Changes of cell-surface thiols and intracellular signaling in human monocytic cell line THP-1 treated with diphenylcyclopropenone

Changes of cell-surface thiols induced by chemical treatment may affect the conformations of membrane proteins and intracellular signaling mechanisms. In our previous study, we found that a non-toxic dose of diphenylcyclopropene (DPCP), which is a potent skin sensitizer, induced an increase of cell-surface thiols in cells of a human monocytic cell line, THP-1. Here, we examined the influence of DPCP on intracellular signaling. First, we confirmed that DPCP induced an increase of cell-surface thiols not only in THP-1 cells, but also in primary monocytes. The intracellular reduced-form glutathione/oxidized-form glutathione ratio (GSH/GSSG ratio) was not affected by DPCP treatment. By means of labeling with a membrane-impermeable thiol-reactive compound, Alexa Fluor 488 C5 maleimide (AFM), followed by two-dimensional gel electrophoresis and analysis by liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS), we identified several proteins whose thiol contents were modified in response to DPCP. These proteins included cell membrane components, such as actin and β-tubulin, molecular chaperones, such as heat shock protein 27A and 70, and endoplasmic reticulum (ER) stress-inducible proteins. Next, we confirmed the expression in DPCP-treated cells of spliced XBP1, a known marker of ER stress. We also detected the phosphorylation of SAPK/JNK and p38 MAPK, which are downstream signaling molecules in the IRE1α-ASK1 pathway, which is activated by ER stress. These data suggested that increase of cell-surface thiols might be associated with activation of ER stress-mediated signaling.     

N-acetyl-p-benzoquinone imine-induced protein thiol modification in isolated rat hepatocytes.

Incubation of isolated rat hepatocytes with N-acetyl-p-benzoquinone imine (NAPQI) or 3,5-dimethyl-N-acetyl-p-benzoquinone imine (3,5-Me2-NAPQI) resulted in a concentration-dependent decrease in the protein thiol content of the mitochondrial, cytosolic and microsomal fractions. On a concentration basis, 3,5-Me2-NAPQI induced a more marked depletion of protein thiols than did NAPQI. Sodium dodecyl sulphate-polyacrylamide gel electrophoretic separation of the proteins of each fraction showed that different proteins had different susceptibilities to modification of their cysteine residues by the quinone imines. A few protein bands showed a decreased protein thiol content following incubation with non-toxic concentrations of quinone imines, whereas other proteins were affected by higher concentrations. Concentrations of quinone imines that were highly cytotoxic induced a general loss of protein thiols. NAPQI-induced protein thiol depletion occurred within 5 min and remained essentially unchanged for at least 30 min. In contrast, protein thiol depletion induced by 3,5-Me2-NAPQI increased over the 30-min time course of the experiment. Toxic concentrations of 3,5-Me2-NAPQI caused the formation of high molecular mass aggregates in all three subcellular fractions after 30 min of incubation. The observed crosslinking was not due to protein disulfide formation. However, no aggregate formation was observed after exposure of hepatocytes to NAPQI. One of the major target proteins of quinone imine-induced protein thiol depletion was a 17 kDa microsomal protein that was identified as the microsomal glutathione S-transferase. Exposure of hepatocytes and isolated liver microsomes to the quinone imines resulted in an up to four-fold increase in the specific activity of the microsomal glutathione S-transferase. In conclusion, our results are consistent with the suggestion of a critical role of protein thiol depletion in quinone imine-induced cytotoxicity.     

Critical role of sulfenic acid formation of thiols in the inactivation of glyceraldehyde-3-phosphate dehydrogenase by nitric oxide.

The relationship between possible modifications of the thiol groups of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by nitric oxide (NO) and modified enzyme activity was examined. There are 16 free thiols, including 4 active site thiols, in a tetramer of GAPDH molecule. NO donors, sodium nitroprusside (SNP), and S-nitroso-N-acetyl-DL-penicillamine (SNAP) decreased the number of free thiols with a concomitant inhibition of GAPDH activity in a concentration- and time-dependent manner. After treatment for 30 min, free thiols were maximally decreased to 8-10 per GAPDH tetramer and enzyme activity was also inhibited to 5-10% of control activity. In the presence of 30 mM dithiothreitol (DTT), these effects were completely blocked. Since similar results were obtained in the case of hydrogen peroxide (H2O2) treatment, which is known to oxidize the thiols, these effects of nitric oxide donors were probably due to modification of thiol groups present in a GAPDH molecule. On the other hand, DTT posttreatment after the treatment of GAPDH with SNP, SNAP, or H2O2 did not completely restore the modified thiols and the inhibited enzyme activity. DTT posttreatment after the 30-min-treatment with these agents restored free thiols to 14 in all treatments. In the case of SNAP treatment, all 4 active sites were restored and enzyme activity reached more than 80% of the control activity, but in two other cases one active site remained modified and enzyme activity was restored to about only 20%. Therefore, all 4 free thiols in the active site seem to be very important for full enzyme activity. DTT posttreatment in the presence of sodium arsenite, which is known to reduce sulfenic acid to thiol, almost completely restored both thiol groups and enzyme activity. These findings suggest that nitric oxide inhibits GAPDH activity by modifications of the thiols which are essential for this activity, and that the modification includes formation of sulfenic acid, which is not restored by DTT. S-nitrosylation, which is one type of thiol modification by NO, occurred when GAPDH was treated with SNAP but not SNP. Analysis of thiol modification showed that SNAP preferentially nitrosylated the active site thiols, the nitrosylation of which fully disappeared by DTT posttreatment. It seems that SNAP nitrosylates the active site thiols of GAPDH to prevent these thiols from oxidizing to sulfenic acid.     

蛋白质巯基及其氧化性修饰的化学检测方法

氧化还原信号转导, 尤其是蛋白质巯基的氧化性修饰, 与人类许多重要疾病关系密切, 是目前国际上热点研究领域之一。然而长期以来, 蛋白质巯基及其氧化修饰的专一、快速检测手段缺乏, 尤其是在活细胞内  源性检测方面几乎是空白, 限制了该领域的发展。本文就近年来用于检测蛋白质巯基及其氧化修饰的化学方法作简要综述, 并就基于巯基修饰发展荧光分子探针及其用于蛋白质组学研究, 氧化还原相关疾病早期诊断作一展望。     

Cytosolic and nuclear protein targets of thiol-reactive electrophiles

Reactive electrophiles formed from toxic drugs and chemicals and by endogenous oxidative stress covalently modify proteins. Although protein covalent binding is thought to initiate a variety of adaptive and toxic responses, the identities of the protein targets are generally unknown, as are protein structural features that confer susceptibility to modification. We have analyzed the protein targets in nuclear and cytoplasmic proteomes from HEK293 cells treated in vitro with two biotin-tagged, thiol-reactive electrophiles, (+)-biotinyl-iodoacetamidyl-3, 6-dioxaoctanediamine (PEO-IAB) and 1-biotinamido-4-(4'-[maleimidoethylcyclohexane]-carboxamido)butane (BMCC). Biotinylated peptides were captured by affinity enrichment using neutravidin beads, and the adducted peptides were then analyzed by multidimensional liquid chromatography-tandem mass spectrometry. A total of 897 adducts were mapped to different cysteine residues in 539 proteins. Adduction was selective and reproducible, and > 90% of all adducted proteins were modified at only one or two sites. A core group of 125 cysteines (14% of the total) was consistently modified by both electrophiles. Selective modification of several protein domain structures and motifs indicates that certain protein families are particularly susceptible to alkylation. This approach can be extended to studies of other protein-damaging oxidants and electrophiles and can provide new insights into targets and consequences of protein damage in toxicity and disease.     

Identification of creatine kinase and alpha-1 antitrypsin as protein targets of alkylation by sulfur mustard

Sulfur mustard (SM) is a toxic chemical warfare agent deployed in several conflicts within the last 100 years and still represents a threat in terroristic attacks and warfare. SM research focuses on understanding the pathophysiology of SM and identifying novel biomarkers of exposure. SM is known to alkylate nucleophilic moieties of endogenous proteins, for example, free thiol groups of cysteine residues. The two-dimensional-thiol-differences in gel electrophoresis (2D-thiol-DIGE) technique is an initial proteomics approach to detect proteins with free cysteine residues. These amino acids are selectively labeled with infrared-maleimide dyes visualized after GE. Cysteine residues derivatized by alkylating agents are no longer accessible for the maleimide–thiol coupling resulting in the loss of the fluorescent signal of the corresponding protein. To prove the applicability of 2D-thiol-DIGE, this technology was exemplarily applied to neat human serum albumin treated with SM, to lysates from human cell culture exposed to SM as well as to human plasma exposed to CEES (chloroethyl ethyl sulfide, an SM analogue). Exemplarily, the most prominent proteins modified by SM were identified by matrix-assisted laser desorption/ionization time-of-flight (tandem) mass spectrometry, MALDI-TOF MS(/MS), as creatine kinase (CK) from human cells and as alpha-1 antitrypsin (A1AT) from plasma samples. Peptides containing the residue Cys²⁸² of CK and Cys²³² of A1AT were unambiguously identified by micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (μLC-ESI MS/HR MS) as being alkylated by SM bearing the specific hydroxyethylthioethyl-(HETE)-moiety. Both peptides might represent potential biomarkers of SM exposure. This is the first report introducing these endogenous proteins as targets of SM alkylation.     

Disruption of glutathione homeostasis causes accumulation of S-glutathionyl proteins in response to exposure to reactive oxygen species in human erythrocytes

Oxidation of protein cysteine residues by disulfide bond formation with glutathione (GSH) is a reversible posttranslational modification following oxidative stress. Although S-glutathionylation seems to play a key role in cellular regulation and protect protein thiols from hyperoxidation, the molecular mechanism that mediates the glutathionyl protein is still unclear. We investigated the effect of disrupting GSH homeostasis on the S-glutathionylation of proteins via exposure to tert-butyl hydroperoxide (BHP) to study the formation of glutathionyl protein in human red blood cells (RBCs). Two independent treatments aimed at disrupting GSH homeostasis were devised to examine the influences of S-glutathionylation on RBC proteins. Glutathionyl proteins were detected transiently in intact RBCs during BHP exposure. Although glutathionyl proteins (220-240 kDa) disappeared immediately in the presence of glucose, they remained for a long time after BHP exposure in RBCs when the GSH-dependent system was disrupted. Furthermore, we identified that the high molecular weight glutathionyl protein is erythroid spectrin using immunodetection. Thus, it was indicated that the protein-bound GSH produced by peroxide exposure is immediately released by the GSH-dependent system in normal RBCs. However, disruption of GSH homeostasis causes accumulation of the glutathionyl protein. We propose that S-glutathionyl spectrin may be a useful biomarker for dysfunctions in GSH homeostasis and oxidative stress in human RBCs.     

Oxidation of proximal protein sulfhydryls by phenanthraquinone, a component of diesel exhaust particles

Diesel exhaust particles (DEP) contain quinones that are capable of catalyzing the generation of reactive oxygen species in biological systems, resulting in induction of oxidative stress. In the present study, we explored sulfhydryl oxidation by phenanthraquinone, a component of DEP, using thiol compounds and protein preparations. Phenanthraquinone reacted readily with dithiol compounds such as dithiothreitol (DTT), 2,3-dimercapto-1-propanol (BAL), and 2,3-dimercapto-1-propanesulfonic acid (DMPS), resulting in modification of the thiol groups, whereas minimal reactivities of this quinone with monothiol compounds such as GSH, 2-mercaptoethanol, and N-acetyl-L-cysteine were seen. The modification of DTT dithiol caused by phenanthraquinone proceeded under anaerobic conditions but was accelerated by molecular oxygen. Phenanthraquinone was also capable of modifying thiol groups in pulmonary microsomes from rats and total membrane preparation isolated from bovine aortic endothelial cells (BAEC), but not bovine serum albumin (BSA), which has a Cys34 as a reactive monothiol group. A comparison of the thiol alkylating agent N-ethylmaleimide (NEM) with that of phenanthraquinone indicates that the two mechanisms of thiol modification are distinct. Studies revealed that thiyl radical intermediates and reactive oxygen species were generated during interaction of phenanthraquinone with DTT. From these findings, it is suggested that phenanthraquinone-mediated destruction of protein sulfhydryls appears to involve the oxidation of presumably proximal thiols and the reduction of molecular oxygen.     

Oxidation and inactivation of SERCA by selective reaction of cysteine residues with amino acid peroxides

The oxidative modification of proteins plays an important role in a wide range of pathological processes and aging. Proteins are modified by numerous biologic oxidants including hydrogen peroxide, peroxynitrite, singlet oxygen, and oxygen- and nitrogen-centered radicals. More recently, an additional class of physiologically important oxidants has been identified, peptide and protein peroxides. The latter react quite rapidly and selectively with protein cysteine residues. The sarco/endoplasmic reticulum Ca-ATPase (SERCA) is reversibly regulated through NO-dependent S-glutathiolation of specific cysteine residues. The irreversible oxidation of these cysteine residues could, therefore, impair NO-dependent muscle relaxation. Here, we show that specific protein-derived (amino acid) peroxides react selectively with a subset of the 22 reduced cysteine residues of SERCA1, including a peptide-containing Cys674 and Cys675, where Cys674 (in SERCA2) represents one of the targets for NO-dependent S-glutathiolation. Out of 11 tested amino acid, peptide, and protein peroxides, those derived from free tryptophan and free tyrosine showed the highest reactivity towards SERCA, while no oxidation under similar experimental conditions was detected through hydrogen peroxide. Among the peroxides from tryptophan, those of free tryptophan showed a significantly higher reactivity as compared to those from N- and C-terminally blocked tryptophan. Quantitative HPLC-MS/MS analysis demonstrated that the highest reactivity of the tryptophan-derived peroxides was observed for Cys774 and Cys938, cysteine residues, which are embedded within the transmembrane domains of SERCA1. This unusual reactivity of transmembrane domains cannot be solely rationalized by the hydrophobicity of the oxidant, as the peroxide from dl-tryptophan shows considerable higher reactivity as compared to the one derived from N-acetyl-tryptophan methyl ester. Our data demonstrate a potential role of peptide- and protein-derived peroxides as important mediators of oxidative stress in vivo, which may cause a selective oxidation of Cys residues leading to inactivation of membrane proteins.     

In vivo detection and characterization of protein adducts resulting from bioactivation of haloethene cysteine S-conjugates by 19F NMR: chlorotrifluoroethene and tetrafluoroethene.

Several haloalkenes are selective nephrotoxins. The bioactivation of nephrotoxic haloalkenes involves hepatic glutathione S-conjugate formation, peptidase-catalyzed metabolism of the glutathione S-conjugates to the corresponding cysteine S-conjugates, uptake of cysteine S-conjugates by the kidneys, and renal cysteine conjugate beta-lyase-catalyzed beta-elimination of a thiol. The haloalkyl and haloalkenyl thiols thus released are unstable and yield reactive intermediates whose interactions with cellular constituents are though to contribute to the observed toxicity of S-conjugates. Tetrafluoroethene and chlorotrifluoroethene are metabolized to the cysteine S-conjugates S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC) and S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFC), respectively. Administration of TFEC (1.0 mmol/kg) or CTFC (1.0 mmol/kg) to rats resulted in acylation of renal proteins, as demonstrated with 19F nuclear magnetic resonance spectroscopy. Single, broad resonances near 41 or 56 ppm were found in spectra of renal proteins from TFEC- or CTFC-treated rats, respectively, and these resonances were not lost on dialysis. Renal protein incubated with 2-chloro-1,1,2-trifluoroethyl-2-nitrophenyl disulfide, a proreactive intermediate that yields 2-chloro-1,1,2-trifluoroethanethiol, showed the same 19F NMR spectrum as was found with CTFC-treated rats. In vitro incubation of various N alpha-blocked amino acids with this proreactive intermediate indicated that only lysine is stably adducted, whereas histidine is transiently acylated. In each case, proteolysis of modified protein converted a single broad NMR resonance to a doublet with little change in chemical shift and with clearly resolved, characteristic H-F couplings. The single, stable amino acid adduct formed with renal proteins of rats given CTFC or TFEC was N epsilon-(chlorofluorothioacetyl)lysine and N epsilon-(difluorothioacetyl)lysine, respectively.     

Redox in redux: Emergent roles for glutathione S-transferase P (GSTP) in regulation of cell signaling and S-glutathionylation

Glutathione (GSH) provides a major source of thiol homeostasis critical to the maintenance of a reduced cellular environment that is conducive to cell survival. Mammals have accumulated a significant cadre of sulfur containing proteins, the interactive significance of which has become clear in recent times. Glutathione transferases (GST) are prevalent in eukaryotes and have been ascribed catalytic functions that involve detoxification of electrophiles through thioether bond formation with the cysteine thiol of GSH. The neutralizing impact of these reactions on products of reactive oxygen has contributed to the significant evolutionary conservation and adaptive functional redundancy of the multifaceted GSH system. Amongst the GSTs, GSTP has been implicated in tumorigenesis and in anticancer drug resistance. Emerging studies indicate that GSTP has ligand binding properties and contributes in the regulation of signaling kinases through direct protein:protein interactions. Furthermore, S-glutathionylation is a post-translational modification of low pK(a) cysteine residues in target proteins. The forward rate of the S-glutathionylation reaction can be influenced by GSTP, whereas the reverse rate is affected by a number of redox sensitive proteins including glutaredoxin, thioredoxin and sulfiredoxin. The functional importance of these reactions in governing how cells respond to oxidative or nitrosative stress exemplifies the broad importance of GSH/GST homeostasis in conditions such as cancer, ageing and neurodegenerative diseases. GSTP has also provided a platform for therapeutic drug development where some agents have completed preclinical testing and are in clinical trial for the management of cancer.     

Deciphering Subtype-Selective Modulations in TRPA1 Biosensor Channels

The transient receptor potential (TRP) proteins are a family of ion channels that act as cellular sensors. Several members of the TRP family are sensitive to oxidative stress mediators. Among them, TRPA1 is remarkably susceptible to various oxidants, and is known to mediate neuropathic pain and respiratory, vascular and gastrointestinal functions, making TRPA1 an attractive therapeutic target. Recent studies have revealed a number of modulators (both activators and inhibitors) that act on TRPA1. Endogenous mediators of oxidative stress and exogenous electrophiles activate TRPA1 through oxidative modification of cysteine residues. Non-electrophilic compounds also activate TRPA1. Certain non-electrophilic modulators may act on critical non-cysteine sites in TRPA1. However, a method to achieve selective modulation of TRPA1 by small molecules has not yet been established. More recently, we found that a novel N-nitrosamine compound activates TRPA1 by S-nitrosylation (the addition of a nitric oxide (NO) group to cysteine thiol), and does so with significant selectivity over other NO-sensitive TRP channels. It is proposed that this subtype selectivity is conferred through synergistic effects of electrophilic cysteine transnitrosylation and molecular recognition of the non-electrophilic moiety on the N-nitrosamine. In this review, we describe the molecular pharmacology of these TRPA1 modulators and discuss their modulatory mechanisms.     

Mitochondrial protein thiol modifications in acetaminophen hepatotoxicity: Effect on HMG-CoA synthase

Acetaminophen (APAP) overdose is the leading cause of drug related liver failure in many countries. N-acetyl-p-benzoquinone imine (NAPQI) is a reactive metabolite that is formed by the metabolism of APAP. NAPQI preferentially binds to glutathione and then cellular proteins. NAPQI binding is considered an upstream event in the pathophysiology, especially when binding to mitochondrial proteins and therefore leads to mitochondrial toxicity. APAP caused a significant increase in liver toxicity 3 h post-APAP administration as measured by increased serum alanine aminotransferase (ALT) levels. Using high-resolution mitochondrial proteomics techniques to measure thiol and protein changes, no significant change in global thiol levels was observed. However, 3-hydroxy-3-methylglutaryl coenzyme A synthase 2 (HMG-CoA synthase) had significantly decreased levels of reduced thiols and activity after APAP treatment. HMG-CoA synthase is a key regulatory enzyme in ketogenesis and possesses a number of critical cysteines in the active site. Similarly, catalase, a key enzyme in hydrogen peroxide metabolism, also showed modification in protein thiol content. These data indicate post-translational modifications of a few selected proteins involved in mitochondrial and cellular regulation of metabolism during liver toxicity after APAP overdose. The pathophysiological relevance of these limited changes in protein thiols remains to be investigated.     

The intracellular redox stress caused by hexavalent chromium is selective for proteins that have key roles in cell survival and thiol redox control

Hexavalent chromium [Cr(VI)] compounds (e.g. chromates) are strong oxidants that readily enter cells where they are reduced to reactive Cr intermediates that can directly oxidize some cell components and can promote the generation of reactive oxygen and nitrogen species. Inhalation is a major route of exposure which directly exposes the bronchial epithelium. Previous studies with non-cancerous human bronchial epithelial cells (BEAS-2B) demonstrated that Cr(VI) treatment results in the irreversible inhibition of thioredoxin reductase (TrxR) and the oxidation of thioredoxins (Trx) and peroxiredoxins (Prx). The mitochondrial Trx/Prx system is somewhat more sensitive to Cr(VI) than the cytosolic Trx/Prx system, and other redox-sensitive mitochondrial functions are subsequently affected including electron transport complexes I and II. Studies reported here show that Cr(VI) does not cause indiscriminant thiol oxidation, and that the Trx/Prx system is among the most sensitive of cellular protein thiols. Trx/Prx oxidation is not unique to BEAS-2B cells, as it was also observed in primary human bronchial epithelial cells. Increasing the intracellular levels of ascorbate, an endogenous Cr(VI) reductant, did not alter the effects on TrxR, Trx, or Prx. The peroxynitrite scavenger MnTBAP did not protect TrxR, Trx, Prx, or the electron transport chain from the effects of Cr(VI), implying that peroxynitrite is not required for these effects. Nitration of tyrosine residues of TrxR was not observed following Cr(VI) treatment, further ruling out peroxynitrite as a significant contributor to the irreversible inhibition of TrxR. Cr(VI) treatments that disrupt the TrxR/Trx/Prx system did not cause detectable mitochondrial DNA damage. Overall, the redox stress that results from Cr(VI) exposure shows selectivity for key proteins which are known to be important for redox signaling, antioxidant defense, and cell survival.     

Characterization of a site-specific PEGylated analog of exendin-4 and determination of the PEGylation site

PEGylation has been a successful strategy for improving the pharmacokinetic and pharmaceutical properties of proteins and peptides. However, PEGylated products also create significant challenges for detailed structural characterization. In this work, a site-specific PEGylation strategy was successfully performed on an exendin-4 analog (Ex4C) through a maleimide method. Tricine–sodium dodecylsulfate polyacrylamide gel electrophoresis (Tricine–SDS-PAGE), analytical reversed phase HPLC (RP-HPLC) and MALDI-TOF were applied to verify the accomplishment of the PEGylation. Peptide mapping was investigated after tryptic digestion, and the PEGylaton site was successfully located on the C-terminal fragment of Ex4C. Amino acid analysis (AAA) of cysteine was then applied to verify the block in the thiol group caused by PEGylation. We believe that the combination of proper enzymatic digestion and amino acid analysis of cysteine provided an easy and convincing way to identify the PEGylation site in this maleimide method.     

Oxygen dependence of oxidative stress. Rate of NADPH supply for maintaining the GSH pool during hypoxia

NADPH supply for oxidized glutathione (GSSG) reduction was studied in hepatocytes under different steady-state O2 concentrations with controlled infusions of diamide, a thiol oxidant. When bis-chloro-nitrosourea (BCNU) was used to inhibit GSSG reductase, the rate of GSH depletion approximated the rate of diamide infusion, showing that diamide reacted preferentially with GSH under these experimental conditions. Under aerobic conditions without BCNU treatment, the GSH and NADPH pools were largely unaffected and little diamide accumulation or protein thiol oxidation occurred with diamide infusion rates up to 5.3 nmol/10(6) cells per min. However, at greater infusion rates, GSH and NADPH decreased, diamide and GSSG concentrations increased, and protein thiols were oxidized. This critical infusion rate was easily discernible and provided a convenient means to assess the capacity of cells to reduce GSSG as a function of O2 concentration. As the O2 concentration was decreased below 15 microM, the critical infusion rate decreased from the aerobic value of 5.3 to less than 2 nmol/10(6) cells per min in anoxic cells; half-maximal change occurred at 5 microM O2. Although cells could not maintain normal thiol and NADPH pools at infusion rates above the critical value, analysis of the rates of thiol depletion showed that the maximal NADPH supply rate for GSSG reduction under aerobic conditions was 7-8 nmol/10(6) cells per min and was affected by hypoxia to the same degree as the critical value. Thus, hypoxia and anoxia impair the capability of cells to supply NADPH for the reduction of thiol oxidants. This could be an important factor in the sensitivity of hypoxic and ischemic tissues to oxidative injury.     

Carbamoylation of peptides and proteins in vitro by S-(N-methylcarbamoyl)glutathione and S-(N-methylcarbamoyl)cysteine, two electrophilic S-linked conjugates of methyl isocyanate

The reactivity toward peptides and proteins of S-(N-methylcarbamoyl)glutathione (SMG), the glutathione conjugate of methyl isocyanate, and the corresponding cysteine adduct, S-(N-methylcarbamoyl)cysteine (SMC), was investigated with the aid of in vitro model systems. Incubation of SMC or a trideuteriomethyl analogue of SMC with either the reduced or oxidized forms of oxytocin afforded similar mixtures of mono-, bis- and tris-N-methylcarbamoylated peptides. Structure elucidation of the mono and bis adducts by fast atom bombardment tandem mass spectrometry indicated that carbamoylation of oxytocin occurred preferentially at Cys-6 and that Cys-1 and/or Tyr-2 were secondary sites of modification. Upon incubation of S-[N-([14C]methyl)carbamoyl]glutathione (14C-SMG) with native bovine serum albumin (BSA), radioactivity became bound covalently to the protein in a time- and concentration-dependent fashion. "Blocking" of the lone Cys-34 thiol group of BSA in the form of a disulfide prior to exposure of the protein to 14C-SMG failed to decrease significantly the extent or time course of this covalent binding. It is concluded that carbamate thioester conjugates of MIC are reactive, carbamoylating entities which can donate the elements of MIC to nucleophilic functionalities on peptides and proteins. Free thiols appear to be preferred sites for such carbamoylation processes, a phenomenon that may have important toxicological consequences in the pathology of tissue lesions induced by MIC and related isocyanates.     

GSH-mediated S-transarylation of a quinone glyceraldehyde-3-phosphate dehydrogenase conjugate

Many cellular proteins with reactive thiols form covalent bonds with electrophiles, thereby modifying their structures and activities. Here, we describe the recovery of a glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), from such an electrophilic attack by 1,2-napthoquinone (1,2-NQ). GAPDH readily formed a covalent bond with 1,2-NQ through Cys152 at a low concentration (0.2 μM) in a cell-free system, but when human epithelial A549 cells were exposed to this quinone at 20 μM, only minimal binding was observed although extensive binding to numerous other cellular proteins occurred. Depletion of cellular glutathione (GSH) with buthionine sulfoximine (BSO) resulted in some covalent modification of cellular GAPDH by 1,2-NQ and a significant reduction of GAPDH activity in the cells. Incubation of native, but not boiled, human GAPDH that had been modified by 1,2-NQ with GSH resulted in a concentration-dependent removal of 1,2-NQ from the GAPDH conjugate, accompanied by partial recovery of lost catalytic activity and formation of a 1,2-NQ-GSH adduct (1,2-NQ-SG). While GAPDH is recognized as a multifunctional protein, our results show that GAPDH also has a unique ability to recover from electrophilic modification by 1,2-NQ through a GSH-dependent S-transarylation reaction.