Peroxisomes as Modulators of Cellular Protein Thiol Oxidation: A New Model System.

AIMS: Peroxisomes are ubiquitous, single-membrane-bounded organelles that contain considerable amounts of enzymes involved in the production or breakdown of hydrogen peroxide (H2O2), a key signaling molecule in multiple biological processes and disease states. Despite this, the role of this organelle in cross-compartmental H2O2 signaling remains largely unclear, mainly because of the difficulty to modulate peroxisomal H2O2 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 H2O2 release. RESULTS: Here, we report the development of a human cell line that can be used to selectively generate H2O2 inside peroxisomes in a time- and dose-controlled manner. In addition, we provide evidence that peroxisome-derived H2O2 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 H2O2 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 H2O2 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.

The peroxisomal import receptor PEX5 functions as a stress sensor, retaining catalase in the cytosol in times of oxidative stress.

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 H2O2-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.

KillerRed as a Tool to Study the Cellular Responses to Peroxisome-Derived Oxidative Stress.

Many biological processes and cell fate decisions are modulated by changes in redox environment. To gain insight into how subcellular compartmentalization of reactive oxygen species (ROS) formation contributes to (site-specific) redox signaling and oxidative stress responses, it is critical to have access to tools that allow tight spatial and temporal control of ROS production. Over the past decade, the use of genetically encoded photosensitizers has attracted growing interest of researchers because these proteins can be easily targeted to various subcellular compartments and allow for controlled release of ROS when excited by light. This chapter provides guidance and practical advice on the use of po-KR, a peroxisomal variant of the phototoxic red fluorescent protein KillerRed, to address fundamental questions about how mammalian cells cope with peroxisome-derived oxidative stress.

Export-deficient monoubiquitinated PEX5 triggers peroxisome removal in SV40 large T antigen-transformed mouse embryonic fibroblasts.

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.

A cost-effective approach to microporate mammalian cells with the Neon Transfection System.

Electroporation is one of the most efficient nonviral methods for transferring exogenous DNA into mammalian cells. However, the relatively high costs of electroporation kits and reagents temper the routine use of this fast and easy to perform technique in many laboratories. Several years ago, a new flexible and easy to operate electroporation device was launched under the name Neon Transfection System. This device uses specialized pipette tips containing gold-plated electrodes as electroporation chamber. Here we report a protocol to regenerate these expensive tips as well as some other Neon kit accessories, thereby reducing the cost of electroporation at least 10-fold.

PEX5, the shuttling import receptor for peroxisomal matrix proteins, is a redox-sensitive protein.

Peroxisome maintenance depends on the import of nuclear-encoded proteins from the cytosol. The vast majority of these proteins is destined for the peroxisomal lumen and contains a C-terminal peroxisomal targeting signal, called PTS1. This targeting signal is recognized in the cytosol by the receptor PEX5. After docking at the peroxisomal membrane and release of the cargo into the organelle matrix, PEX5 is recycled to the cytosol through a process requiring monoubiquitination of an N-terminal, cytosolically exposed cysteine residue (Cys11 in the human protein). At present, the reason why a cysteine, and not a lysine residue, is the target of ubiquitination remains unclear. Here, we provide evidence that PTS1 protein import into human fibroblasts is a redox-sensitive process. We also demonstrate that Cys11 in human PEX5 functions as a redox switch that regulates PEX5 activity in response to intracellular oxidative stress. Finally, we show that exposure of human PEX5 to oxidized glutathione results in a ubiquitination-deficient PEX5 molecule, and that substitution of Cys11 by a lysine can counteract this effect. In summary, these findings reveal that the activity of PEX5, and hence PTS1 import, is controlled by the redox state of the cytosol. The potential physiological implications of these findings are discussed.

Mitochondria are targets for peroxisome-derived oxidative stress in cultured mammalian cells.

Many cellular processes are driven by spatially and temporally regulated redox-dependent signaling events. Although mounting evidence indicates that organelles such as the endoplasmic reticulum and mitochondria can function as signaling platforms for oxidative stress-regulated pathways, little is known about the role of peroxisomes in these processes. In this study, we employ targeted variants of the genetically encoded photosensitizer KillerRed to gain a better insight into the interplay between peroxisomes and cellular oxidative stress. We show that the phototoxic effects of peroxisomal KillerRed induce mitochondria-mediated cell death and that this process can be counteracted by targeted overexpression of a select set of antioxidant enzymes, including peroxisomal glutathione S-transferase kappa 1, superoxide dismutase 1, and mitochondrial catalase. We also present evidence that peroxisomal disease cell lines deficient in plasmalogen biosynthesis or peroxisome assembly are more sensitive to KillerRed-induced oxidative stress than control cells. Collectively, these findings confirm and extend previous observations suggesting that disturbances in peroxisomal redox control and metabolism can sensitize cells to oxidative stress. In addition, they lend strong support to the ideas that peroxisomes and mitochondria share a redox-sensitive relationship and that the redox communication between these organelles is not only mediated by diffusion of reactive oxygen species from one compartment to the other. Finally, these findings indicate that mitochondria may act as dynamic receivers, integrators, and transmitters of peroxisome-derived mediators of oxidative stress, and this may have profound implications for our views on cellular aging and age-related diseases.

Intraperoxisomal redox balance in mammalian cells: oxidative stress and interorganellar cross-talk.

Reactive oxygen species (ROS) are at once unsought by-products of metabolism and critical regulators of multiple intracellular signaling cascades. In nonphotosynthetic eukaryotic cells, mitochondria are well-investigated major sites of ROS generation and related signal initiation. Peroxisomes are also capable of ROS generation, but their contribution to cellular oxidation-reduction (redox) balance and signaling events are far less well understood. In this study, we use a redox-sensitive variant of enhanced green fluorescent protein (roGFP2-PTS1) to monitor the state of the peroxisomal matrix in mammalian cells. We show that intraperoxisomal redox status is strongly influenced by environmental growth conditions. Furthermore, disturbances in peroxisomal redox balance, although not necessarily correlated with the age of the organelle, may trigger its degradation. We also demonstrate that the mitochondrial redox balance is perturbed in catalase-deficient cells and upon generation of excess ROS inside peroxisomes. Peroxisomes are found to resist oxidative stress generated elsewhere in the cell but are affected when the burden originates within the organelle. These results suggest a potential broader role for the peroxisome in cellular aging and the initiation of age-related degenerative disease.

Peroxisome dynamics in cultured mammalian cells.

Despite the identification and characterization of various proteins that are essential for peroxisome biogenesis, the origin and the turnover of peroxisomes are still unresolved critical issues. In this study, we used the HaloTag technology as a new approach to examine peroxisome dynamics in cultured mammalian cells. This technology is based on the formation of a covalent bond between the HaloTag protein--a mutated bacterial dehalogenase which is fused to the protein of interest--and a synthetic haloalkane ligand that contains a fluorophore or affinity tag. By using cell-permeable ligands of distinct fluorescence, it is possible to image distinct pools of newly synthesized proteins, generated from a single genetic HaloTag-containing construct, at different wavelengths. Here, we show that peroxisomes display an age-related heterogeneity with respect to their capacity to incorporate newly synthesized proteins. We also demonstrate that these organelles do not exchange their protein content. In addition, we present evidence that the matrix protein content of pre-existing peroxisomes is not evenly distributed over new organelles. Finally, we show that peroxisomes in cultured mammalian cells, under basal growth conditions, have a half-life of approximately 2 days and are mainly degraded by an autophagy-related mechanism. The implications of these findings are discussed.

Small G proteins in peroxisome biogenesis: the potential involvement of ADP-ribosylation factor 6.

BACKGROUND: Peroxisomes execute diverse and vital functions in virtually every eukaryote. New peroxisomes form by budding from pre-existing organelles or de novo by vesiculation of the ER. It has been suggested that ADP-ribosylation factors and COPI coatomer complexes are involved in these processes. RESULTS: Here we show that all viable Saccharomyces cerevisiae strains deficient in one of the small GTPases which have an important role in the regulation of vesicular transport contain functional peroxisomes, and that the number of these organelles in oleate-grown cells is significantly upregulated in the arf1 and arf3 null strains compared to the wild-type strain. In addition, we provide evidence that a portion of endogenous Arf6, the mammalian orthologue of yeast Arf3, is associated with the cytoplasmic face of rat liver peroxisomes. Despite this, ablation of Arf6 did neither influence the regulation of peroxisome abundance nor affect the localization of peroxisomal proteins in cultured fetal hepatocytes. However, co-overexpression of wild-type, GTP hydrolysis-defective or (dominant-negative) GTP binding-defective forms of Arf1 and Arf6 caused mislocalization of newly-synthesized peroxisomal proteins and resulted in an alteration of peroxisome morphology. CONCLUSION: These observations suggest that Arf6 is a key player in mammalian peroxisome biogenesis. In addition, they also lend strong support to and extend the concept that specific Arf isoform pairs may act in tandem to regulate exclusive trafficking pathways.

Comparison of the PTS1- and Rab8b-binding properties of Pex5p and Pex5Rp/TRIP8b.

Tetratricopeptide (TPR)-domain proteins are involved in various cellular processes. The TPR domain is known to be responsible for interaction with other proteins commonly recognizing sequence motifs at the C-termini. One such TPR-protein, TRIP8b, was originally identified in rat as an interaction partner of Rab8b, and its human orthologue as a protein related to the peroxisomal targeting signal 1 (PTS1) receptor Pex5p (Pex5Rp). Somewhat later, the mouse orthologue was reported to bind the hyperpolarization-activated, cyclic nucleotide-regulated HCN channels, and, very recently, the rat orthologue was shown to interact with latrophilin 1, the calcium-independent receptor of alpha-latrotoxin. Here we employed various methodological approaches to investigate and compare the binding specificities of the human PTS1 receptor Pex5p and the related protein Pex5Rp/TRIP8b towards a subset of targets, including Rab8b and various C-termini resembling PTS1. The results show that the TPR domains of Pex5p and Pex5Rp/TRIP8b have distinct but overlapping substrate specificities. This suggests that selectivity in the recognition of substrates by the TPR domains of Pex5p and Pex5Rp/TRIP8b is a matter of considerable complexity, and that no single determinant appears to be sufficient in unambiguously defining a binding target for either protein. This idea is further corroborated by our observations that changes in the surrounding residues or the conformational state of one of the binding partners can profoundly alter their binding activities. The implications of these findings for the possible peroxisome-related functions of Pex5Rp/TRIP8b are discussed.

Trypanosoma brucei glycosomal ABC transporters: identification and membrane targeting.

Trypanosomes contain unique peroxisome-like organelles designated glycosomes which sequester enzymes involved in a variety of metabolic processes including glycolysis. We identified three ABC transporters associated with the glycosomal membrane of Trypanosoma brucei. They were designated GAT1-3 for Glycosomal ABC Transporters. These polypeptides are so-called half-ABC transporters containing only one transmembrane domain and a single nucleotide-binding domain, like their homologues of mammalian and yeast peroxisomes. The glycosomal localization was shown by immunofluorescence microscopy of trypanosomes expressing fusion constructs of the transporters with Green Fluorescent Protein. By expression of fluorescent deletion constructs, the glycosome-targeting determinant of two transporters was mapped to different fragments of their respective primary structures. Interestingly, these fragments share a short sequence motif and contain adjacent to it one--but not the same--of the predicted six transmembrane segments of the transmembrane domain. We also identified the T. brucei homologue of peroxin PEX19, which is considered to act as a chaperonin and/or receptor for cytosolically synthesized proteins destined for insertion into the peroxisomal membrane. By using a bacterial two-hybrid system, it was shown that glycosomal ABC transporter fragments containing an organelle-targeting determinant can interact with both the trypanosomatid and human PEX19, despite their low overall sequence identity. Mutated forms of human PEX19 that lost interaction with human peroxisomal membrane proteins also did not bind anymore to the T. brucei glycosomal transporter. Moreover, fragments of the glycosomal transporter were targeted to the peroxisomal membrane when expressed in mammalian cells. Together these results indicate evolutionary conservation of the glycosomal/peroxisomal membrane protein import mechanism.

Analysis of human Pex19p's domain structure by pentapeptide scanning mutagenesis.

Pex19p, a primarily cytosolic protein, is essential for the biogenesis of numerous peroxisomal membrane proteins (PMPs); however, its precise function is unclear. Pex19p might function as a PMP-specific chaperone, a cycling PMP-receptor protein, a PMP membrane insertion factor, or an association/dissociation factor of membrane-associated protein complexes. Alternatively, Pex19p might act as a multifunctional peroxin and participate in a number of these activities. Here, we have employed transposon mutagenesis to generate a library of human pex19 alleles coding for Pex19p variants containing random in-frame pentapeptide insertions. A total of 87 different variants were characterized to identify functionally important regions. These studies revealed that Pex19p has a tripartite domain structure consisting of: (i) an amino-terminal domain that binds to Pex3p and is essential for docking at the peroxisome membrane; (ii) a central domain that competes with Pex5p and Pex13p for binding to Pex14p and may play a role in the assembly of PTS-receptor docking complexes; and (iii) a carboxy-terminal domain that interacts with multiple PMPs including Pex3p, Pex11pbeta, Pex12p, Pex13p, Pex16p, and Pex26p. Whether the latter interactions constitute the chaperone or transport functions (or both), remains to be determined. Finally, our observation that Pex19p contains two distinct binding sites for Pex3p suggests that the peroxin may bind PMPs in multiple places and for multiple purposes.

Potential role for Pex19p in assembly of PTS-receptor docking complexes.

Human Pex19p binds a broad spectrum of peroxisomal membrane proteins (PMPs). It has been proposed that this peroxin may: (i) act as a cycling PMP receptor protein, (ii) facilitate the insertion of newly synthesized PMPs into the peroxisomal membrane, or (iii) function as a chaperone to associate and/or dissociate complexes comprising integral PMPs already in the peroxisomal membrane. We previously demonstrated that human Pex19p binds peroxisomal integral membrane proteins at regions distinct from their sorting sequences. Here we demonstrate that a mutant of Pex13p that fails to bind to Pex19p nevertheless targets to and integrates into the peroxisomal membrane. In addition, through in vitro biochemical analysis, we show that Pex19p competes with Pex5p and Pex13p for binding to Pex14p, supporting a role for this peroxin in regulating assembly/disassembly of membrane-associated protein complexes. To further examine the molecular mechanism underlying this competition, six evolutionarily conserved amino acids in the Pex5p/Pex13p/Pex19p binding domain of Pex14p were subjected to site-directed mutagenesis and the corresponding mutants functionally analyzed. Our results indicate that the physically overlapping binding sites of Pex14p for Pex5p, Pex13p, and Pex19p are functionally distinct, suggesting that competition occurs through induction of structural changes, rather than through direct competition. Importantly, we also found that amino acid substitutions resulting in a strongly reduced binding affinity for Pex13p affect the peroxisomal localization of Pex14p.

Analysis of mammalian peroxin interactions using a non-transcription-based bacterial two-hybrid assay.

In recent years, substantial progress has been made in the identification of proteins involved in peroxisome biogenesis. However, with the exception of the peroxisome-targeting signal receptors and the receptor docking proteins, the function of most of these proteins, called peroxins, remains largely unknown. One step toward elucidating the function of a protein is to identify its interacting partners. We have used a non-transcription-based bacterial two-hybrid system to analyze the interactions among a set of 12 mammalian peroxins and a yeast protein three-hybrid system to investigate whether proteins that interact with the same peroxin and have overlapping binding sites cooperate or compete for this site. Here we report a detailed interaction map of these peroxins and demonstrate that (i) farnesylation, and not the CAAX motif, of Pex19p strongly enhances its affinity for Pex13p; (ii) the CAAXmotif, and not farnesylation, of Pex19p strongly enhances its affinity for Pex11pbeta; and (iii) the C(3)HC(4) RING (really interesting new gene) finger domain of Pex12p does not alter the binding properties of Pex5p for the C-terminal peroxisome-targeting signal PTS1. Finally, we show that the Pex5p-Pex13p interaction is bridged by Pex14p and that the latter molecule exists predominantly as a dimer in vivo. Collectively, as demonstrated in this work with peroxins, these results indicate that the bacterial two-hybrid system is an attractive complementary approach to the conventional transcription-based yeast two-hybrid system for studying protein-protein interactions.