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The cocrystal structure of the human serum albumin HSA-[[Delta].sup.12]-[PGJ.sub.2]-myristate complex was analyzed by X-ray crystallography to study the molecular details of the binding of metabolite [PGD.sub.2] to albumin. The results from crystallographic and mass spectrometric analyses for the HSA-[[Delta].sup.12]-[PGJ.sub.2] complex provide intriguing new insights into the molecular details related to interaction between electrophilic ligand and its primary producer and transporter.

Several studies are conducted to understand the molecular mechanisms associated with the constitutive expression of the glutathione S-transferase (GST) isozymes during differentiation of the Caco-2 cells in humans. The expression of the GST isozymes is shown to be highly regulated by the Kelch-like ECH-associated protein 1 (Keap1).

The redox potential of the protein-bound dopamine (DA) is determined and a novel mechanism is developed for the oxidative modification of the protein, in which the DA-cysteine adduct causes oxidative modification of the DA-bound protein in the presence of [Cu.sup.2+]. The results have shown that the DA-modified protein might be involved in redox modification under oxidative stress, whereby DA covalently binds to cysteine residues, generating the redox-active cysteinyl-DA adduct that causes the metal-catalyzed oxidation of protein.

Results indicate that 15-deoxy-delta(sup)12,14-prostaglandin J(sub)2 may modulate proteasome-dependent regulation of cellular functions, which in turn may be involved in the accumulation of p53 and subsequent induction of apoptosis induced by 15-deoxy-delta(sup)12,14-prostaglandin J(sub)2.

4-Oxo-2-nonenal (ONE), an aldehyde originating from the peroxidation of ω6 polyunsaturated fatty acids, preferentially reacts with the cysteine residues of protein. Despite the fact that there has been significant recent interest in the protein reactivity and biological activity of ONE, the structural basis of the ONE-cysteine adducts remain to be established. In the present study, to gain a structural insight into the sulfhydryl modification by ONE, we characterized reaction products that originated from the initial ONE-cysteine Michael adducts. N-Acetyl-l-cysteine (10 mM) was incubated with an equimolar concentration of ONE in 0.1 M phosphate buffer (pH 7.4) at 37 °C. Within 1 h of incubation, the reaction of N-acetyl-l-cysteine with ONE resulted in the formation of two (C-2 and C-3) Michael addition products possessing a carbonyl functionality. Subsequent incubation of the reaction mixture resulted in their disappearance and concomitant formation of advanced reaction products, including a minor product IIIand major products IVa, IVb, and V. Product IIIwas identified to be a thiomorpholine derivative, 4-acetyl-5-hydroxyl-6-(2-oxoheptyl)thiomorpholine-3-carboxylic acid, which might have originated from the C-2 Michael addition product. The major products were identified to be the novel 2-cyclopentenone derivatives, that is, 2-(acetylamino)-3-[(3-butyl-4-oxocyclopent-2-en-1-yl)sulfanyl]propionic acid (IVaand its isomer IVb) and 2-(acetylamino)-3-[(4-butyl-5-oxocyclopent-3-en-1-yl)sulfanyl]propionic acid (V), which might be generated through the base-catalyzed cyclization of the C-2 and C-3 Michael addition products, respectively. The furan derivative, which has been reported as the end product of the Michael adducts, was found to be formed only under acidic conditions. Thus, this study identified the novel ONE-cysteine adducts, including the most prominent 2-cyclopentenone derivatives, that originated from the initial Michael adducts. [ABSTRACT FROM AUTHOR]

Prostaglandin D[SUB2](PGD[SUB2]), a major cyclooxygenase product in a variety of tissues and cells, readily undergoes dehydration to yield electrophilic PGs, such as 15-deoxy-Δ[SUP12,14]-PGJ[SUB2] (15d-PGJ[SUB2]). We have previously shown that 15d-PGJ[SUB2] potently induces apoptosis of SH-SYSY human neuroblastoma cells via accumulation of the tumor suppressor gene product p53. In the study presented here, we investigated the molecular mechanisms involved in the 15d-PGJ[SUB2]-induced accumulation of p53. It was observed that 15d-PGJ[SUB2] potently induced p53 protein expression but scarcely induced p53 gene expression. In addition, exposure of the cells to 15d-PGJ[SUB2] resulted in an accumulation of ubiquitinated proteins and in a significant inhibition of proteasome activities, suggesting that 15d-PGJ[SUB2] acted on the ubiquitin- proteasome pathway, a regulatory mechanism of p53 turnover. The effects of 15d-PGJ[SUB2] on the protein turnover were attributed to its electrophilic feature, based on the observations that (i) the reduction of the double bond in the cyclopentenone ring of 15d-PGJ[SUB2] virtually abolished the effects on protein turnover, (ii) overexpression of an endogenous redox regulator, thioredoxin 1, significantly retarded the inhibition of proteasome activities and accumulations of p53 and ubiquitinated proteins induced by 15d-PGJ[SUB2], and (iii) treatment of SH-SY5Y cells with biotinylated 15d-PGJ[SUB2] indeed resulted in the formation of a 15d-PGJ[SUB2]-proteasome conjugate. These data suggest that the modulation of proteasome activity may be involved in the mechanism responsible for the accumulation of p53 and subsequent induction of apoptotic cell death induced by 15d-PGJ[SUB2]. [ABSTRACT FROM AUTHOR]

Human serum albumin (HSA), the most abundant protein in plasma, has a very unique function, catalyzing the conversion of prostaglandin J2 (PGJ2), a dehydration product of PGD2, to yield Δ12-PGJ2. These PGD2 metabolites are actively transported into cells and accumulated in the nuclei, where they act as potent inducers of cell growth inhibition and cell differentiation, and exhibit their own unique spectrum of biological effects. The facts that (i) arachidonic acid metabolites bind to human serum albumin (HSA) and the metabolism of these molecules is altered as a result of binding, (ii) HSA catalyzes the transformation of PGJ2 into Δ12-PGJ2, and (iii) Δ12-PGJ2 is stable in serum suggest that HSA may bind and stabilize Δ12 PGJ2 in a specific manner. A molecular interaction analysis using surface plasmon resonance (Biacore) indeed suggested the presence of a specific Δ12-PGJ2-binding site in HSA. To investigate the molecular details of the binding of this PGD2 metabolite to albumin, we analyzed the cocrystal structure of the HSA-Δ12-PGJ2-myristate complex by X-ray crystallography and found that two Δ12-PGJ2 molecules bind to a primary site in subdomain lB of the protein. The electron density results suggested that one of the two Δ12-PGJ2 molecules that specifically bind to the site covalently interacted with a histidine residue (His146). Using nano-LC-MS/MS analysis of the HSA-Δ12-PGJ2 complex, the formation of an unusual Δ12-PGJ2-histidine adduct at His146 was confirmed. Thus, our crystallographic and mass spectrometric analyses of the HSA-Δ12-PGJ2 complex provided intriguing new insights into the molecular details of how this electrophilic ligand interacts with its primary producer and transporter. [ABSTRACT FROM AUTHOR]

Dopamine (DA) is an unstable neurotransmitter that readily oxidizes to the DA quinone and forms reactive oxygen species, such as superoxide and hydrogen peroxide. The oxidized dopamine also forms thiol conjugates with sulfhydryl groups on cysteine, glutathione, and proteins. In the present study, we determined the redox potential of the protein-bound DA and established a novel mechanism for the oxidative modification of the protein, in which the DA-cysteine adduct generated in the DA-modified protein causes oxidative modification of the DA-bound protein in the presence of Cu2+. Exposure of a sulfhydryl enzyme, glyceraldehyde-3-phosphate dehydrogenase, to DA resulted in a significant loss of sulfhydryl groups and the formation of the DA-cysteine adduct. When the DA-modified protein was incubated with Cu2+, we observed aggregation and degradation of the DA-bound protein and concomitant formation of a protein carbonyl, a marker of an oxidatively modified protein. Furthermore, we analyzed the carbonyl products generated during the Cu2+-catalyzed oxidation of the DA-modified protein and revealed the production of glutamic and aminoadipic semialdehydes, consisting of the protein carbonyls generated. The cysteinyl-DA residue generated in the DA-modified protein was suggested to represent a redox-active adduct, based on the observations that the cysteinyl-DA adduct, 5-S-cysteinyldopamine, produced by the reaction of cysteine with DA, gave rise to the oxidative modification of bovine serum albumin in the presence of Cu2+. These data suggest that the DA-modified protein may be involved in redox alteration under oxidative stress, whereby DA covalently binds to cysteine residues, generating the redox-active cysteinyl-DA adduct that causes the metal-catalyzed oxidation of protein. [ABSTRACT FROM AUTHOR]

Polyphenols have received a significant amount of attention in disease prevention because of their unique chemical and biological properties. However, the underlying molecular mechanism for their beneficial effects remains unclear. We have now identified a polyphenol as a source of innate epitopes detected in natural IgM and established a unique gain-of-function mechanism in the formation of innate epitopes by polyphenol via the polymerization of proteins. Upon incubation with bovine serum albumin (BSA) under physiological conditions, several polyphenols converted the protein into the innate epitopes recognized by the IgM Abs. Interestingly, piceatannol, a naturally occurring hydroxylated analogue of a red wine polyphenol, resveratrol, mediated the modification of BSA, whose polymerized form was specifically recognized by the IgMs. The piceatannol-mediated polymerization of the protein was associated with the formation of a lysine-derived cross-link, dehydrolysinonorleucine. In addition, an oxidatively deaminated product, α-aminoadipic semialdehyde, was detected as a potential precursor for the cross-link in the piceatannol-treated BSA, suggesting that the polymerization of the protein might be mediated by the oxidation of a lysine residue by piceatannol followed by a Schiff base reaction with the ε-amino group of an unoxidized lysine residue. The results of this study established a novel mechanism for the formation of innate epitopes by small dietary molecules and support the notion that many of the beneficial effects of polyphenols could be attributed, at least in part, to their lysyl oxidase-like activity. They also suggest that resveratrol may have beneficial effects on human health because of its conversion to piceatannol.

De novo synthesis of possible candidates for the Inagami-Tamura endogenous digitalis-like factor (EDLF) was achieved to validate a previously proposed structure. Our synthetic approach involves a highly regio- and diastereoselective Mizoroki-Heck reaction and a Friedel-Crafts-type cyclodehydration to construct steroidal tetracycle 14 as a versatile common intermediate leading to seven 2,14β-dihydroxyestradiol analogues 1a-c, 2a-c, and 3 as possible candidates. By comparing the potency of inhibitory activity against Na + /K + -ATPase between the synthesized candidates and the EDLF, it was found that the proposed structure is not likely to be a true structure of the Inagami-Tamura EDLF.

Advanced glycation end products (AGEs) make up a heterogeneous group of molecules formed from the nonenzymatic reaction of reducing sugars with the free amino groups of proteins. The abundance of AGEs in a variety of age-related diseases, including diabetic complications and atherosclerosis, and their pathophysiological effects suggest the existence of innate defense mechanisms. Here we examined the presence of serum proteins that are capable of binding glycated bovine serum albumin (AGEs-BSA), prepared upon incubation of BSA with dehydroascorbate, and identified complement component C1q subcomponent subunit A as a novel AGE-binding protein in human serum. A molecular interaction analysis showed the specific binding of C1q to the AGEs-BSA. In addition, we identified DNA-binding regions of C1q, including a collagen-like domain, as the AGE-binding site and established that the amount of positive charge on the binding site was the determining factor. C1q indeed recognized several other modified proteins, including acylated proteins, suggesting that the binding specificity of C1q might be ascribed, at least in part, to the electronegative potential of the ligand proteins. We also observed that C1q was involved in the AGEs-BSA-activated deposition of complement proteins, C3b and C4b. In addition, the AGEs-BSA mediated the proteolytic cleavage of complement protein 5 to release C5a. These findings provide the first evidence of AGEs as a new ligand recognized by C1q, stimulating the C1q-dependent classical complement pathway.

Acrolein shows a facile reactivity with the ε-amino group of lysine to form N(ε)-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) as the major product. In addition, FDP-lysine generated in the acrolein-modified protein could function as an electrophile, reacting with thiol compounds, to form an irreversible thioether adduct. In the present study, to establish the utility of this irreversible conjugate, we attempted to use it as an immunogen to raise a monoclonal antibody (mAb), which specifically recognized protein-bound thiol compounds. Using the glutathione (GSH) conjugate of the acrolein-modified protein as an immunogen, we raised the mAb 2C4, which cross-reacted with the GSH conjugate of acrolein-modified proteins. Specificity studies revealed that mAb 2C4 recognized both the GSH conjugate of an acrolein-lysine adduct, FDP-lysine, and oxidized GSH (GSSG). In addition, mAb 2C4 cross-reacted not only with the GSH conjugates of the acrolein-modified protein but also with the GSH-treated, oxidized protein (S-glutathiolated protein), suggesting that the antibody significantly recognized the protein-bound GSH as the epitope. An immunohistochemical analysis of the atherosclerotic lesions from the human aorta showed that immunoreactive materials with mAb 2C4 were indeed present in the macrophage-derived foam cells and migrating smooth muscles. In addition, using mAb 2C4, we analyzed the GSH-treated, oxidized low-density lipoproteins by agarose gel electrophoresis under reducing or nonreducing conditions followed by immunoblot analysis and found that the majority of the GSH was irreversibly incorporated into the proteins. The results of this study not only showed the utility of the antibody raised against the GSH conjugate of the acrolein-modified proteins but also suggested that the irreversible binding of GSH and other redox molecules to the oxidized LDL might represent the process common to the modification of LDL during atherogenesis.

Acrolein, a ubiquitous pollutant in the environment, is endogenously formed through oxidation reactions and is believed to be involved in cytopathological effects observed during oxidative stress. Acrolein exerts these effects because of its facile reactivity with biological materials, particularly proteins. In the present study, we quantitatively analyzed the acrolein-specific adducts generated during lipid peroxidation-modification of proteins and identified the acrolein adduct most abundantly generated in the in vitro oxidized low-density lipoproteins (LDL). Taking advantage of the fact that the acrolein-lysine adducts, N(ε)-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) and N(ε)-(3-methylpyridinium)lysine (MP-lysine), have stable core structures resistant to the acid hydrolysis condition of proteins, we examined the formation of these adducts in proteins using high performance liquid chromatography with online electrospray ionization tandem mass spectrometry. However, only MP-lysine was detected as a minor product in the iron/ascorbate-mediated oxidation of polyunsaturated fatty acids in the presence of proteins and in the oxidized low-density lipoproteins (LDL). However, using a reductive amination-based pyridylamination method, we analyzed the acrolein-specific adducts with a carbonyl functionality and found that acrolein modification of the protein produced a number of carbonylated amino acids, including an acrolein-histidine adduct. On the basis of the chemical and spectroscopic evidence, this adduct was identified as N(τ)-(3-propanal)histidine. More notably, N(τ)-(3-propanal)histidine appeared to be one of the major adducts generated in the oxidized LDL. These data suggest that acrolein generated during lipid peroxidation may primarily react with histidine residues of proteins to form N(τ)-(3-propanal)histidine.

Various lines of evidence indicate that the oxidative modification of protein and the subsequent accumulation of the degenerated proteins have been found in cells and tissues during aging, oxidative stress, and in a variety of pathological states. The critical agents that give rise to this protein degeneration may be represented by aldehydes. Although the covalent modification of proteins by aldehydes alone has been well-studied, the effect of reactive oxygen species, such as H2O2, upon aldehyde modification of the protein has received little attention. We have now established a unique protein modification in which H2O2 and, to a lesser extent, alkyl hydroperoxides mediate the binding of alkanals to the lysine residues of protein to generate structurally unusual N-acylation products. Upon the reaction of a lysine-containing peptide, N(alpha)-benzoylglycyl-lysine, with hexanal in the presence of H2O2, a product containing one molecule of hexanal per peptide was detected. On the basis of the chemical and spectroscopic evidence, the product was identified to be the acylation product, N(epsilon)-hexanoyllysine. H2O2 mediated the N-acylation of the lysine derivative by the saturated aldehydes of 1-6 carbons in length. The H2O2-mediated acylation of the protein was immunochemically confirmed by reaction of the proteins with hexanal in the presence of H2O2. Furthermore, the enhanced N-acylations (N-acetylation and N-hexanoylation) were also observed in the kidney of rats exposed to ferric nitrilotriacetate, a well-characterized inducer of oxidative stress. Mechanistic studies using a phosphonium lysine derivative suggest a Baeyer-Villiger-like reaction proceeding through peroxide addition to the aldehyde Schiff base. These data suggest that the hydroperoxides, including H2O2, might be involved not only in the oxidative modification of protein but also in the covalent binding of the saturated aldehydes to proteins under oxidative stress.

Electrophilic molecules are endogenously generated and are causally involved in many pathophysiological effects. Prostaglandin D (20 (PGD (2)), a major cyclooxygenase product in a variety of tissues, readily undergoes dehydration to yield the cyclopentenone-type PGs of the J (2)-series such as 15-deoxy-Delta (12,14)-PGJ (2) (15d-PGJ (2)). 15d-PGJ (2) is an electrophile, which can covalently react via the Michael addition reaction with nucleophiles, such as the free sulfhydryls of glutathione and cysteine residues in cellular proteins that play an important role in the control of the redox cell-signaling pathways. Covalent binding of 15d-PGJ (2) to cellular proteins may be one of the mechanisms by which 15d-PGJ (2) induces a cellular response involved in most of the pathophysiological effects associated with inflammation. In the present perspective, we provide a comprehensive summary of 15d-PGJ (2) as an electrophilic mediator of cellular responses.

A proteomic approach was used to identify 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) protein targets in human neuroblastoma SH-SY5Y cells. By using biotinylated 15d-PGJ2, beta-actin was found as the major adducted protein; at least 12 proteins were also identified as minor biotin-positive spots, falling in different functional classes, including glycolytic enzymes (enolase and lactate dehydrogenase), redox enzymes (biliverdin reductase), and a eukaryotic regulatory protein (14-3-3gamma). 15d-PGJ2 induced marked morphological changes in the actin filament network and in particular promoted F-actin depolymerization as confirmed by Western blot analysis. By using a mass spectrometric approach, we found that 15d-PGJ2 reacts with isolated G-actin in a 1:1 stoichiometric ratio and selectively binds the Cys374 site through a Michael adduction mechanism. Computational studies showed that the covalent binding of 15d-PGJ2 induces a significant unfolding of actin structure and in particular that 15d-PGJ2 distorts the actin subdomains 2 and 4, which define the nucleotide binding sites impeding the nucleotide exchange. The functional effect of 15d-PGJ2 on G-actin was studied by polymerization measurement: in the presence of 15d-PGJ2, a lower amount of F-actin forms, as followed by the increase in pyrenyl-actin fluorescence intensity, as the major effect of increasing 15d-PGJ2 concentrations occurs on the maximum extent of actin polymerization, whereas it is negligible on the initial rate of reaction. In summary, the results here reported give an insight into the role of 15d-PGJ2 as a cytotoxic compound in neuronal cell dysfunction. Actin is the main protein cellular target of 15d-PGJ2, which specifically binds through a Michael adduction to Cys374, leading to a protein conformational change that can explain the disruption of the actin cytoskeleton, F-actin depolymerization, and impairment of G-actin polymerization.

Ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], a seleno-organic compound showing glutathione peroxidase-like activity, is one of the promising synthetic antioxidants. In the present study, we investigated the electrophilic potential of this antioxidant and established the mechanism of the cysteine-targeted oxidation of protein. In addition, using ebselen as an electrophilic probe, we characterized the cysteine residues required for posttranslational modification into an electrophile sensor protein in the phase 2 detoxification response. Ebselen showed a potent antioxidant effect against the spontaneous and 4-hydroxy-2-nonenal-stimulated production of intracellular reactive oxygen species in rat liver epithelial RL34 cells. Meanwhile, upon in vitro incubation with a redox-active sulfhydryl protein (thioredoxin), ebselen showed a strong electrophilic potential of mediating the formation of selenenylsulfide and intra- and intermolecular disulfide linkages within the protein. By taking advantage of this antioxidant and electrophilic property of ebselen, we characterized posttranslational modification of Kelch-like ECH-associated protein 1 (Keap1), an electrophile sensor protein, which represses the ability of the transcription factor NF-E2-related factor 2 (Nrf2) upon induction of the phase 2 detoxification response. Ebselen potently induced the gene expression of a series of phase 2 enzymes in rat liver epithelial RL34 cells, which was associated with the formation of a high molecular weight complex of Keap1. Furthermore, a cysteine residue in Keap1, C151, was found to be uniquely required not only for the formation of the complex but also for the induction of the phase 2 response by ebselen. Thus, this unique antioxidant and electrophilic property of ebselen giving rise to the cysteine-targeted oxidation enabled us to evaluate the role of sensor cysteines in redox regulation of protein function under electrophile stress.