Eva-Maria Hanschmann

Linkage of inflammation and oxidative stress via release of glutathionylated peroxiredoxin-2, which acts as a danger signal

Significance Inflammation often complicates diseases associated with oxidative stress. This study shows that inflammatory macrophages release proteins with specific forms of cysteine oxidation to disulfides, particularly glutathionylation. Redox proteomics identified peroxiredoxin 2 (PRDX2) as a protein released in glutathionylated form by inflammation both in vivo and in vitro. Extracellular PRDX2 then triggers the production of TNF-α. These data indicate that redox-dependent mechanisms, in an oxidative cascade, can induce inflammation. The mechanism by which oxidative stress induces inflammation and vice versa is unclear but is of great importance, being apparently linked to many chronic inflammatory diseases. We show here that inflammatory stimuli induce release of oxidized peroxiredoxin-2 (PRDX2), a ubiquitous redox-active intracellular enzyme. Once released, the extracellular PRDX2 acts as a redox-dependent inflammatory mediator, triggering macrophages to produce and release TNF-α. The oxidative coupling of glutathione (GSH) to PRDX2 cysteine residues (i.e., protein glutathionylation) occurs before or during PRDX2 release, a process central to the regulation of immunity. We identified PRDX2 among the glutathionylated proteins released in vitro by LPS-stimulated macrophages using mass spectrometry proteomic methods. Consistent with being part of an inflammatory cascade, we find that PRDX2 then induces TNF-α release. Unlike classical inflammatory cytokines, PRDX2 release does not reflect LPS-mediated induction of mRNA or protein synthesis; instead, PRDX2 is constitutively present in macrophages, mainly in the reduced form, and is released in the oxidized form on LPS stimulation. Release of PRDX2 is also observed in human embryonic kidney cells treated with TNF-α. Importantly, the PRDX2 substrate thioredoxin (TRX) is also released along with PRDX2, enabling an oxidative cascade that can alter the –SH status of surface proteins and thereby facilitate activation via cytokine and Toll-like receptors. Thus, our findings suggest a model in which the release of PRDX2 and TRX from macrophages can modify the redox status of cell surface receptors and enable induction of inflammatory responses. This pathway warrants further exploration as a potential novel therapeutic target for chronic inflammatory diseases.

Both Thioredoxin 2 and Glutaredoxin 2 Contribute to the Reduction of the Mitochondrial 2-Cys Peroxiredoxin Prx3 *

The proteins from the thioredoxin family are crucial actors in redox signaling and the cellular response to oxidative stress. The major intracellular source for oxygen radicals are the components of the respiratory chain in mitochondria. Here, we show that the mitochondrial 2-Cys peroxiredoxin (Prx3) is not only substrate for thioredoxin 2 (Trx2), but can also be reduced by glutaredoxin 2 (Grx2) via the dithiol reaction mechanism. Grx2 reduces Prx3 exhibiting catalytic constants (Km, 23.8 μmol·liter−1; Vmax, 1.2 μmol·(mg·min)−1) similar to Trx2 (Km, 11.2 μmol·liter−1; Vmax, 1.1 μmol·(mg·min)−1). The reduction of the catalytic disulfide of the atypical 2-Cys Prx5 is limited to the Trx system. Silencing the expression of either Trx2 or Grx2 in HeLa cells using specific siRNAs did not change the monomer:dimer ratio of Prx3 detected by a specific 2-Cys Prx redox blot. Only combined silencing of the expression of both proteins led to an accumulation of oxidized protein. We further demonstrate that the distribution of Prx3 in different mouse tissues is either linked to the distribution of Trx2 or Grx2. These results introduce Grx2 as a novel electron donor for Prx3, providing further insights into pivotal cellular redox signaling mechanisms.

Thioredoxin and glutaredoxin system proteins-immunolocalization in the rat central nervous system.

BACKGROUND The oxidoreductases of the thioredoxin (Trx) family of proteins play a major role in the cellular response to oxidative stress. Redox imbalance is a major feature of brain damage. For instance, neuronal damage and glial reaction induced by a hypoxic-ischemic episode is highly related to glutamate excitotoxicity, oxidative stress and mitochondrial dysfunction. Most animal models of hypoxia-ischemia in the central nervous system (CNS) use rats to study the mechanisms involved in neuronal cell death, however, no comprehensive study on the localization of the redox proteins in the rat CNS was available. METHODS The aim of this work was to study the distribution of the following proteins of the thioredoxin and glutathione/glutaredoxin (Grx) systems in the rat CNS by immunohistochemistry: Trx1, Trx2, TrxR1, TrxR2, Txnip, Grx1, Grx2, Grx3, Grx5, and γ-GCS, peroxiredoxin 1 (Prx1), Prx2, Prx3, Prx4, Prx5, and Prx6. We have focused on areas most sensitive to a hypoxia-ischemic insult: Cerebellum, striatum, hippocampus, spinal cord, substantia nigra, cortex and retina. RESULTS AND CONCLUSIONS Previous studies implied that these redox proteins may be distributed in most cell types and regions of the CNS. Here, we have observed several remarkable differences in both abundance and regional distribution that point to a complex interplay and crosstalk between the proteins of this family. GENERAL SIGNIFICANCE We think that these data might be helpful to reveal new insights into the role of thiol redox pathways in the pathogenesis of hypoxia-ischemia insults and other disorders of the CNS. This article is part of a Special Issue entitled Human and Murine Redox Protein Atlases.

Crucial function of vertebrate glutaredoxin 3 (PICOT) in iron homeostasis and hemoglobin maturation

Vertebrate glutaredoxin 3 (PICOT) is essential for the maturation of the heme cofactor of hemoglobin through its essential functions in iron homeostasis. The data suggest an evolutionarily conserved role of cytosolic monothiol multidomain Grxs in cellular iron metabolism pathways.

Monothiol Glutaredoxin-1 Is an Essential Iron-Sulfur Protein in the Mitochondrion of African Trypanosomes

African trypanosomes encode three monothiol glutaredoxins (1-C-Grx). 1-C-Grx1 occurs exclusively in the mitochondrion, and 1-C-Grx2 and -3 are predicted to be mitochondrial and cytosolic proteins, respectively. All three 1-C-Grx are expressed in both the mammalian bloodstream and the insect procyclic form of Trypanosoma brucei, with the highest levels found in stationary phase and starving parasites. In the rudimentary mitochondrion of bloodstream cells, 1-C-Grx1 reaches concentrations above 200 μm/subunit. Recombinant T. brucei 1-C-Grx1 exists as a noncovalent homodimer, whereas 1-C-Grx2 and 1-C-Grx3 are monomeric proteins. In vitro, dimeric 1-C-Grx1 coordinated an H2O2-sensitive [2Fe-2S] cluster that required GSH as an additional ligand. Both bloodstream and procyclic trypanosomes were refractory to down-regulation of 1-C-Grx1 expression by RNA interference. In procyclic parasites, the 1-c-grx1 alleles could only be deleted if an ectopic copy of the gene was expressed. A 5–10-fold overexpression of 1-C-Grx1 in both parasite forms did not yield a growth phenotype under optimal culture conditions. However, exposure of these cells to the iron chelator deferoxamine or H2O2, but not to iron or menadione, impaired cell growth. Treatment of wild-type bloodstream parasites with deferoxamine and H2O2 caused a 2-fold down- and up-regulation of 1-C-Grx1, respectively. The results point to an essential role of the mitochondrial 1-C-Grx1 in the iron metabolism of these parasites.

Cold Atmospheric Plasma Treatment Induces Anti-Proliferative Effects in Prostate Cancer Cells by Redox and Apoptotic Signaling Pathways

One of the promising possibilities of the clinical application of cold plasma, so-called cold atmospheric plasma (CAP), is its application on malignant cells and cancer tissue using its anti-neoplastic effects, primarily through the delivery of reactive oxygen and nitrogen species (ROS, RNS). In this study, we investigated the impact of CAP on cellular proliferation and consecutive molecular response mechanisms in established prostate cancer (PC) cell lines. PC cells showed a significantly reduced cell growth following CAP treatment as a result of both an immediate increase of intracellular peroxide levels and through the induction of apoptosis indicated by annexin V assay, TUNEL assay, and the evaluation of changes in nuclear morphology. Notably, co-administration of N-acetylcysteine (NAC) completely neutralized CAP effects by NAC uptake and rapid conversion to glutathione (GSH). Vitamin C could not counteract the CAP induced effects on cell growth. In summary, relatively short treatments with CAP of 10 seconds were sufficient to induce a significant inhibition of cancer proliferation, as observed for the first time in urogenital cancer. Therefore, it is important to understand the mode of CAP related cell death and clarify and optimize CAP as cancer therapy. Increased levels of peroxides can alter redox-regulated signaling pathways and can lead to growth arrest and apoptosis. We assume that the general intracellular redox homeostasis, especially the levels of cellular GSH and peroxidases such as peroxiredoxins affect the outcome of the CAP treatment.

Cysteine oxidation targets peroxiredoxins 1 and 2 for exosomal release through a novel mechanism of redox-dependent secretion

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.

Segment-specific overexpression of redoxins after renal ischemia and reperfusion: protective roles of glutaredoxin 2, peroxiredoxin 3, and peroxiredoxin 6

The disruption of redox control, i.e., oxidative stress, is one of the most destructive causes of ischemia-reperfusion (IR) injury. Thioredoxin (Trx) family proteins play a major role in the cellular response to oxidative stress. Here, we systematically investigated the levels and tissue distribution of 15 members of this family (Trx and TrxR 1 and 2, Nrx, Prx 1-6, and Grx 1-3 and 5) in mouse kidneys after induction of IR by comparing control, clamped, and contralateral organs. After IR, levels of various redoxins were quantified. Immunohistochemical analysis revealed segment-specific alterations induced by the ischemic insult. Grx2, Prx3, and Prx6 were highly expressed in proximal tubule cells. Overexpression of these proteins in HEK293 and HeLa cells subjected to hypoxia and reoxygenation revealed higher survival and proliferation rates and lower oxidative damage compared to controls. Furthermore, we report for the first time the accumulation of Grx1 at the apical side of distal convoluted cells and the specific secretion of Grx1 into the urine after IR. The differences in both the basal equipment and the segment-specific responses of the antioxidant proteins may contribute to the distinct susceptibilities and regeneration processes of the various segments of the nephron to the IR insult.

Redox regulation of cytoskeletal dynamics during differentiation and de-differentiation.

BACKGROUND The cytoskeleton, unlike the bony vertebrate skeleton or the exoskeleton of invertebrates, is a highly dynamic meshwork of protein filaments that spans through the cytosol of eukaryotic cells. Especially actin filaments and microtubuli do not only provide structure and points of attachments, but they also shape cells, they are the basis for intracellular transport and distribution, all types of cell movement, and--through specific junctions and points of adhesion--join cells together to form tissues, organs, and organisms. SCOPE OF REVIEW The fine tuned regulation of cytoskeletal dynamics is thus indispensible for cell differentiation and all developmental processes. Here, we discussed redox signalling mechanisms that control this dynamic remodeling. Foremost, we emphasised recent discoveries that demonstrated reversible thiol and methionyl switches in the regulation of actin dynamics. MAJOR CONCLUSIONS Thiol and methionyl switches play an essential role in the regulation of cytoskeletal dynamics. GENERAL SIGNIFICANCE The dynamic remodeling of the cytoskeleton is controlled by various redox switches. These mechanisms are indispensible during development and organogenesis and might contribute to numerous pathological conditions. This article is part of a Special Issue entitled Redox regulation of differentiation and de-differentiation.

Identification of a dithiol-disulfide switch in collapsin response mediator protein 2 (CRMP2) that is toggled in a model of neuronal differentiation

Background: Cytosolic glutaredoxin 2 (Grx2) is essential for neuronal development in zebrafish; collapsin response mediator protein 2 (CRMP2) was identified as Grx2 substrate. Results: Oxidation of CRMP2 by hydrogen peroxide induces an intermolecular disulfide, changes in α-helical content, and hydrophobicity. Conclusion: The dithiol-disulfide redox switch defines two conformations of CRMP2. Significance: This switch may be functional during axonal outgrowth. Vertebrate-specific glutaredoxin 2 (Grx2) is expressed in at least two isoforms, mitochondrial Grx2a and cytosolic Grx2c. We have previously shown that cytosolic Grx2 is essential for embryonic development of the brain. In particular, we identified collapsin response mediator protein 2 (CRMP2/DPYSL2), a mediator of the semaphorin-plexin signaling pathway, as redox-regulated target of Grx2c and demonstrated that this regulation is required for normal axonal outgrowth. In this study, we demonstrate the molecular mechanism of this regulation, a specific and reversible intermolecular Cys-504-Cys-504 dithiol-disulfide switch in homotetrameric CRMP2. This switch determines two conformations of the quaternary CRMP2 complex that controls axonal outgrowth and thus neuronal development.