Cysteine Oxidation Targets Peroxiredoxins 1 and 2 for Exosomal Release through a Novel Mechanism of Redox-Dependent Secretion
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Nonclassical protein secretion is of major importance as a number of cytokines and inflammatory mediators are secreted via this route. Current evidence indicates that there are several mechanistically distinct methods of nonclassical secretion. We have shown recently that peroxiredoxin (Prdx) 1 and Prdx2 are released by various cells upon exposure to inflammatory stimuli such as lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α). The released Prdx then acts to induce production of inflammatory cytokines. However, Prdx1 and 2 do not have signal peptides and therefore must be secreted by alternative mechanisms, as has been postulated for the inflammatory mediators interleukin-1β (IL-1β) and high mobility group box-1 (HMGB1). We show here that circulating Prdx1 and 2 are present exclusively as disulfide-linked homodimers. Inflammatory stimuli also induce in vitro release of Prdx1 and 2 as disulfide-linked homodimers. Mutation of cysteines Cys51 or Cys172 (but not Cys70) in Prdx2, and Cys52 or Cys173 (but not Cys71 or Cys83) in Prdx1 prevented dimer formation and this was associated with inhibition of their TNF-α-induced release. Thus, the presence and oxidation of key cysteine residues in these proteins are a prerequisite for their secretion in response to TNF-α, and this release can be induced with an oxidant. By contrast, the secretion of the nuclear-associated danger signal HMGB1 is independent of cysteine oxidation, as shown by experiments with a cysteine-free HMGB1 mutant. Release of Prdx1 and 2 is not prevented by inhibitors of the classical secretory pathway, instead, both Prdx1 and 2 are released in exosomes from both human embryonic kidney (HEK) cells and monocytic cells. Serum Prdx1 and 2 also are associated with the exosomes. These results describe a novel pathway of protein secretion mediated by cysteine oxidation that underlines the importance of redox-dependent signaling mechanisms in inflammation.
Peroxiredoxins (Prdxs) are a family of antioxidant proteins that protects cells from oxidative damage via reduction of peroxides using thioredoxin (TXN) and, in some cases glutaredoxin, as the electron donor and also functions as molecular chaperones and regulators of signal transduction (1,2). A number of studies have shown that Prdxs are secreted from cancer or virus-infected cells (3), are present in biological fluids such as bronchiolar lavage (4) and serum (5) as well as on the membrane of lymphocytes from rheumatoid arthritis patients (6). Prdxs are also known as natural killer enhancing factors (NKEF) due to early observations that these proteins could enhance natural killer cell cytotoxicity against tumor cells (7). Soon after, the antioxidant properties of both NKEF-A and B (Prdx I and II respectively) were demonstrated in erythrocytes (8). The association of oxidative stress with inflammation has been known for some time, but the precise relationship and mechanisms by which these two phenomena are related are not well understood.
In a previous study using redox proteomics, a technique originally developed to identify intracellular glutathionylated proteins (9), we identified Prdx2 among the proteins released from mouse macrophages in response to LPS under specific forms of cysteine oxidation, including formation of disulfide-linked homodimers and mixed disulfides with glutathione (glutathionylation) (10). The released Prdx2 then acts to induce production of inflammatory cytokines, like a classical inflammatory danger signal (11). Studies by others also have shown that Prdx1, 5 and 6 also induce inflammatory cytokines in vitro (12,13).
However, Prdxs (except Prdx4) do not have signal peptides and, therefore, must be secreted by alternative, nonclassical mechanisms as postulated for IL-1β (14) and HMGB1 (15). Here we test the hypothesis that cysteine oxidation is a prerequisite for the release of Prdx1 and 2. For this purpose we set up an experimental model where Prdx release is induced from HEK 293T cells upon stimulation with TNF-α. Previous studies have shown that HEK 293T cells respond to TNF-α (but not LPS) by secreting IL-8 (16); hence this system was used as a model of a proinflammatory stimulus. We then expressed different cysteine mutants of Prdx1 and 2 to investigate the role of specific cysteines in the secretion of these proteins, both in response to LPS and an oxidant, menadione. Release of a classical cytokine, IL-8, was used for comparison. We also investigated whether Prdx1 and 2 were released in exosomes and whether an inhibitor of the classical secretory pathway, brefeldin A (BFA), affected their secretion. The results presented here confirm that the presence and oxidation of specific cysteines in Prdx1 and 2 are an essential requirement for their secretion through the exosomal pathway.
Materials and Methods
LPS from E. coli 055:B5, glyburide, nocodazole, brefeldin A (BFA), menadione and N-ethylmaleimide (NEM) were all purchased from Sigma-Aldrich (St. Louis, MO, USA).
Cloning and Mutation of Human Prdx2 and Human Prdx1 in pcDNA6
Oligonucleotides used in the cloning and mutagenesis of human Prdx1, human Prdx2 and rat HMGB-1.
Cloning and Mutation of Rat HMGB-1 in pcDNA6
Rat HMGB-1 was amplified from rat cDNA (a generous gift from Huan Yang, The Feinstein Institute for Medical Research, Manhasset, NY, USA) by PCR using Pfu-DNA polymerase (Promega) using primers designed to insert restriction sites for HindIII and XhoI. Inserts were restricted using HindIII and XhoI and ligated into pcDNA6 (Life Technologies [Thermo Fisher Scientific]). All cysteines (C23S, C45S and C106S) were first changed to serine residues using the QuikChange Lightning site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA) and the oligonucleotides shown in Table 1. Double and triple cysteine mutations were achieved by additional rounds of site-directed mutagenesis. The mutations of all constructs were verified by sequencing.
Culture and Transient Transfection of 293T Cells
293T cells were routinely cultured in-Dulbecco modified Eagle medium (DMEM) (Sigma-Aldrich) supplemented with 10% fetal bovine serum and penicillin and streptomycin. Transient transfections were performed using 25-kDa linear polyethylenimine (PEI) (purchased from Polysciences Inc., Warrington, PA, USA). Stock solutions of PEI were prepared in water at a concentration of 1 mg/mL, and the pH adjusted to 7.0. The transfection conditions used are essentially those as described previously (17). Briefly, HEK 293T cells were plated at a density of 0.5 × 106 cells/well in 6-well plates and transfected with 5 µg of DNA. The transfection complex was formed at a DNA-PEI ratio of 1:3 in OPTI-MEM (Life Technologies [Thermo Fisher Scientific]), with a 30 min incubation at room temperature prior to addition to the cells. Twenty-four hours later, culture media were replaced with 1 mL of serum-free DMEM containing 50 ng/mL of recombinant human TNF-α (PeproTech, Rocky Hill, NJ, USA). Cells were cultured for a further 24 h before collecting supernatants and lysing cells in 200 of lysis buffer (10 mmol/L Tris HCl, 150 mmol/L NaCl, 1 mmol/L ethylene glycol tetraacetic acid (EGTA), 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 1% NP-40, pH 7.4). NEM was added to all supernatants and cell lysates to a final concentration of 50 mmol/L immediately after collection and incubated at room temperature for 15 min. In some experiments, cell viability was determined at the end of the experiment using the CellTiter-Blue assay (Promega) according to the manufacturer’s instructions.
Culture of THP-1 Cells
THP-1 monocytic cells were routinely cultured in RPMI (Sigma-Aldrich) supplemented with 10% fetal bovine serum and penicillin and streptomycin. For stimulation of THP-1 cells with LPS, cells were treated with 100 ng/mL of LPS in serum-free RPMI and cultured for 24 h before collecting cells and supernatants by centrifugation. NEM was again added to all supernatants and cell lysates to a final concentration of 50 mmol/L immediately after collection.
293T cell culture supernatants or cleared cell lysates were applied without prior dilution to 12% acrylamide sodium dodecyl sulphate (SDS) gels and transferred to nitrocellulose membranes (Millipore) by electroblotting. After blocking in 5% bovine serum albumin (BSA) in PBS containing 0.05% Tween 20, membranes were probed with one of the following antibodies: polyclonal rabbit anti-human Prdx2 or Prdx1 antibody, mouse anti-His (Life Technologies [Thermo Fisher Scientific]), mouse anti-V5 (Life Technologies [Thermo Fisher Scientific]), mouse anti-Prdx4 (Santa Cruz Biotechnology Inc., Dallas, TX, USA), mouse anti-Prdx5 (Santa Cruz Biotechnology), rabbit anti-HSP70 (Sigma-Aldrich), mouse anti-actin (Sigma-Aldrich) followed by detection with anti-rabbit-IgG-horse-radish peroxidase conjugate (Sigma-Aldrich) or anti-mouse IgG horseradish peroxidase conjugate (Stratagene, Agilent Technologies). In some experiments, membranes were stripped using Restore stripping buffer (Pierce Scientific [Thermo Fisher Scientific]) before blocking and reprobing with an antibody as described above. Western blots were developed using advanced chemiluminescence (ECL) reagents (GE Healthcare, Amersham, UK) and exposed to autoradiography using Hyperfilm (GE Healthcare). Films were developed using an AGFA Curix 60 developer (Agfa HealthCare, Brentford, Middlesex, UK).
Cell Viability Assays
Cell viability was measured using the CellTiter-Glo assay (Promega) according to the manufacturer’s instructions. To confirm that this assay was sensitive enough to determine even relatively small levels of cellular toxicity, varying numbers of 293T cells were plated, incubated at 37°C for 24 h to allow cells to adhere, then cell viability measured.
The concentration of IL-8 secreted by HEK 293T cells was determined using as commercially available enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA).
Purification of Recombinant hPrdx2 from HEK293T Cells
HEK 293T cells transiently transfected with pcDNA6-hPrdx2 were lysed in ice-cold lysis buffer (50 mmol/L sodium phosphate pH 8.0, 300 mmol/L NaCl, 0.01% Tween20, Triton 100), centrifuged to remove cell debris and applied to a His Gravi-Trap Talon column (GE Healthcare). Columns were washed extensively with washing buffer (50 mmol/L sodium phosphate pH 8.0, 300 mmol/L NaCl, 0.01% Tween20, 50 mmol/L imidazole) and eluted with elution buffer (50 mmol/L sodium phosphate pH 8.0, 300 mmol/L NaCl, 0.01% Tween20, 300 mmol/L imidazole). The eluted Prdx2 was buffer exchanged into PBS (pH 7.4) using a PD10 desalting column (GE Healthcare).
Preparation of Exosomes
Serum-free medium (1 ml) from cultured cells was collected, treated with NEM to a final concentration of 50 mmol/L and incubated at room temperature for 15 min, then centrifuged at 2,000g for 10 min to remove cells and cell debris. Supernatants were then centrifuged at 100,000g at 4°C for 2 h, washed with PBS, and centrifuged again for 1 h and the resulting supernatants collected. Pellets (exosomes) were resuspended in SDS-PAGE sample buffer (50 mmol/L Tris-Cl [pH 6.8], 100 mmol/L dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol) and subjected to 12% SDS-PAGE under nonreducing conditions for Western blot analysis as described above.
Collection of Human Plasma
Human peripheral blood was collected from healthy donors, all of whom gave written informed consent, under the approval of the local Research Governance and Ethics Committee. Blood was collected in vacutainers containing EDTA. Immediately after collection, blood was centrifuged at 1,000g to pellet cells. The plasma was removed and NEM added to a final concentration of 50 mmol/L. For preparation of exosomes from plasma, samples were centrifuged at 10,000g for 30 min at 4°C. The supernatants were then centrifuged at 100,000g at 4°C for 2 h, and the resulting pellets washed with PBS, before centrifuging at 100,000g at 4°C for 1 h. Pellets were lysed in SDS sample buffer for subsequent analysis by Western blotting.
All supplementary materials are available online at https://doi.org/www.molmed.org.
TNF-α- and Oxidant-Induced Release of Prdx2 Homodimer
TNF-α Induces Release of Prdx1 and 2 but Not Prdx4 or 5
Stability of the Monomer in Cell Culture Conditions
Disulfide-Mediated Dimerization Is Essential for Release of Prdx1 and 2
An appreciable amount of monomer was observed in the cell supernatants for the first time, but only when disulfide bond formation was prevented by mutation of Cys51 or Cys172. One possibility was that overexpression of these mutant proteins was causing protein aggregation and consequent cellular toxicity. To determine whether this was the case and whether cellular location was affected by the mutation, we expressed wild-type and C51S Prdx2 as GFP-fusion proteins for analysis by confocal microscopy. wild-type GFP-Prdx2 was visible as punctate cytoplasmic staining, in contrast to the GFP control, which showed diffuse staining throughout the cytoplasm and nucleus (Supplementary Figures 1A, B). The C51S mutant GFP-fusion was present in much larger aggregates within the cytoplasm (Supplementary Figure 1C). Staining of endogenous Prdx2 by immunofluorescent labeling confirmed that the punctate cytoplasmic staining observed for the wild-type recombinant Prdx2 accurately reflected the localization of the endogenous protein (Supplementary Figure 1D).
Prdx1 and 2 Dimers Are Secreted via a Nonclassical Pathway and Are Associated with the Exosomes
Prdx2 Is Present as Dimers in Human Plasma
To determine whether this mechanism also occurs in vivo, we analyzed Prdx2 in human plasma. Prdx2 was detected in human plasma by Western blotting using specific antibodies as shown in Figures 7E and F. The majority of Prdx2 was present as oligomers, with some dimer present. Upon treatment of the samples with DTT, the oligomeric form disappeared and most of the protein was reduced to monomers (Figure 7E). We could also show that, similar to the conditioned medium from cultured cells, the Prdx2 present in plasma was associated with exosomes (see Figure 7F).
Here we show a novel redox-regulated mechanism for the extracellular release of Prdx1 and 2 in response to inflammatory stimuli. The lack of a signal peptide in these proteins meant that classical secretion was unlikely and this was confirmed by the fact that release of these proteins was unaffected by BFA. The aim of the study was to test the hypothesis that cysteine oxidation is implicated in the release of Prdx1 and 2. The observation that only the dimer is present in the supernatant raised the possibility that secreted Prdx, whether in monomeric or dimeric form, would be oxidized after its release in our culture conditions. However, when recombinant Prdx2 originally present both as monomer and dimer was added to cultured cells, we could still detect both monomer and dimer. Thus, if a mixture of monomer and dimer was secreted, this would be detected in the supernatant.
Prdx1 and 2 are typical 2-Cys Prdxs, which rely on two specific cysteine residues for their catalytic activity, with a peroxidatic cysteine that is oxidized to a sulfenic acid by H2O2 and a resolving one which then reduces it by forming a disulfide bridge (Figure 5A). The peroxidatic cysteines are Cys52 or 51, while the resolving ones are Cys173 or 172, in Prdx1 and 2 respectively. Prdx2 has one (Cys70) and Prdx1 has two (Cys71 and Cys83) additional cysteines. Consistent with the mechanism shown in Figure 5A, we found that mutation of the peroxidatic or resolving cysteines prevented dimerization, while mutation of the additional cysteine had no effect on dimerization. Intriguingly, mutation of either the peroxidatic or the resolving cysteines in both Prdx1 and 2 prevented secretion from cells
The one exception to this was the C51S mutation of Prdx2, which was observed in the supernatant in the monomeric form. It is possible, however, that the appearance of the Prdx2 monomer in the supernatants of cells expressing the C51S mutant is an artifact of recombinant expression and aggregation of this protein. This is supported by experiments showing that, when expressed as a GFP-fusion protein, the C51S mutant is present in large aggregates in the cytoplasm, while the wild-type protein is present as uniformly distributed punctate staining in the cytoplasm. Secretion of misfolded or overexpressed proteins is a phenomenon previously described for various proteins and is thought to represent a means by which the cytoplasm can be cleared of aggregated or accumulated protein (24,25). As there does not appear to be any difference in the extent of expression for the wild-type and C51S mutant, it seems more likely that the release of the monomer in the case of the C51S mutant is due to the lack of dimerization which seems to cause aggregation within the cells. Of note, the corresponding C52S mutant in Prdx1 did not behave in this manner, with the overall evidence suggesting that mutations which prevented dimerization also prevented secretion. It is clear from the preceding discussion that, although mutation of individual cysteines is the only way of determining their importance, there are some limitations to this approach. However, with the exception of the experiments with the C51S mutant, the evidence points to a role for cysteine oxidation in the secretion of Prdx1 and 2.
There is a growing list of proteins that are secreted by nonclassical pathways (26), and our finding might define a class of them whose secretion is mediated by the redox state of the protein. Interestingly, dimerization via the formation of covalent dimers is also essential for the secretion of fibroblast growth factor (FGF) 1 (27), as point mutation of Cys30 inhibits export of this protein (28). FGF1 is also one of a group of nonclassically secreted proteins that are not released spontaneously, but that require cell stress to be secreted (26). However, the similarities between release of FGF1 and Prdx 1 and 2 end there. In fact, although FGF1 homodimers are absolutely necessary for secretion, they form only one part of a much larger multiprotein complex that is required for release (29,30). Export from the cell then occurs via translocation through the cell membrane (31) and not via exosomal release, as shown here for Prdx1 and 2.
It is difficult to say whether glutathionylation is important for this process. Our previous work has shown that both Prdx1 and 2 are released from mouse macrophage cells in response to LPS as glutathionylated dimers (10). One possibility is that the glutathionylation releases monomers and dimers from larger multimeric structures that, as such, would normally be retained in the cell. In fact, Prdx1 dimers can organize in decamers (34) and it was shown that, while normally 97% of the protein is in this form, glutathionylation completely eliminates these decamers, releasing monomers and dimers (35). Although the decameric form was not investigated specifically in this study, it is likely that the dimers that are secreted are released from these higher-order oligomers.
The mechanism of release of Prdx1 and 2 is distinct from that for Il-1β, which can be inhibited by glyburide or nocodazole. It is unsurprising that glyburide does not inhibit the release of Prdxs, as this compound inhibits activation of the NLRP3 inflammasome, hence inhibiting the processing of pro-IL1 to mature IL-1β for secretion, while Prdx1 and 2 do not undergo proteolytic processing during secretion. However, secretion of IL-1β also can be inhibited by cytoskeletal inhibitors such as nocodazole (36), demonstrating the involvement of microtubules in the exocytosis of the vesicles containing IL-1β (36). It appears from the results shown here that microtubules are unimportant for the release of Prdx1 and 2 and the major determining factor in their release is dimerization via cysteine oxidation. The fact that the oxidant menadione also induces the release of Prdx1 and 2 suggest that oxidation per se is sufficient to cause their release.
The redox regulation of these Prdxs is of particular importance as the proinflammatory activity of both Prdx1 (11,12) and Prdx2 (10,13) has been demonstrated previously. The fact that these Prdxs are released in a redox-regulated manner in response to inflammatory stimuli as well as oxidant treatment is the first evidence for the potential generation of inflammatory mediators by cellular redox changes. The redox enzyme TXN also is released from RAW cells in response to LPS in an oxidized (glutathionylated) form (10) and also is released during infection and inflammation and has immunoregulatory effects (37). Similarly to the Prdxs shown here, secretion of TXN occurs by a nonclassical pathway (38), but intriguingly, secretion is independent of redox state (39).
Other proinflammatory molecules secreted in exosomes or vesicles include HSP70 (21) and HMGB-1 (40). HSP70 is released from cells in exosomes (23), but there is no evidence that its association with exosomes is associated with changes in its redox status. Although redox changes are important in controlling the intracellular localization of HMGB-1 in terms of nuclear versus cytoplasmic localization (41), extracellular secretion of HMGB-1 is independent of redox state.
We have uncovered a novel mechanism for nonclassical secretion of inflammatory mediators that relies on cysteine oxidation and might potentially define a “redox secretome.” The presence of Prdx1 and 2 in healthy human plasma suggests that redox changes, by controlling their secretion, regulate their role in inflammation. This may be particularly important in pathologies such as ischemia-reperfusion injury, where an inflammatory response is observed in the absence of infection or autoimmunity, and where a redox imbalance may act as the trigger of inflammation. A comprehensive definition of the redox secretome in pathological conditions might open new perspectives in terms of pharmacological targets or biomarkers for several diseases.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
Funded by the EU, European Regional Development Fund, France (Channel) England (PeReNE Project), and by the Brighton and Sussex Medical School.
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