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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, H 2 O 2 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Hydrogen peroxide (H 2 O 2 ), a natural metabolite commonly found in aerobic organisms, plays a crucial role in numerous cellular signaling processes. One of the key organelles involved in the cell's metabolism of H 2 O 2 is the peroxisome. In this review, we first provide a concise overview of the current understanding of H 2 O 2 as a molecular messenger in thiol redox signaling, along with the role of peroxisomes as guardians and modulators of cellular H 2 O 2 balance. Next, we direct our focus toward the recently identified primary protein targets of H 2 O 2 originating from peroxisomes, emphasizing their importance in unraveling the complex interplay between peroxisomal H 2 O 2 and cell signaling. We specifically focus on three areas: signaling through peroxiredoxin redox relay complexes, calcium signaling, and phospho-signaling. Finally, we highlight key research directions that warrant further investigation to enhance our comprehension of the molecular and biochemical mechanisms linking alterations in peroxisomal H 2 O 2 metabolism with disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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.

Peroxisomes are functionally specialized organelles that harbor multiple hydrogen peroxide (H 2 O 2 )-producing and -degrading enzymes. Given that this oxidant functions as a major redox signaling agent, peroxisomes have the intrinsic ability to mediate and modulate H 2 O 2 -driven processes, including autophagy. However, it remains unclear whether changes in peroxisomal H 2 O 2 (po-H 2 O 2 ) emission impact the autophagic process and to which extent peroxisomes with a disturbed H 2 O 2 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-H 2 O 2 can be modulated. We demonstrate that (i) po-H 2 O 2 can oxidatively modify multiple selective autophagy receptors and core autophagy proteins, (ii) neither modest nor robust levels of po-H 2 O 2 emission act as a prime determinant of pexophagy, and (iii) high levels of po-H 2 O 2 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 H 2 O 2 release can escape pexophagy and downregulate autophagic activity, thereby perpetuating the accumulation of damaged and toxic cellular debris.

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 H 2 O 2 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 H 2 O 2 by the combined use of (i) a previously developed cell line in which peroxisomal H 2 O 2 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 H 2 O 2 metabolism modulate the cellular sulfenome is key to our understanding of how these organelles act as redox signaling hubs in health and disease., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The involvement of peroxisomes in cellular hydrogen peroxide (H 2 O 2 ) metabolism has been a central theme since their first biochemical characterization by Christian de Duve in 1965. While the role of H 2 O 2 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 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, new approaches are urgently needed. In this study, we have combined a previously developed Flp-In T-REx 293 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. By using this approach, we identified more than 400 targets of peroxisome-derived 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 (e.g., peroxiredoxins, annexins, and tubulins) 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 (e.g., transcription factors) 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 valuable insight into how peroxisomes may be integrated into the cellular H 2 O 2 signaling network., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lismont, Revenco, Li, Costa, Lenaerts, Hussein, De Bie, Knoops, Van Veldhoven, Derua and Fransen.)

Peroxisomes harbor numerous enzymes that can produce or degrade hydrogen peroxide (H 2 O 2 ). 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 H 2 O 2 -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 H 2 O 2 metabolism and (selective) autophagy. After introducing the general concepts of (selective) autophagy, we critically examine the emerging roles of H 2 O 2 as one of the key modulators of the lysosome-dependent catabolic program. In addition, we explore possible relationships among peroxisome functioning, cellular H 2 O 2 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 H 2 O 2 metabolism contribute to autophagy-related disorders., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with one of the authors, MF., (Copyright © 2021 Li, Lismont, Revenco, Hussein, Costa and Fransen.)

The calcineurin inhibitors (CNI) cyclosporine A and tacrolimus comprise the basis of immunosuppressive regimes in all solid organ transplantation. However, long-term or high exposure to CNI leads to histological and functional renal damage (CNI-associated nephrotoxicity). In the kidney, proximal tubule cells are the only cells that metabolize CNI and these cells are believed to play a central role in the origin of the toxicity for this class of drugs, although the underlying mechanisms are not clear. Several studies have reported oxidative stress as an important mediator of CNI-associated nephrotoxicity in response to CNI exposure in different available proximal tubule cell models. However, former models often made use of supra-therapeutic levels of tissue drug exposure. In addition, they were not shown to express the relevant enzymes (e.g., CYP3A5) and transporters (e.g., P-glycoprotein) for the metabolism of CNI in human proximal tubule cells. Moreover, the used methods for detecting ROS were potentially prone to false positive results. In this study, we used a novel proximal tubule cell model established from human allograft biopsies that demonstrated functional expression of relevant enzymes and transporters for the disposition of CNI. We exposed these cells to CNI concentrations as found in tissue of stable solid organ transplant recipients with therapeutic blood concentrations. We measured the glutathione redox balance in this cell model by using organelle-targeted variants of roGFP2, a highly sensitive green fluorescent reporter protein that dynamically equilibrates with the glutathione redox couple through the action of endogenous glutaredoxins. Our findings provide evidence that CNI, at concentrations commonly found in allograft biopsies, do not alter the glutathione redox balance in mitochondria, peroxisomes, and the cytosol. However, at supra-therapeutic concentrations, cyclosporine A but not tacrolimus increases the ratio of oxidized/reduced glutathione in the mitochondria, suggestive of imbalances in the redox environment., Competing Interests: The authors have declared that no competing interests exist.

Peroxisomes are highly dynamic subcellular compartments with important functions in lipid and ROS metabolism. Impaired peroxisomal function can lead to severe metabolic disorders with developmental defects and neurological abnormalities. Recently, a new group of disorders has been identified, characterised by defects in the membrane dynamics and division of peroxisomes rather than by loss of metabolic functions. However, the contribution of impaired peroxisome plasticity to the pathophysiology of those disorders is not well understood. Mitochondrial fission factor (MFF) is a key component of both the peroxisomal and mitochondrial division machinery. Patients with MFF deficiency present with developmental and neurological abnormalities. Peroxisomes (and mitochondria) in patient fibroblasts are highly elongated as a result of impaired organelle division. The majority of studies into MFF-deficiency have focused on mitochondrial dysfunction, but the contribution of peroxisomal alterations to the pathophysiology is largely unknown. Here, we show that MFF deficiency does not cause alterations to overall peroxisomal biochemical function. However, loss of MFF results in reduced import-competency of the peroxisomal compartment and leads to the accumulation of pre-peroxisomal membrane structures. We show that peroxisomes in MFF-deficient cells display alterations in peroxisomal redox state and intra-peroxisomal pH. Removal of elongated peroxisomes through induction of autophagic processes is not impaired. A mathematical model describing key processes involved in peroxisome dynamics sheds further light into the physical processes disturbed in MFF-deficient cells. The consequences of our findings for the pathophysiology of MFF-deficiency and related disorders with impaired peroxisome plasticity are discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

Peroxisomes have the intrinsic ability to produce and scavenge hydrogen peroxide (H 2 O 2 ), a diffusible second messenger that controls diverse cellular processes by modulating protein activity through cysteine oxidation. Current evidence indicates that H 2 O 2 , a molecule whose physicochemical properties are similar to those of water, traverses cellular membranes through specific aquaporin channels, called peroxiporins. Until now, no peroxiporin-like proteins have been identified in the peroxisomal membrane, and it is widely assumed that small molecules such as H 2 O 2 can freely permeate this membrane through PXMP2, a non-selective pore-forming protein with an upper molecular size limit of 300-600 Da. By employing the CRISPR-Cas9 technology in combination with a Flp-In T-REx 293 cell line that can be used to selectively generate H 2 O 2 inside peroxisomes in a controlled manner, we provide evidence that PXMP2 is not essential for H 2 O 2 permeation across the peroxisomal membrane, neither in control cells nor in cells lacking PEX11B, a peroxisomal membrane-shaping protein whose yeast homologue facilitates the permeation of molecules up to 400 Da. During the course of this study, we unexpectedly noted that inactivation of PEX11B leads to partial localization of both peroxisomal membrane and matrix proteins to mitochondria and a decrease in peroxisome density. These findings are discussed in terms of the formation of a functional peroxisomal matrix protein import machinery in the outer mitochondrial membrane., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Hydrogen peroxide (H 2 O 2 ), a non-radical reactive oxygen species generated during many (patho)physiological conditions, is currently universally recognized as an important mediator of redox-regulated processes. Depending on its spatiotemporal accumulation profile, this molecule may act as a signaling messenger or cause oxidative damage. The focus of this review is to comprehensively evaluate the evidence that peroxisomes, organelles best known for their role in cellular lipid metabolism, also serve as hubs in the H 2 O 2 signaling network. We first briefly introduce the basic concepts of how H 2 O 2 can drive cellular signaling events. Next, we outline the peroxisomal enzyme systems involved in H 2 O 2 metabolism in mammals and reflect on how this oxidant can permeate across the organellar membrane. In addition, we provide an up-to-date overview of molecular targets and biological processes that can be affected by changes in peroxisomal H 2 O 2 metabolism. Where possible, emphasis is placed on the molecular mechanisms and factors involved. From the data presented, it is clear that there are still numerous gaps in our knowledge. Therefore, gaining more insight into how peroxisomes are integrated in the cellular H 2 O 2 signaling network is of key importance to unravel the precise role of peroxisomal H 2 O 2 production and scavenging in normal and pathological conditions., Competing Interests: The authors declare no conflict of interest.

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 .

Disturbances in cellular redox balance have been associated with pro-aging mechanisms and increased risk for various chronic disease states. Multiple lines of evidence indicate that peroxisomes are central players in cellular redox metabolism. Nevertheless, the potential role of this organelle as intracellular redox signaling platform has been largely overlooked for a long time. Fortunately, this situation is now changing. This review provides a snapshot of the current progress in the field, with an emphasis on the situation in mammals. We first briefly introduce the basics of redox biology and how reactive oxygen and nitrogen species can drive cellular signaling events. Next, we discuss current evidence linking peroxisome (dys)function to redox signaling, both in health and disease. We also highlight what is currently known about the downstream targets of peroxisome-derived oxidants. In addition, we present an extensive list of proteins that are involved in peroxisome functioning and have been identified as being responsive to oxidative stress in large scale redox proteomics studies. Finally, we address how changes in peroxisomal redox state may impact on functional mechanisms underlying inter-organelle communication. Gaining more insight into these mechanisms is key to our understanding of how peroxisomes are embedded in cellular signaling networks implicated in aging and diseases such as cancer, diabetes, and neurodegenerative disorders.

Accumulating evidence indicates that peroxisome functioning, catalase localization, and cellular oxidative balance are intimately interconnected. Nevertheless, it remains largely unclear why modest increases in the cellular redox state especially interfere with the subcellular localization of catalase, the most abundant peroxisomal antioxidant enzyme. This study aimed at gaining more insight into this phenomenon. Therefore, we first established a simple and powerful approach to study peroxisomal protein import and protein-protein interactions in living cells in response to changes in redox state. By employing this approach, we confirm and extend previous observations that Cys-11 of human PEX5, the shuttling import receptor for peroxisomal matrix proteins containing a C-terminal peroxisomal targeting signal (PTS1), functions as a redox switch that modulates the protein's activity in response to intracellular oxidative stress. In addition, we show that oxidative stress affects the import of catalase, a non-canonical PTS1-containing protein, more than the import of a reporter protein containing a canonical PTS1. Furthermore, we demonstrate that changes in the local redox state do not affect PEX5-substrate binding and that human PEX5 does not oligomerize in cellulo, not even when the cells are exposed to oxidative stress. Finally, we present evidence that catalase retained in the cytosol can protect against H 2 O 2 -mediated redox changes in a manner that peroxisomally targeted catalase does not. Together, these findings lend credit to the idea that inefficient catalase import, when coupled with the role of PEX5 as a redox-regulated import receptor, constitutes a cellular defense mechanism to combat oxidative insults of extra-peroxisomal origin., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Over the past decades, peroxisomes have emerged as key regulators in overall cellular lipid and reactive oxygen species metabolism. In mammals, these organelles have also been recognized as important hubs in redox-, lipid-, inflammatory-, and innate immune-signaling networks. To exert these activities, peroxisomes must interact both functionally and physically with other cell organelles. This review provides a comprehensive look of what is currently known about the interconnectivity between peroxisomes and mitochondria within mammalian cells. We first outline how peroxisomal and mitochondrial abundance are controlled by common sets of cis - and trans -acting factors. Next, we discuss how peroxisomes and mitochondria may communicate with each other at the molecular level. In addition, we reflect on how these organelles cooperate in various metabolic and signaling pathways. Finally, we address why peroxisomes and mitochondria have to maintain a healthy relationship and why defects in one organelle may cause dysfunction in the other. Gaining a better insight into these issues is pivotal to understanding how these organelles function in their environment, both in health and disease., Competing Interests: The authors declare no conflict of interest.

Background: Persons with multiple sclerosis (PwMS) are often characterized by increased motor fatigability, which is a performance change on an objectively measured criterion after any type of voluntary muscle contractions. This review summarizes the existing literature to determine which protocols and outcome measures are best to detect or study motor fatigability and the underlying mechanisms in MS., Methods: Two electronic databases, PubMed and Web of Science, were searched for relevant articles published until August 2016 with a combination of multiple sclerosis, fatigability, muscle fatigue, and motor fatigue., Results: A total of 48 articles were retained for data extraction. A variety of fatigability protocols were reported; protocols showed differences in type (isometric vs concentric), duration (15 to 180 s), and number of contractions (fixed or until exhaustion). Also, 12 articles reported motor fatigability during functional movements, predominantly assessed by changes in walking speed; 11 studies evaluated the mechanisms underlying motor fatigability, using additional electrical nerve or transcranial magnetic stimulation. Three articles reported psychometrics of the outcomes., Conclusions: The disparity of protocols and outcome measures to study different aspects of motor fatigability in PwMS impedes direct comparison between data. Most protocols use maximal single-joint isometric contractions, with the advantage of high standardization. Because there is no head-to-head comparison of the different protocols and only limited information on psychometric properties of outcomes, there is currently no gold standard to assess motor fatigability. The disability level, disease phenotype, and studied limb may influence the assessment of motor fatigability in PwMS.

To gain additional insight into how specific cell organelles may participate in redox signaling, it is essential to have access to tools and methodologies that are suitable to monitor spatiotemporal differences in the levels of different reactive oxygen species (ROS) and the oxidation state of specific redox couples. Over the years, the use of genetically encoded fluorescent redox indicators with a ratiometric readout has constantly gained in popularity because they can easily be targeted to various subcellular compartments and monitored in real time in single cells. Here we provide step-by-step protocols and tips for the successful use of roGFP2, a redox-sensitive variant of the enhanced green fluorescent protein, to monitor changes in glutathione redox balance and hydrogen peroxide homeostasis in the cytosol, peroxisomes, and mitochondria of mammalian cells.

The initiation and progression of many human diseases are mediated by a complex interplay of genetic, epigenetic, and environmental factors. As all diseases begin with an imbalance at the cellular level, it is essential to understand how various types of molecular aberrations, metabolic changes, and environmental stressors function as switching points in essential communication networks. In recent years, peroxisomes have emerged as important intracellular hubs for redox-, lipid-, inflammatory-, and nucleic acid-mediated signaling pathways. In this review, we focus on how nature and nurture modulate peroxisome biogenesis and function in mammalian cells. First, we review emerging evidence that changes in peroxisome activity can be linked to the epigenetic regulation of cell function. Next, we outline how defects in peroxisome biogenesis may directly impact cellular pathways involved in the development of disease. In addition, we discuss how changes in the cellular microenvironment can modulate peroxisome biogenesis and function. Finally, given the importance of peroxisome function in multiple aspects of health, disease, and aging, we highlight the need for more research in this still understudied field., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Reduction-oxidation or "redox" reactions are an integral part of a broad range of cellular processes such as gene expression, energy metabolism, protein import and folding, and autophagy. As many of these processes are intimately linked with cell fate decisions, transient or chronic changes in cellular redox equilibrium are likely to contribute to the initiation and progression of a plethora of human diseases. Since a long time, it is known that mitochondria are major players in redox regulation and signaling. More recently, it has become clear that also peroxisomes have the capacity to impact redox-linked physiological processes. To serve this function, peroxisomes cooperate with other organelles, including mitochondria. This review provides a comprehensive picture of what is currently known about the redox interplay between mitochondria and peroxisomes in mammals. We first outline the pro- and antioxidant systems of both organelles and how they may function as redox signaling nodes. Next, we critically review and discuss emerging evidence that peroxisomes and mitochondria share an intricate redox-sensitive relationship and cooperate in cell fate decisions. Key issues include possible physiological roles, messengers, and mechanisms. We also provide examples of how data mining of publicly-available datasets from "omics" technologies can be a powerful means to gain additional insights into potential redox signaling pathways between peroxisomes and mitochondria. Finally, we highlight the need for more studies that seek to clarify the mechanisms of how mitochondria may act as dynamic receivers, integrators, and transmitters of peroxisome-derived mediators of oxidative stress. The outcome of such studies may open up exciting new avenues for the community of researchers working on cellular responses to organelle-derived oxidative stress, a research field in which the role of peroxisomes is currently highly underestimated and an issue of discussion.

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and transported by the shuttling receptor PEX5 to the peroxisomal membrane docking/translocation machinery, where they are translocated into the organelle matrix. Under certain experimental conditions this protein import machinery has the remarkable capacity to accept already oligomerized proteins, a property that has heavily influenced current models on the mechanism of peroxisomal protein import. However, whether or not oligomeric proteins are really the best and most frequent clients of this machinery remain unclear. In this work, we present three lines of evidence suggesting that the peroxisomal import machinery displays a preference for monomeric proteins. First, in agreement with previous findings on catalase, we show that PEX5 binds newly synthesized (monomeric) acyl-CoA oxidase 1 (ACOX1) and urate oxidase (UOX), potently inhibiting their oligomerization. Second, in vitro import experiments suggest that monomeric ACOX1 and UOX are better peroxisomal import substrates than the corresponding oligomeric forms. Finally, we provide data strongly suggesting that although ACOX1 lacking a peroxisomal targeting signal can be imported into peroxisomes when co-expressed with ACOX1 containing its targeting signal, this import pathway is inefficient.

Peroxisomes are ubiquitous cell organelles essential for human health. To maintain a healthy cellular environment, dysfunctional and superfluous peroxisomes need to be selectively removed. Although emerging evidence suggests that peroxisomes are mainly degraded by pexophagy, little is known about the triggers and molecular mechanisms underlying this process in mammalian cells. In this study, we show that PEX5 proteins fused to a bulky C-terminal tag trigger peroxisome degradation in SV40 large T antigen-transformed mouse embryonic fibroblasts. In addition, we provide evidence that this process is autophagy-dependent and requires monoubiquitination of the N-terminal cysteine residue that marks PEX5 for recycling. As our findings also demonstrate that the addition of a bulky tag to the C terminus of PEX5 does not interfere with PEX5 monoubiquitination but strongly inhibits its export from the peroxisomal membrane, we hypothesize that such a tag mimics a cargo protein that cannot be released from PEX5, thus keeping monoubiquitinated PEX5 at the membrane for a sufficiently long time to be recognized by the autophagic machinery. This in turn suggests that monoubiquitination of the N-terminal cysteine of peroxisome-associated PEX5 not only functions to recycle the peroxin back to the cytosol, but also serves as a quality control mechanism to eliminate peroxisomes with a defective protein import machinery.