes are functionally specialized organelles that harbor multiple hydrogen peroxide (H2O2)-producing and -degrading enzymes. Given that this oxidant functions as a major redox signaling agent, peroxisomes have the intrinsic ability to mediate and modulate H2O2-driven processes, including autophagy. However, it remains unclear whether changes in peroxisomal H2O2 (po-H2O2) emission impact the autophagic process and to which extent peroxisomes with a disturbed H2O2 metabolism are selectively eliminated through a process called "pexophagy". To address these issues, we generated and validated HEK-293 and HeLa pexophagy reporter cell lines in which the production of po-H2O2 can be modulated. We demonstrate that (i) po-H2O2 can oxidatively modify multiple selective autophagy receptors and core autophagy proteins, (ii) neither modest nor robust levels of po-H2O2 emission act as a prime determinant of pexophagy, and (iii) high levels of po-H2O2 impair autophagic flux by oxidative inhibition of enzymes involved in LC3II formation. Unexpectedly, our analyses also revealed that the autophagy receptor optineurin can be recruited to peroxisomes, thereby triggering pexophagy. In summary, these findings lend support to the idea that, during cellular and organismal aging, peroxisomes with enhanced H2O2 release can escape pexophagy and downregulate autophagic activity, thereby perpetuating the accumulation of damaged and toxic cellular debris.

Identification of Peroxisome-Derived Hydrogen Peroxide-Sensitive Target Proteins Using a YAP1C-Based Genetic Probe.

As the reversible oxidation of protein cysteine thiols is an important mechanism in signal transduction, it is essential to have access to experimental approaches that allow for spatiotemporal indexing of the cellular sulfenome in response to local changes in H2O2 levels. Here, we provide a step-by-step guide for enriching and identifying the sulfenome of mammalian cells at the subcellular level in response to peroxisome-derived H2O2 by the combined use of (i) a previously developed cell line in which peroxisomal H2O2 production can be induced in a time- and dose-dependent manner; (ii) YAP1C, a genetically encoded yeast AP-1-like transcription factor-based probe that specifically reacts with S-sulfenylated cysteines and traps them through mixed disulfide bonds; and (iii) mass spectrometry. Given that this approach includes differential labeling of reduced and reversibly oxidized cysteine residues, it can also provide additional information on the positions of the modified cysteines. Gaining more in-depth insight into the complex nature of how alterations in peroxisomal H2O2 metabolism modulate the cellular sulfenome is key to our understanding of how these organelles act as redox signaling hubs in health and disease.

Assessment of the Peroxisomal Redox State in Living Cells Using NADPH- and NAD+/NADH-Specific Fluorescent Protein Sensors.

The pyridine nucleotides NAD(H) and NADP(H) are key molecules in cellular metabolism, and measuring their levels and oxidation states with spatiotemporal precision is of great value in biomedical research. Traditional methods to assess the redox state of these metabolites are intrusive and prohibit live-cell quantifications. This obstacle was surpassed by the development of genetically encoded fluorescent biosensors enabling dynamic measurements with subcellular resolution in living cells. Here, we provide step-by-step protocols to monitor the intraperoxisomal NADPH levels and NAD+/NADH redox state in cellulo by using targeted variants of iNAP1 and SoNar, respectively.

Peroxisome-Derived Hydrogen Peroxide Modulates the Sulfenylation Profiles of Key Redox Signaling Proteins in Flp-In T-REx 293 Cells.

The involvement of peroxisomes in cellular hydrogen peroxide (H2O2) metabolism has been a central theme since their first biochemical characterization by Christian de Duve in 1965. While the role of H2O2 substantially 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 H2O2 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, new approaches are urgently needed. In this study, we have combined a previously developed Flp-In T-REx 293 cell system in which peroxisomal H2O2 production can be modulated with a yeast AP-1-like-based sulfenome mining strategy to inventory protein thiol targets of peroxisome-derived H2O2 in different subcellular compartments. By using this approach, we identified more than 400 targets of peroxisome-derived H2O2 in peroxisomes, the cytosol, and mitochondria. We also observed that the sulfenylation kinetics profiles of key targets belonging to different protein families (e.g., peroxiredoxins, annexins, and tubulins) can vary considerably. In addition, we obtained compelling but indirect evidence that peroxisome-derived H2O2 may oxidize at least some of its targets (e.g., transcription factors) through a redox relay mechanism. In conclusion, given that sulfenic acids function as key intermediates in H2O2 signaling, the findings presented in this study provide valuable insight into how peroxisomes may be integrated into the cellular H2O2 signaling network.

Mitochondrial and metabolic remodelling in human skin fibroblasts in response to glucose availability.

Cell culture conditions highly influence cell metabolism in vitro. This is relevant for preclinical assays, for which fibroblasts are an interesting cell model, with applications in regenerative medicine, diagnostics and therapeutic development for personalized medicine, and the validation of ingredients for cosmetics. Given these cells' short lifespan in culture, we aimed to identify the best cell culture conditions and promising markers to study mitochondrial health and stress in normal human dermal fibroblasts (NHDF). We tested the effect of reducing glucose concentration in the cell medium from high glucose (HGm) to a more physiological level [low glucose medium (LGm)], or its complete removal and replacement by galactose [medium that forces oxidative phosphorylation (OXPHOSm)], always in the presence of glutamine and pyruvate. We have demonstrated that only with OXPHOSm was it possible to observe the selective inhibition of mitochondrial adenosine triphosphate (ATP) production. This reliance on mitochondrial ATP was accompanied by changes in oxygen consumption rate and extracellular acidification rate, oxidation of citric acid cycle substrates, fatty acids, lactate, and other substrates, increased mitochondrial network extension and polarization, the increased protein content of voltage-dependent anion channel (VDAC) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha and changes in several key transcripts related to energy metabolism. LGm did not promote significant metabolic changes in NHDF, although mitochondrial network extension and VDAC protein content were increased compared to HGm-cultured cells. Our results indicate that short-term adaptation to OXPHOSm is ideal for studying mitochondrial health and stress in NHDF.

The Peroxisome-Autophagy Redox Connection: A Double-Edged Sword?

Peroxisomes harbor numerous enzymes that can produce or degrade hydrogen peroxide (H2O2). Depending on its local concentration and environment, this oxidant can function as a redox signaling molecule or cause stochastic oxidative damage. Currently, it is well-accepted that dysfunctional peroxisomes are selectively removed by the autophagy-lysosome pathway. This process, known as "pexophagy," may serve a protective role in curbing peroxisome-derived oxidative stress. Peroxisomes also have the intrinsic ability to mediate and modulate H2O2-driven processes, including (selective) autophagy. However, the molecular mechanisms underlying these phenomena are multifaceted and have only recently begun to receive the attention they deserve. This review provides a comprehensive overview of what is known about the bidirectional relationship between peroxisomal H2O2 metabolism and (selective) autophagy. After introducing the general concepts of (selective) autophagy, we critically examine the emerging roles of H2O2 as one of the key modulators of the lysosome-dependent catabolic program. In addition, we explore possible relationships among peroxisome functioning, cellular H2O2 levels, and autophagic signaling in health and disease. Finally, we highlight the most important challenges that need to be tackled to understand how alterations in peroxisomal H2O2 metabolism contribute to autophagy-related disorders.

Peroxisomal Dysfunction and Oxidative Stress in Neurodegenerative Disease: A Bidirectional Crosstalk.

Peroxisomes are multifunctional organelles best known for their role in cellular lipid and hydrogen peroxide metabolism. In this chapter, we review and discuss the diverse functions of this organelle in brain physiology and neurodegeneration, with a particular focus on oxidative stress. We first briefly summarize what is known about the various nexuses among peroxisomes, the central nervous system, oxidative stress, and neurodegenerative disease. Next, we provide a comprehensive overview of the complex interplay among peroxisomes, oxidative stress, and neurodegeneration in patients suffering from primary peroxisomal disorders. Particular examples that are discussed include the prototypic Zellweger spectrum disorders and X-linked adrenoleukodystrophy, the most prevalent peroxisomal disorder. Thereafter, we elaborate on secondary peroxisome dysfunction in more common neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, and multiple sclerosis. Finally, we highlight some issues and challenges that need to be addressed to progress towards therapies and prevention strategies preserving, normalizing, or improving peroxisome activity in patients suffering from neurodegenerative conditions.

Single nanomolar doxorubicin exposure triggers compensatory mitochondrial responses in H9c2 cardiomyoblasts.

Dose-dependent and cumulative cardiotoxicity associated with doxorubicin (DOX) is the main limitation of anticancer therapy. Pediatric cancer survivors are particularly vulnerable, and no effective prevention measures are available. The aim of the present study was to investigate the persistent effects of nanomolar DOX concentrations and determine whether a pretreatment would induce mitochondrial adaptations in H9c2 cardiomyoblasts. H9c2 cells were incubated with DOX (10 and 25 nM) for 24 h, followed by 9 days of recovery in drug-free medium. We found that the sub-therapeutic DOX treatment induced persistent hypertrophy and dose-dependent cell cycle arrest in G2/M. Glycolytic activity, indirectly based on extracellular acidification rate, and basal respiration were significantly decreased in DOX-treated cells compared to controls, although both groups showed similar maximal respiration. Additionally, nanomolar DOX pretreatment resulted in upregulation of mitochondrial DNA transcripts accompanied by a decrease in DNA methyltransferase 1 (DNMT1) and global methylation levels. Finally, the pretreatment with DOX ameliorated H9c2 cells resistance against a subsequent exposure to DOX. These results suggest that nanomolar DOX pretreatment induced a beneficial and possibly epigenetic-based mitochondrial adaptation, raising the possibility that an early sub-therapeutic DOX treatment can be used as a preconditioning and protective approach during anticancer therapies.