Formation of mixed disulfides between glutathione and the cysteines of some proteins (glutathionylation) has been suggested as a mechanism through which protein functions can be regulated by the redox status. The aim of this study was to identify the proteins of T cell blasts that undergo glutathionylation under oxidative stress. To this purpose, we radiolabeled cellular glutathione with 35 S, exposed T cells to oxidants (diamide or hydrogen peroxide), and performed nonreducing, two-dimensional electrophoresis followed by detection of labeled proteins by phosphorimaging and their identification by mass spectrometry techniques. We detected several proteins previously not recognized to be glutathionylated, including cytoskeletal proteins (vimentin, myosin, tropomyosin, cofilin, profilin, and the already known actin), enzymes (enolase, aldolase, 6-phosphogluconolactonase, adenylate kinase, ubiquitinconjugating enzyme, phosphoglycerate kinase, triosephosphate isomerase, and pyrophosphatase), redox enzymes (peroxiredoxin 1, protein disulfide isomerase, and cytochrome c oxidase), cyclophilin, stress proteins (HSP70 and HSP60), nucleophosmin, transgelin, galectin, and fatty acid binding protein. Based on the presence of several protein isoforms in control cells, we suggest that enolase and cyclophilin are heavily glutathionylated under basal conditions. We studied the effect of glutathionylation on some of the enzymes identified in the present study and found that some of them (enolase and 6-phosphogluconolactonase) are inhibited by glutathionylation, whereas the enzymatic activity of cyclophilin (peptidylprolyl isomerase) is not. These findings suggest that protein glutathionylation might be a common mechanism for the global regulation of protein functions.

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 redoxdependent 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.cysteine oxidation | thiol oxidation | redox proteomics

SummaryThioredoxin (Trx) is a ubiquitous intracellular protein disulfide oxidoreductase with a CXXC active site that can be released by various cell types upon activation. We show here that Trx is chemotactic for monocytes, polymorphonuclear leukocytes, and T lymphocytes, both in vitro in the standard micro Boyden chamber migration assay and in vivo in the mouse air pouch model. The potency of the chemotactic action of Trx for all leukocyte populations is in the nanomolar range, comparable with that of known chemokines. However, Trx does not increase intracellular Ca 2 ϩ and its activity is not inhibited by pertussis toxin. Thus, the chemotactic action of Trx differs from that of known chemokines in that it is G protein independent. Mutation of the active site cysteines resulted in loss of chemotactic activity, suggesting that the latter is mediated by the enzyme activity of Trx. Trx also accounted for part of the chemotactic activity released by human T lymphotropic virus (HTLV)-1-infected cells, which was inhibited by incubation with anti-Trx antibody. Since Trx production is induced by oxidants, it represents a link between oxidative stress and inflammation that is of particular interest because circulating Trx levels are elevated in inflammatory diseases and HIV infection.

Taken together, these in vivo and in vitro findings support a model whereby HIV encephalitis is sustained by virus replication in microglial cells, a process amplified by recruitment of mononuclear cells via HIV-induced MCP-1.

The cytokine erythropoietin (EPO) protects the heart from ischemic injury, in part by preventing apoptosis. However, EPO administration can also raise the hemoglobin concentration, which, by increasing oxygen delivery, confounds assignment of cause and effect. The availability of EPO analogs that do not bind to the dimeric EPO receptor and lack erythropoietic activity, e.g., carbamylated EPO (CEPO), provides an opportunity to determine whether EPO possesses direct cardioprotective activity. In vivo, cardiomyocyte loss after experimental myocardial infarction (MI) of rats (40 min of occlusion with reperfusion) was reduced from Ϸ57% in MI-control to Ϸ45% in animals that were administered CEPO daily for 1 week (50 g͞kg of body weight s.c.) with the first dose administered intravenously 5 min before reperfusion. CEPO did not increase the hematocrit, yet it prevented increases in left ventricular (LV) end-diastolic pressure, reduced LV wall stress in systole and diastole, and improved LV response to dobutamine infusion compared with vehicle-treated animals. In agreement with the cardioprotective effect observed in vivo, staurosporineinduced apoptosis of adult rat or mouse cardiomyocytes in vitro was also significantly attenuated (Ϸ35%) by CEPO, which is comparable with the effect of EPO. These data indicate that prevention of cardiomyocyte apoptosis, in the absence of an increase in hemoglobin concentration, explains EPO's cardioprotection. Nonerythropoietic derivatives such as CEPO, devoid of the undesirable effects of EPO, e.g., thrombogenesis, could represent safer and more effective alternatives for treatment of cardiovascular diseases, such as MI and heart failure. Furthermore, these findings expand the activity spectrum of CEPO to tissues outside the nervous system. apoptosis ͉ cardioprotection ͉ cytokine ͉ tissue injury E rythropoietin (EPO) protects the brain and the spinal cord from ischemic and traumatic injury (1, 2), the peripheral nerve from diabetic damage (3), the kidney from ischemic (4, 5) or toxic insults (6), and the heart from acute ischemia, either permanent (7-9) or with reperfusion (10). Current data suggest that the observed protective effects of EPO depend on an antiapoptotic effect of this cytokine (7, 10). In the brain, EPO also greatly reduces the inflammatory response after ischemiareperfusion (11). It is notable that in several acute models, e.g., brain ischemia, a single dose of EPO that does not increase the Hb concentration nevertheless confers neuroprotection. However, in other in vivo injury models, including cardiac ischemia with reperfusion and diabetic neuropathy, injury develops gradually, and multiple doses of EPO appear superior but also increase the Hb concentration. The possibility that part of the benefit obtained with EPO in these models may depend on the increased oxygen-carrying capacity of the blood cannot be excluded. However, cardiac protection has clearly been demonstrated after only a single dose (8, 10) when evaluated 1-3 days after infarction before any increase i...

Proteins can form reversible mixed disulfides with glutathione (GSH). It has been hypothesized that protein glutathionylation may represent a mechanism of redox regulation, in a fashion similar to that mediated by protein phosphorylation. We investigated whether GSH has a signaling role in the response of HL60 cells to hydrogen peroxide (H 2O2), in addition to its obvious antioxidant role. We identified early changes in gene expression induced at different times by H 2O2 treatment, under conditions that increase protein glutathionylation and minimal toxicity. We then investigated the effect of prior GSH depletion by buthionine sulfoximine and diethylmaleate on this response. The analysis revealed 2,016 genes regulated by H 2O2. Of these, 215 genes showed GSHdependent expression changes, classifiable into four clusters displaying down-or up-regulation by H 2O2, either potentiated or inhibited by GSH depletion. The modulation of 20 selected genes was validated by real-time RT-PCR. The biological process categories overrepresented in the largest cluster (genes whose upregulation was inhibited by GSH depletion) were NF-B activation, transcription, and DNA methylation. This cluster also included several cytokine and chemokine ligands and receptors, the redox regulator thioredoxin interacting protein, and the histone deacetylase sirtuin. The cluster of genes whose up-regulation was potentiated by GSH depletion included two HSPs (HSP40 and HSP70) and the AP-1 transcription factor components Fos and FosB. This work demonstrates that GSH, in addition to its antioxidant and protective function against oxidative stress, has a specific signaling role in redox regulation.chemokines ͉ cytokines ͉ hydrogen peroxide ͉ oxidative stress G lutathione (GSH) protects the cell from damage induced by high levels of reactive oxygen species (ROS), defined as oxidative stress (1). Its main antioxidant activity consists of the detoxification of peroxides, partly with the aid of various GSH peroxidases. Its role as a major thiol antioxidant is demonstrated by the fact that various conditions of oxidative stress are exacerbated by GSH depletion, for instance by inhibitors of its synthesis, and ameliorated by the addition of GSH or its precursors, including N-acetylcysteine (NAC). Experimentally, thiol antioxidants, including GSH and NAC, have been used as tools to investigate the role of ROS in biological systems.In addition to scavenging ROS, GSH can form mixed disulfides with proteins, a phenomenon known as protein glutathionylation (2-4). This can occur by various reactions, either by thiol͞disulfide exchange between protein cysteines and oxidized GSH (GSH disulfide, GSSG), by direct oxidation, or through the NO-mediated formation of S-nitrosothiols (3, 4).The effect of protein glutathionylation is generally considered deleterious in the framework of oxidative stress, because it is one of the many forms of thiol oxidation induced by ROS. However, according to the more recent concept of redox regulation, several protein cysteines can be d...

Immature plasmacytoid dendritic cells are the principal alpha interferon-producing cells (IPC), responsible for primary antiviral immunity. IPC express surface molecules CD4, CCR5, and CXCR4, which are known coreceptors required for human immunodeficiency virus (HIV) infection. Here we show that IPC are susceptible to and replicate HIV type 1 (HIV-1). Importantly, viral replication is triggered upon activation of IPC with CD40 ligand, a signal physiologically delivered by CD4 T cells. Immunohistochemical staining of tonsil from HIV-infected individuals reveals HIV p24+ IPC, consistent with in vivo infection of these cells. IPC exposed in vitro to HIV produce alpha interferon, which partially inhibits viral replication. Nevertheless, IPC efficiently transmit HIV-1 to CD4 T-cells, and such transmission is also augmented by CD40 ligand activation. IPC produce RANTES/CCL5 and MIP-1α/CCL3 when exposed to HIV in vitro. IPC also induce naïve CD4 T cells to proliferate and would therefore preferentially infect these cells. These results indicate that IPC may play an important role in the dissemination of HIV

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