Protein glutathionylation in erythrocytes.
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Biomedical subjects
Daniela Giustarini
Publications and source records attributed to Daniela Giustarini.
The pro-oxidant role of protein SH groups of hemoglobin in rat erythrocytes exposed to menadione.
Menadione is selectively toxic to erythrocytes. Although GSH is considered a primary target of menadione, intraerythrocyte thiolic alterations consequent to menadione exposure are only partially known. In this study alterations of GSH and protein thiols (PSH) and their relationship with methemoglobin formation were investigated in human and rat red blood cells (RBC) exposed to menadione. In both erythrocyte types, menadione caused a marked increase in methemoglobin associated with GSH depletion and increased oxygen consumption. However, in human RBC, GSH formed a conjugate with menadione, whereas, in rat RBC it was converted to GSSG, concomitantly with a loss of protein thiols (corresponding to menadione arylation), and an increase in glutathione-protein mixed disulfides (GS-SP). Such differences were related to the presence of highly reactive cysteines, which characterize rat hemoglobin (cys beta125). In spite of the greater thiol oxidation in rat than in human RBC, methemoglobin formation and the rate of oxygen consumption elicited by menadione in both species were rather similar. Moreover, in repeated experiments under N2 or CO-blocked heme, it was found that menadione conjugation (arylation) in both species was not dependent on the presence of oxygen or the status of heme. Therefore, we assumed that GSH (human RBC) and protein (rat RBC) arylation was equally responsible for increased oxygen consumption and Hb oxidation. Moreover, thiol oxidation of rat RBC was strictly related to methemoglobin formation.
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Blood glutathione disulfide: in vivo factor or in vitro artifact?
BACKGROUND: The reported mean concentration of glutathione disulfide (GSSG) in human blood/erythrocytes varies widely (1 to >500 micromol/L), as does that of reduced glutathione (GSH) to a lesser extent. We have identified and investigated possible pitfalls in measurement of both GSH and GSSG. METHODS: We measured GSH and GSSG using a spectrophotometer with a modification of the GSH recycling method; the same samples were also measured by reversed-phase HPLC after derivatization of thiols (dithiothreitol was used to reduce disulfides) with monobromobimane. The thiol-bimane adduct was measured by a fluorescence detector. RESULTS: Measured GSH/GSSG concentrations were affected by the following: (a) oxidation of thiols in acidified samples; (b) oxidation after restoring neutral-alkaline pH; (c) oxidation during acid deproteinization; (d) shift in the GSH/GSSG equilibrium because of irreversible blocking of free thiols; and (e) reaction of electrophiles with amino groups. In particular, oxidation during sample deproteinization with acid influenced and produced artifacts (30-150 micromol/L GSSG was produced by this procedure); this phenomenon was directly correlated with the presence of oxygenated hemoglobin, being minimized by both oxygen deprivation and incubation in an atmosphere of 5% carbon monoxide. CONCLUSIONS: GSSG is present in healthy human blood at low concentrations (2-6 micromol/L), and most published data on GSSG may be affected by artifacts.
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Proteins as biomarkers of oxidative/nitrosative stress in diseases: the contribution of redox proteomics.
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) contribute to the pathogenesis and/or progression of several human diseases. Proteins are important molecular signposts of oxidative/nitrosative damage. However, it is generally unresolved whether the presence of oxidatively/nitrosatively modified proteins has a causal role or simply reflects secondary epiphenomena. Only direct identification and characterization of the modified protein(s) in a given pathophysiological condition can decipher the potential roles played by ROS/RNS-induced protein modifications. During the last few years, mass spectrometry (MS)-based technologies have contributed in a significant way to foster a better understanding of disease processes. The study of oxidative/nitrosative modifications, investigated by redox proteomics, is contributing to establish a relationship between pathological hallmarks of disease and protein structural and functional abnormalities. MS-based technologies promise a contribution in a new era of molecular medicine, especially in the discovery of diagnostic biomarkers of oxidative/nitrosative stress, enabling early detection of diseases. Indeed, identification and characterization of oxidatively/nitrosatively modified proteins in human diseases has just begun.
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S-nitrosation versus S-glutathionylation of protein sulfhydryl groups by S-nitrosoglutathione.
S-Nitrosation of protein sulfhydryl groups is an established response to oxidative/nitrosative stress. The transient nature and reversibility of S-nitrosation, as well as its specificity, render this posttranslational modification an attractive mechanism of regulation of protein function and signal transduction, in analogy to S-glutathionylation. Several feasible mechanisms for protein S-nitrosation have been proposed, including transnitrosation by S-nitrosothiols, such as S-nitrosoglutathione (GSNO), where the nitrosonium moiety is directly transferred from one thiol to another. The reaction between GSNO and protein sulfhydryls can also produce a mixed disulfide by S-glutathionylation, which involves the nucleophilic attack of the sulfur of GSNO by the protein thiolate anion. In this study, we have investigated the possible occurrence of S-glutathionylation during reaction of GSNO with papain, creatine phosphokinase, glyceraldehyde-3-phosphate dehydrogenase, alcohol dehydrogenase, bovine serum albumin, and actin. Our results show that papain, creatine phosphokinase, and glyceraldehyde-3-phosphate dehydrogenase were significantly both S-nitrosated and S-glutathionylated by GSNO, whereas alcohol dehydrogenase, bovine serum albumin, and actin appeared nearly only S-nitrosated. The susceptibility of the modified proteins to denitrosation and deglutathionylation by reduced glutathione was also investigated.
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S-glutathionylation: from redox regulation of protein functions to human diseases.
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) play an integral role in the modulation of several physiological functions but can also be potentially destructive if produced in excessive amounts. Protein cysteinyl thiols appear especially sensitive to ROS/RNS attack. Experimental evidence started to accumulate recently, documenting that S-glutathionylation occurs in a number of physiologically relevant situations, where it can produce discrete modulatory effects on protein function. The increasing evidence of functional changes resulting from this modification, and the growing number of proteins shown to be S-glutathionylated both in vitro and in vivo support this contention, and confirm this as an attractive area of research. S-glutathionylated proteins are now actively investigated with reference to problems of biological interest and as possible biomarkers of human diseases associated with oxidative/nitrosative stress.
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