Fast-reacting Thiols in Rat Hemoglobins Can Intercept Damaging Species in Erythrocytes More Efficiently Than Glutathione

Rossi, Ranieri; Barra, Donatella; Bellelli, Andrea; Boumis, G.; Canofeni, Silvia; Simplicio, P. Di; Lusini, Lorenzo; Pascarella, Stefano; Amiconi, Gino

doi:10.1074/jbc.273.30.19198

Cited by 64 publications

(54 citation statements)

References 48 publications

(34 reference statements)

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“…6). Only PSSG concentrations increased after diamide treatment, a finding that is consistent with previous findings in rat blood (14,22 ) and is attributable to the presence of highly reactive sulfhydryl groups in rat hemoglobin, which react first with diamide and then with GSH, forming PSSG.…”

Section: Measurement Of Gssg and Pssg To Reveal Oxidative Stresssupporting

confidence: 79%

Oxidized Forms of Glutathione in Peripheral Blood as Biomarkers of Oxidative Stress

et al. 2006

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Background: Reduced glutathione (GSH) and its redox forms, glutathione disulfide (GSSG) and glutathionylated proteins (PSSG), are biomarkers of oxidative stress, but methodologic artifacts can interfere with their measurement. We evaluated the importance of correct sample handling during the preanalytical phase for GSH, GSSG, and PSSG measurement. Methods: We used human blood for in vitro experiments with oxidants [tert-butylhydroperoxide (t-BOOH), diamide, and menadione]. For in vivo experiments, we used rats in which we cannulated the jugular and femoral veins for both oxidant administration and blood collection. We measured GSH, GSSG, and PSSG with HPLC with or without sample pretreatment with N-ethylmaleimide (NEM) to prevent artifacts. We also measured malondialdehyde (MDA) with HPLC, and protein carbonyls (PCO) with spectrophotometric procedures. Results: When methodologic artifacts were prevented by pretreatment with NEM, GSSG results increased up to 3-fold over the basal concentrations, even in the presence of 5 mol/L t-BOOH or diamide and 20 mol/L menadione. PSSG increased by ϳ50% at 20 mol/L t-BOOH or diamide and at 50 mol/L menadione. PCO and MDA remained unchanged. In vivo oxidation treatments elicited immediate and significant increases in GSSG and PSSG over basal values (up to 200-fold), whereas PCO and MDA showed only slight variation 120 or 180 min after treatment. Conclusions: With the use of artifact-free measurement methods, GSH, GSSG, and PSSG are potentially powerful and reliable biomarkers of oxidative stress status

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Section: Measurement Of Gssg and Pssg To Reveal Oxidative Stresssupporting

confidence: 79%

Oxidized Forms of Glutathione in Peripheral Blood as Biomarkers of Oxidative Stress

et al. 2006

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“…When the residue corresponding to Thr-102 is mutated to Ala in VHR (S131A) and PTP1 (S222A), remarkable decreases in activity were observed without affecting substrate binding and formation of the covalent catalytic intermediate (20,21). Thus, interaction of Cys-95 with OG1 of Thr-102 and also its exposure to solvent both appear important for its catalysis (19). In vitro, the active site cysteine undergoes reversible oxidation and inactivation via conversion of the cysteine thiolate to sulfenic acid (SOH).…”

Section: Resultsmentioning

confidence: 99%

“…Hydrogen bonding interaction and stabilization of the cysteinyl anion to OG/OG1 of Ser/Thr is likely to reduce the pK a of the thiol, thereby allowing it to participate in catalysis (19). When the residue corresponding to Thr-102 is mutated to Ala in VHR (S131A) and PTP1 (S222A), remarkable decreases in activity were observed without affecting substrate binding and formation of the covalent catalytic intermediate (20,21).…”

Section: Resultsmentioning

confidence: 99%

Structure of Human Dual Specificity Protein Phosphatase 23, VHZ, Enzyme-Substrate/Product Complex

Agarwal

Burley²,

Swaminathan

2008

Journal of Biological Chemistry

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Protein phosphorylation plays a crucial role in mitogenic signal transduction and regulation of cell growth and differentiation. Dual specificity protein phosphatase 23 (DUSP23) or VHZ mediates dephosphorylation of phospho-tyrosyl (pTyr) and phospho-seryl/threonyl (pSer/pThr) residues in specific proteins. In vitro, it can dephosphorylate p44ERK1 but not p54SAPK-␤ and enhance activation of c-Jun N-terminal kinase (JNK) and p38. Human VHZ, the smallest of the catalytically active protein-tyrosine phosphatases (PTP) reported to date (150 residues), is a class I Cys-based PTP and bears the distinctive active site signature motif HCXXGXXRS(T). We present the crystal structure of VHZ determined at 1.93 Å resolution. The polypeptide chain adopts the typical ␣␤␣ PTP fold, giving rise to a shallow active site cleft that supports dual phosphorylated substrate specificity. Within our crystals, the Thr-135-Tyr-136 from a symmetry-related molecule bind in the active site with a malate ion, where they mimic the phosphorylated TY motif of the MAPK activation loop in an enzyme-substrate/product complex. Analyses of intermolecular interactions between the enzyme and this pseudo substrate/product along with functional analysis of Phe-66, Leu-97, and Phe-99 residues provide insights into the mechanism of substrate binding and catalysis in VHZ.Protein phosphatases and kinases together help control a host of cellular functions (1). Reversible phosphorylation of tyrosine, serine, and threonine side chains represents a fundamental mode of cellular signal transduction, regulating cell growth and differentiation. Protein phosphatases, unlike the protein kinase superfamily, can be classified as members of separate superfamilies that are mechanistically and structurally distinct from one another (2). Based on the substrate specificity toward phospho-seryl/threonyl (pSer/pThr) 2 or tyrosyl residues (pTyr), they are divided into two families: protein serine/threonine phosphatases and protein-tyrosine phosphatases (PTPs). The so-called dual specificity phosphatases (DSPs) dephosphorylate both pTyr and pSer/pThr and belong to class I cysteine-based PTPs. Cys-based PTPs and DSPs share the conserved signature motif HCXXGXXRS(T) and exhibit similar mechanisms of catalysis, involving a two-step process that proceeds via formation of a cysteinyl-phosphate enzyme-substrate intermediate (3).Mitogen-activated protein kinase (MAPK) activation requires phosphorylation at threonine and tyrosine with a characteristic TY/TXY motif found within the enzyme activation loop. This reversible process is mediated by either MAPK phosphatases (MKPs) or dual specificity MKPs (DSPs) (4). DSPs control MAPK signaling via dephosphorylation and exhibit feedback regulation (5). There are ϳ65 human genes encoding various DSPs with low sequence similarity with respect to one another (2). The gene encoding dual specificity protein phosphatase 23 (DUSP23) is located on human chromosome 1q23 (6). DUSP23 (originally erroneously termed DUSP25) encodes a 150-residue protein tha...

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“…Conversely, rat Hb was shown to produce large amounts of Hb-SSG: after treatment with diamide, all GSH was bound to Hb. This is attributable to the peculiarity of rat Hb (25 ), which is characterized by a highly reactive cysteine in position ␤125, in addition to cysteine ␤93, which is common to most mammalian Hbs. In diabetes, where oxidative stress is increased, we have measured enhanced Hb-SSG (ϩ648%); in addition, we have also evaluated the correlation of obtained values with other indicators of oxidative stress, i.e., protein carbonyls, isoprostanes, and malondialdehyde.…”

Section: Clinical Chemistry 49 No 2 2003mentioning

confidence: 99%

Protein Glutathionylation in Erythrocytes

et al. 2003

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Signal ratios generated by wild-type probe reacted with PCR products from the control construct were all close to unity (i.e., there was no cross-reaction), as were those from wild-type PCR products reacted with the control sequence probe. End-point dilution of the control construct confirmed the spectrophotometric estimate of 10 11 RNA copies/L. Assay of the final construct for DNA copies showed that this contained ϳ10 5 DNA copies/mL (ϳ10 Ϫ6 of the RNA copies). It was intended that the RNA construct be added to the final assay at a concentrations of 100 RNA copies/reaction. At this dilution, the DNA contaminants would be diluted to Ͻ0.001 copies/reaction; thus it was decided not to risk the integrity of the RNA construct by the use of more vigorous DNase treatment.Quantitative molecular assays, particularly quantitative PCR, have become an important tool in medical research and routine clinical investigation, particularly for the assay of microbial genomes in clinical samples (9 ) and for the quantification of gene expression in mammalian cells (10 ). In such assay systems, accurate quantification depends on standardization and control of all steps in the protocol. In the case of quantitative PCR, this includes controls for nucleic acid extraction efficiency, amplification efficiency, and nonspecific inhibition of the reaction (11 ). Additionally, there is a need for accurately quantified internal standards with which to compare unknowns to obtain absolute nucleic acid copy numbers in the sample.The method described here allows molecular constructs for use as internal controls in quantitative molecular assays to be synthesized in a simple two-step PCR protocol. The simplicity of the procedure allows controls to be synthesized in laboratories that do not have facilities available for molecular biology procedures beyond PCR, such as diagnostic laboratories that use molecular detection methods. The method has been applied to the control of a quantitative PCR for MV RNA but could also be applied to the generation of DNA targets. Controls may be synthesized that are appropriate to any source of nucleic acid (e.g., mRNA in the quantification of microbial or mammalian gene expression) that is being detected or quantified by PCR or any other molecular method. Analysis of antioxidant molecules is potentially important to understanding the role of oxidative stress (1-6 ) in disease, as oxidative damage is accompanied or preceded by their depletion.Reduced glutathione (GSH) is ubiquitous and abundant; it can be oxidized to its disulfide form (GSSG) in response to an oxidative perturbation. Usually, however, this species is rapidly reduced by the action of glutathione reductase (7 ). If GSSG accumulates within the cell, it can create protein-glutathione adducts via thiol-disulfide exchange reactions. Thus, in addition to the ratio of GSH to GSSG, the content of glutathionylated proteins (GSSPs) can indicate oxidative stress. The analysis of GSSPs has potential advantages over measurements of GSH and GSSG because GSSPs ar...

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Fast-reacting Thiols in Rat Hemoglobins Can Intercept Damaging Species in Erythrocytes More Efficiently Than Glutathione

Cited by 64 publications

References 48 publications

Oxidized Forms of Glutathione in Peripheral Blood as Biomarkers of Oxidative Stress

Oxidized Forms of Glutathione in Peripheral Blood as Biomarkers of Oxidative Stress

Structure of Human Dual Specificity Protein Phosphatase 23, VHZ, Enzyme-Substrate/Product Complex

Protein Glutathionylation in Erythrocytes

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