Erythropoietin (EPO) promotes neuronal survival after hypoxia and other metabolic insults by largely unknown mechanisms. Apoptosis and necrosis have been proposed as mechanisms of cellular demise, and either could be the target of actions of EPO. This study evaluates whether antiapoptotic mechanisms can account for the neuroprotective actions of EPO. Systemic administration of EPO (5,000 units͞kg of body weight, i.p.) after middle-cerebral artery occlusion in rats dramatically reduces the volume of infarction 24 h later, in concert with an almost complete reduction in the number of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling of neurons within the ischemic penumbra. In both pure and mixed neuronal cultures, EPO (0.1-10 units͞ml) also inhibits apoptosis induced by serum deprivation or kainic acid exposure. Protection requires pretreatment, consistent with the induction of a gene expression program, and is sustained for 3 days without the continued presence of EPO. EPO (0.3 units͞ml) also protects hippocampal neurons against hypoxia-induced neuronal death through activation of extracellular signal-regulated kinases and protein kinase Akt-1͞protein kinase B. The action of EPO is not limited to directly promoting cell survival, as EPO is trophic but not mitogenic in cultured neuronal cells. These data suggest that inhibition of neuronal apoptosis underlies short latency protective effects of EPO after cerebral ischemia and other brain injuries. The neurotrophic actions suggest there may be longer-latency effects as well. Evaluation of EPO, a compound established as clinically safe, as neuroprotective therapy in acute brain injury is further supported. E rythropoietin (EPO) was first characterized as a hematopoietic growth factor (1) and has been in clinical use by millions of patients over the last decade for the treatment of anemia. The observation that EPO and its receptor are expressed in rodent and human brain tissue (2-4), as well as by cultured neurons (5-8) and astrocytes (3,7,9), and that EPO has effects on neuronal cells (5), expanded the biological role of EPO beyond hematopoiesis. EPO gene expression in the brain is regulated by hypoxia-inducible factor-1 (1) that is activated by a variety of stressors, including hypoxia. Several independent research groups have reported that EPO protects cultured neurons against glutamate toxicity (6, 10) and reduces ischemic neuronal damage and neurological dysfunction in rodent models of stroke (6,(11)(12)(13). We recently reported that systemic administration of EPO is neuroprotective not only in animal models of cerebral ischemia, but also for mechanical trauma, excitotoxins, and neuroinflammation (11). Marked changes in EPO and EPOreceptor (EPOR) gene expression have been reported to occur in brain tissue after ischemic injury (6, 12). Specificity and biological relevance of these changes have been demonstrated by the observation that neutralization of endogenous EPO with soluble EPOR augments ischemic brain damage (13). Thus, it seems that E...

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.

Erythropoietin (EPO) is a tissue-protective cytokine preventing vascular spasm, apoptosis, and inflammatory responses. Although best known for its role in hematopoietic lineages, EPO also affects other tissues, including those of the nervous system. Enthusiasm for recombinant human erythropoietin (rhEPO) as a potential neuroprotective therapeutic must be tempered, however, by the knowledge it also enlarges circulating red cell mass and increases platelet aggregability. Here we examined whether erythropoietic and tissue-protective activities of rhEPO might be dissociated by a variation of the molecule. We demonstrate that asialoerythropoietin (asialoEPO), generated by total enzymatic desialylation of rhEPO, possesses a very short plasma half-life and is fully neuroprotective. In marked contrast with rhEPO, this molecule at doses and frequencies at which rhEPO exhibited erythropoiesis, did not increase the hematocrit of mice or rats. AsialoEPO appeared promptly within the cerebrospinal fluid after i.v. administration; intravenously administered radioiodine-labeled asialoEPO bound to neurons within the hippocampus and cortex in a pattern corresponding to the distribution of the EPO receptor. Most importantly, asialoEPO exhibits a broad spectrum of neuroprotective activities, as demonstrated in models of cerebral ischemia, spinal cord compression, and sciatic nerve crush. These data suggest that nonerythropoietic variants of rhEPO can cross the blood–brain barrier and provide neuroprotection.

To identify proteins undergoing glutathionylation (formation of protein-glutathione mixed disulfides) in human T cell blasts, we radiolabeled the glutathione pool with 35 S, exposed cells to the oxidant diamide, and analyzed cellular proteins by two-dimensional electrophoresis. One of the proteins undergoing glutathionylation was identified by molecular weight, isoelectric point, and immunoblotting as thioredoxin (Trx). Incubation of recombinant human Trx with glutathione disulfide or S-nitrosoglutathione led to the formation of glutathionylated Trx, identified by matrixassisted laser desorption ionization-time-of-flight mass spectrometry. The glutathionylation site was identified as Cys-72. Glutathionylation of rhTrx abolished its enzymatic activity as insulin disulfide reductase in the presence of NADPH and Trx reductase. Activity was, however, regained with sigmoidal kinetics, indicating a process of autoactivation due to the ability of Trx to deglutathionylate itself. These data suggest that the intracellular glutathione͞glutathione disulfide ratio, an indicator of the redox state of the cell, can regulate Trx functions reversibly through thiol-disulfide exchange reactions. T hioredoxin (Trx; ref. 1), a ubiquitous redox protein, is an essential cofactor electron donor for ribonucleotide reductase, but also has many other cellular functions, including regulation of transcription factors, apoptosis, and antioxidant activity and can act exogenously as a redox active growth factor (1, 2). The catalytic activity of Trx resides in its active site where the two redox active Cyss (Cys-31 and Cys-34 in human Trx) undergo reversible oxidation͞reduction. In addition to the conserved Cys residues in the active site, three additional structural Cys residues (Cys-61, Cys-68, and Cys-72) are present in the structure of human Trx.The present paper describes the experiments that led us to the conclusion that Trx can undergo glutathionylation in T cells exposed to oxidative stress. Protein glutathionylation, the formation of a disulfide between a Cys in a protein and the Cys in the tripeptide glutathione (GSH), is a modification that can be induced in cells by oxidative stress. Glutathionylation can occur by direct oxidation of a protein and GSH, by a thiol-disulfide exchange between a protein Cys and oxidized glutathione (GSSG), and also with the intermediacy of S-nitrosoglutathione (GSNO; refs. 3 and 4). Glutathionylation of proteins is reversible, as those proteins can be reduced by glutaredoxins (5, 6), and the process serves to regulate protein functions by the redox state of the cell (i.e., by the GSSG͞GSH ratio; refs. 7-9).To demonstrate the glutathionylation of Trx, we first labeled the intracellular GSH pool of the T-cell blasts with [ 35 S]Cys and analyzed the cell lysate by two dimensional electrophoresis under nonreducing conditions. A spot was found in the autoradiographic protein map with IP͞M r corresponding to those of Trx. In the second part of the study, we incubated recombinant human Trx in the presence or ...

Protein glutathionylation is a post-translational modification consisting of the formation of a mixed disulfide between protein cysteines and glutathione (GSH). To identify proteins undergoing glutathionylation in primary rat hepatocytes and in human HepG2 hepatoma cells, we radiolabeled the intracellular GSH pool with L-[(35)S] cysteine. Cells were then exposed to oxidative stress. Proteins were separated by two-dimensional gel electrophoresis under nonreducing conditions, and glutathionylated proteins were located by autoradiography and identified by mass spectrometry after tryptic digestion. Several proteins previously not known to undergo glutathionylation were thus recognized. Among the identified proteins some are the same or belong to the same functional class as those we have already identified in a previous paper on T cell blasts (actin, nucleophosmin, phosphogluconolactonase, myosin, profilin, cyclophilin A, stress 70 protein, ubiquitin in HepG2 cells and actin, peroxiredoxin 5, cytochrome C oxidase, heat shock cognate 70 in hepatocytes) while others are newly recognized (Ran specific GTPase activating protein, histidine triad nucleotide binding protein 2 in HepG2 cells and enoyl CoA hydratase in hepatocytes). The technique described proved equally applicable to a variety of cell types.

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We investigated the in vitro metabolism of two (nitrooxy)butyl ester nitric oxide (NO) donor derivatives of flurbiprofen and ferulic acid, [1,1Ј-biphenyl]-4-acetic acid-2-fluoro-␣-methyl-4-(nitrooxy)butyl ester (HCT 1026) and 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid 4-(nitrooxy)butyl ester (NCX 2057), respectively, in rat blood plasma and liver subcellular fractions compared with (nitrooxy)butyl alcohol (NOBA) and glyceryl trinitrate (GTN). HCT 1026 and NCX 2057 undergo rapid ubiquitous carboxyl ester hydrolysis to their respective parent compounds and NOBA. The nitrate moiety of this latter is subsequently metabolized to inorganic nitrogen oxides (NOx), predominantly in liver cytosol by glutathione S-transferase (GST) and to a lesser extent in liver mitochondria. If, however, in liver cytosol, the carboxyl ester hydrolysis is prevented by an esterase inhibitor, the metabolism at the nitrate moiety level does not occur.In blood plasma, HCT 1026 and NCX 2057 are not metabolized to NOx, whereas a slow but sustained NO generation in deoxygenated whole blood as detected by electron paramagnetic resonance indicates the involvement of erythrocytes in the bioactivation of these compounds. Differently from NOBA, GTN is also metabolized in blood plasma and more quickly metabolized by different GST isoforms in liver cytosol. The cytosolic GST-mediated denitration of these organic nitrates in liver limits their interaction with other intracellular compartments to possible generation of NO and/or their subsequent availability and bioactivation in the systemic circulation and extrahepatic tissues. We show the possibility of modulating the activity of hepatic cytosolic enzymes involved in the metabolism of (nitrooxy)butyl ester compounds, thus increasing the therapeutic potential of this class of compounds.The therapeutic potential of organic nitrates has been known for more than 120 years since the use of glyceryl trinitrate (GTN) in the treatment of angina pectoris. Moreover, the pharmaceutical development of organic nitrates containing adjunct pharmacophores was reported over 40 years ago, and these were observed to manifest biological properties beyond those of the parent compound (Hodosan et al., 1969). However, there has been an explosion of activity in the area of hybrid nitrates over the past decade, stimulated by a growing realization that nitrates may represent new therapeutic agents in different areas (Keeble and Moore, 2002). Despite this, the metabolism of several organic nitrates is still to be elucidated. The exact mechanism whereby nitric oxide (NO) is generated from organic nitrates is still unknown, and several mechanisms have been proposed and rejected. Although nonenzymatic pathways involving endogenous sulfhydryl groups (Ignarro et al., 1980) or hemoglobin (Bennett et al., 1986;Cosby et al., 2003) have been suggested to mediate the biotransformation to NO, the attention has also shifted to an enzyme-catalyzed mechanism (Needleman, 1976). Several enzymes have been proposed to be directly or i...

Using redox proteomics techniques to characterize the thiol status of proteins in human T lymphocytes, we identified cyclophilin A (CypA) as a specifically oxidized protein early after mitogen activation. CypA is an abundantly expressed cytosolic protein, target of the immunosuppressive drug cyclosporin A (CsA), for which a variety of functions has been described. In this study, we could identify CypA as a protein undergoing glutathionylation in vivo. Using MALDI-MS we identified Cys52 and Cys62 as targets of glutathionylation in T lymphocytes, and, using bioinformatic tools, we defined the reasons for the susceptibility of these residues to the modification. In addition, we found by circular dichroism spectroscopy that glutathionylation has an important impact on the secondary structure of CypA. Finally, we suggest that glutathionylation of CypA may have biological implications and that CypA may play a key role in redox regulation of immunity.