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.

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.

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.

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.