Molecular Cloning, Expression and Enzymatic Characterization of Tetrahymena thermophila Glutathione-S-Transferase Mu 34.

Glutathione-S-transferase enzymes (GSTs) are essential components of the phase II detoxification system and protect organisms from oxidative stress induced by xenobiotics and harmful toxins such as 1-chloro-2,4-dinitrobenzene (CDNB). In Tetrahymena thermophila, the TtGSTm34 gene was previously reported to be one of the most responsive GST genes to CDNB treatment (LD50 = 0.079 mM). This study aimed to determine the kinetic features of recombinantly expressed and purified TtGSTm34 with CDNB and glutathione (GSH). TtGSTm34-8xHis was recombinantly produced in T. thermophila as a 25-kDa protein after the cloning of the 660-bp full-length ORF of TtGSTm34 into the pIGF-1 vector. A three-dimensional model of the TtGSTm34 protein constructed by the AlphaFold and PyMOL programs confirmed that it has structurally conserved and folded GST domains. The recombinant production of TtGSTm34-8xHis was confirmed by SDS‒PAGE and Western blot analysis. A dual-affinity chromatography strategy helped to purify TtGSTm34-8xHis approximately 3166-fold. The purified recombinant TtGSTm34-8xHis exhibited significantly high enzyme activity with CDNB (190 µmol/min/mg) as substrate. Enzyme kinetic analysis revealed Km values of 0.68 mM with GSH and 0.40 mM with CDNB as substrates, confirming its expected high affinity for CDNB. The optimum pH and temperature were determined to be 7.0 and 25 °C, respectively. Ethacrynic acid inhibited fully TtGSTm34-8xHis enzyme activity. These results imply that TtGSTm34 of T. thermophila plays a major role in the detoxification of xenobiotics, such as CDNB, as a first line of defense in aquatic protists against oxidative damage.

Selective activation of TRPA1 ion channels by nitrobenzene skin sensitizers DNFB and DNCB.

2, 4-dinitrofluorobenzene (DNFB) and 2, 4-dinitrochlorobenzene (DNCB) are well known as skin sensitizers that can cause dermatitis. DNFB has shown to more potently sensitize skin; however, how DNFB and DNCB cause skin inflammation at a molecular level and why this difference in their sensitization ability is observed remain unknown. In this study, we aimed to identify the molecular targets and mechanisms on which DNFB and DNCB act. We used a fluorescent calcium imaging plate reader in an initial screening assay before patch-clamp recordings for validation. Molecular docking in combination with site-directed mutagenesis was then carried out to investigate DNFB and DNCB binding sites in the TRPA1 ion channel that may be selectively activated by these tow sensitizers. We found that DNFB and DNCB selectively activated TRPA1 channel with EC50 values of 2.3 ± 0.7 µM and 42.4 ± 20.9 µM, respectively. Single-channel recordings revealed that DNFB and DNCB increase the probability of channel opening and act on three residues (C621, E625, and Y658) critical for TRPA1 activation. Our findings may not only help explain the molecular mechanism underlying the dermatitis and pruritus caused by chemicals such as DNFB and DNCB, but also provide a molecular tool 7.5-fold more potent than the current TRPA1 activator allyl isothiocyanate (AITC) used for investigating TRPA1 channel pharmacology and pathology.

Artemisia anomala Herba Alleviates 2,4-Dinitrochlorobenzene-Induced Atopic Dermatitis-Like Skin Lesions in Mice and the Production of Pro-Inflammatory Mediators in Tumor Necrosis Factor Alpha-/Interferon Gamma-Induced HaCaT Cells.

Artemisia anomala S. Moore is a perennial herbaceous plant classified as Asteraceae of the genus Artemisia. Many species of Artemisia have been used as medicinal materials. Artemisia anomala S. Moore has been widely used in China to treat inflammatory diseases. However, the mechanism of its action on the keratinocyte inflammatory response is poorly understood. Here, we investigated the anti-inflammatory reaction of Artemisia anomala S. Moore ethanol extract (EAA) using human keratinocyte (HaCaT) cells, which involved investigating the nuclear factor kappa B (NF-κB), signal transducer, and activator of transcription-1 (STAT-1), as well as mitogen-activated protein kinase (MAPK) signaling pathways and atopic dermatitis-like skin lesions in mice. We elucidated the anti-inflammatory effects of EAA on tumor necrosis factor-α/interferon-γ (TNF-α/IFN-γ)-treated human keratinocyte cells and 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis (AD)-like mice. The levels of chemokines and cytokines (IL-8, IL-6, TARC, and RANTES) were determined by an enzyme-linked immunosorbent assay. The NF-κB, STAT-1, and MAPK signaling pathways in HaCaT cells were analyzed by western blotting. Thickening of the mice dorsal and ear skin was measured and inflammatory cell infiltration was observed by hematoxylin and eosin staining. Results showed that EAA suppressed IL-8, IL-6, TARC, and RANTES production. EAA inhibited nuclear translocation of NFκB and STAT-1, as well as reduced the levels of phosphorylated ERK MAPKs. EAA improved AD-like skin lesions in DNCB-treated mice. These findings suggest that EAA possesses stronger anti-inflammatory properties and can be useful as a functional food or candidate agent for AD.

The Interaction of Human Glutathione Transferase GSTA1-1 with Reactive Dyes.

Human glutathione transferase A1-1 (hGSTA1-1) contributes to developing resistance to anticancer drugs and, therefore, is promising in terms of drug-design targets for coping with this phenomenon. In the present study, the interaction of anthraquinone and diazo dichlorotriazine dyes (DCTD) with hGSTA1-1 was investigated. The anthraquinone dye Procion blue MX-R (PBMX-R) appeared to interact with higher affinity and was selected for further study. The enzyme was specifically and irreversibly inactivated by PBMX-R, following a biphasic pseudo-first-order saturation kinetics, with approximately 1 mol of inhibitor per mol of the dimeric enzyme being incorporated. Molecular modeling and protein chemistry data suggested that the modified residue is the Cys112, which is located at the entrance of the solvent channel at the subunits interface. The results suggest that negative cooperativity exists upon PBMX-R binding, indicating a structural communication between the two subunits. Kinetic inhibition analysis showed that the dye is a competitive inhibitor towards glutathione (GSH) and mixed-type inhibitor towards 1-chloro-2,4-dinitrobenzene (CDNB). The present study results suggest that PBMX-R is a useful probe suitable for assessing by kinetic means the drugability of the enzyme in future drug-design efforts.

Inhibition of thioredoxin reductase (TrxR) triggers oxidative stress-induced apoptosis in filarial nematode Setaria cervi channelized through ASK-1-p38 mediated caspase activation.

Inhibition of an imperative antioxidant enzyme with subsequent death is a victorious and widely accepted strategy to combat various infectious diseases. Among different antioxidant enzymes, thioredoxin reductase (TrxR) is an exclusive one. Studies have revealed that direct inhibition of TrxR by different classes of chemical moieties promptly results in the death of an organism. Especially the structural as well as biochemical modifications of the enzyme upon inhibition project serious threat towards the subject organism. Herein, an attempt was made to inhibit TrxR of filarial species by administering Auranofin, 1 chloro 2,4 dinitrobenzene (CDNB), Curcumin, and a novel carbamo dithioperoxo(thioate) derivative (4a). Our study has revealed that inhibition of TrxR resulted in the induction of the classical CED pathway of apoptosis along with the intrinsic and extrinsic pathways of apoptosis (Caspase mediated) routed through the ASK-1/p38 axis. Druggability analysis of filarial TrxR for the selected compounds was performed in silico through molecular docking studies. Therefore, this study attempts to decipher the mechanism of apoptosis induction following TrxR inhibition. The safety of those four compounds in terms of dose and toxicity was taken under consideration. Thitherto, the mechanism of TrxR mediated initiation of cell death in filarial parasite has remained undercover, and therefore, it is a maiden report on the characterization of apoptosis induction upon TrxR inhibition which will eventually help in generating effective antifilarial drugs in the future.

Investigation of the Substrate-Binding Site of a Prostaglandin E Synthase in Bombyx mori.

Prostaglandin E synthase (PGES) catalyzes the conversion of prostaglandin H2 to prostaglandin E2 in the presence of glutathione (GSH) in mammals. Amid the limited knowledge on prostaglandin and its related enzymes in insects, we recently identified PGES from the silkworm Bombyx mori (bmPGES) and determined its crystal structure complexed with GSH. In the current study, we investigated the substrate-binding site of bmPGES by site-directed mutagenesis and X-ray crystallography. We found that the residues Tyr107, Val155, Met159, and Glu203 are located in the catalytic pockets of bmPGES, and mutagenesis of each residue reduced the bmPGES activity. Our results suggest that these four residues contribute to the catalytic activity of bmPGES. Overall, this structure-function study holds implications in controlling pests by designing rational and efficient pesticides.

Synthesis of chitosan derivatives with organoselenium and organosulfur compounds: Characterization, antimicrobial properties and application as biomaterials.

In this study, Schiff bases of chitosan (CS) were synthesized using citronellal, citral, and their derivatives containing selenium and sulfur. Organoselenium and organosulfur compounds show attractive biological and pharmaceutical activities, which can be beneficial to CS-based materials. From the characterization analyses, it was found that the CS-derivatives containing organoselenium and organosulfur compounds exhibited the highest conversion degrees (23 and 28%). Biological assays were conducted using films prepared by the blending of CS-derivatives and poly(vinyl alcohol). The antimicrobial evaluation indicated that the film prepared with the sulfur-containing CS was the most active against the tested pathogens (Escherichia coli, Staphylococcus aureus, and Candida albicans) since it reduced considerably their counts (42.5%, 17.4%, and 18.7%). Finally, in vivo assays revealed that this film attenuates atopic dermatitis-like symptoms in mice by suppressing the increase of myeloperoxidase (MPO) activity and reactive species (RS) levels induced by 2,4-dinitrochlorobenzene (DNCB). In summary, CS-derivatives containing chalcogens, mainly organosulfur, are potential candidates for biomedical applications such as for the treatment of chronic skin diseases.

Correlating the structure and reactivity of a contact allergen, DNCB, and its analogs to sensitization potential.

We report a study that seeks to find a correlation between the overall sensitization potential quantified by the expression of IL-8 by stimulated monocytes and the chemical structure of a model contact allergen, 2,4-dinitrochlorobenzene (DNCB). We show that structure and reactivity of the chemical compounds play an important role in activation of the monocytes and subsequent inflammation in tissue. However, we observed a non-linear correlation between the rate of reaction and biological activity indicating a required balance of stability and reactivity.

Selective Disruption of Mitochondrial Thiol Redox State in Cells and In Vivo.

Mitochondrial glutathione (GSH) and thioredoxin (Trx) systems function independently of the rest of the cell. While maintenance of mitochondrial thiol redox state is thought vital for cell survival, this was not testable due to the difficulty of manipulating the organelle's thiol systems independently of those in other cell compartments. To overcome this constraint we modified the glutathione S-transferase substrate and Trx reductase (TrxR) inhibitor, 1-chloro-2,4-dinitrobenzene (CDNB) by conjugation to the mitochondria-targeting triphenylphosphonium cation. The result, MitoCDNB, is taken up by mitochondria where it selectively depletes the mitochondrial GSH pool, catalyzed by glutathione S-transferases, and directly inhibits mitochondrial TrxR2 and peroxiredoxin 3, a peroxidase. Importantly, MitoCDNB inactivates mitochondrial thiol redox homeostasis in isolated cells and in vivo, without affecting that of the cytosol. Consequently, MitoCDNB enables assessment of the biomedical importance of mitochondrial thiol homeostasis in reactive oxygen species production, organelle dynamics, redox signaling, and cell death in cells and in vivo.

Anti-Atopic Effect of Acorn Shell Extract on Atopic Dermatitis-Like Lesions in Mice and Its Active Phytochemicals.

To investigate the potential effects of acorn shells on atopic dermatitis (AD), we utilized oxazolone (OX)- or 2,4-dinitrochlorobenzene (DNCB)-induced AD-like lesion mouse models. Our research demonstrates that Acorn shell extract (ASE) improved the progression of AD-like lesions, including swelling, which were induced by oxazolone on Balb/c mouse ears. Additionally, ASE significantly decreased the ear thickness (OX: 0.42 ± 0.01 mm, OX-ASE: 0.32 ± 0.02 mm) and epidermal thickness (OX: 75.3 ± 32.6 µm, OX-ASE: 46.1 ± 13.4 µm). The continuous DNCB-induced AD mouse model in SKH-1 hairless mice demonstrated that ASE improved AD-like symptoms, including the recovery of skin barrier dysfunction, Immunoglobulin E hyperproduction (DNCB: 340.1 ± 66.8 ng/mL, DNCB-ASE: 234.8 ± 32.9 ng/mL) and an increase in epidermal thickness (DNCB: 96.4 ± 21.9 µm, DNCB-ASE: 52.4 ± 16.3 µm). In addition, we found that ASE suppressed the levels of AD-involved cytokines, such as Tumor Necrosis Factor α, IL-1ß, IL-25 and IL-33 in both animal models. Furthermore, gallic acid and ellagic acid isolated from ASE suppressed ß-hexosaminidase release and IL-4 expression in RBL-2H3 cells. The acorn shell and its active phytochemicals have potential as a new remedy for the improvement of atopic dermatitis and other inflammatory diseases.

Viologen-modified electrodes for protection of hydrogenases from high potential inactivation while performing H2 oxidation at low overpotential.

In this work we present a viologen-modified electrode providing protection for hydrogenases against high potential inactivation. Hydrogenases, including O2-tolerant classes, suffer from reversible inactivation upon applying high potentials, which limits their use in biofuel cells to certain conditions. Our previously reported protection strategy based on the integration of hydrogenase into redox matrices enabled the use of these biocatalysts in biofuel cells even under anode limiting conditions. However, mediated catalysis required application of an overpotential to drive the reaction, and this translates into a power loss in a biofuel cell. In the present work, the enzyme is adsorbed on top of a covalently-attached viologen layer which leads to mixed, direct and mediated, electron transfer processes; at low overpotentials, the direct electron transfer process generates a catalytic current, while the mediated electron transfer through the viologens at higher potentials generates a redox buffer that prevents oxidative inactivation of the enzyme. Consequently, the enzyme starts the catalysis at no overpotential with viologen self-activated protection at high potentials.

Prednisolone-loaded coatable polyvinyl alcohol/alginate hydrogel for the treatment of atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease resulting pruritic, erythema, edema, excoriation, and thickening of the skin, and thus leads to significant impairment in the patient's life. The objective of this study is to develop a steroid drug [prednisolone (PS)]-loaded coatable hydrogel for the treatment of AD. PS-loaded hydrogel was composed of PVA entrapped in mildly crosslinked alginate (PS-loaded PVA/ALG hydrogel). The PS concentration to be loaded in the hydrogel that takes AD efficacy without cell necrosis was determined from the cytotoxicity test using human dermal fibroblasts. The in vivo therapeutic effects for AD of the PS-loaded PVA/ALG hydrogel were evaluated using 2, 4-dinitrochlorobenzene (DNCB)-induced AD Balb/c mouse model. The PS-loaded hydrogel has an appropriate viscosity for easy application and provides moisturizing effect on the skin, as well as anti-inflammatory effect by the sustained drug release for effective AD treatment. From the animal study, the PS-loaded PVA/ALG hydrogel showed effective suppressions of various AD symptoms such as ear edema, pruritus, high IgE levels, epidermal swelling, and mast cell infiltration. Our findings suggest that the PS-loaded coatable PVA/ALG hydrogel may be a promising therapeutic system for the treatment of AD.

Therapeutic and technological potential of 7-chloro-4-phenylselanyl quinoline for the treatment of atopic dermatitis-like skin lesions in mice.

This study investigated the main effects of the oral treatment with 7-chloro-4-phenylselanyl quinoline (4-PSQ) on symptoms, inflammatory and oxidative parameters in an atopic dermatitis (AD) model in BALB/c mice. In addition, the possibility of antioxidant property of 4-PSQ improves the potential of a biofilm (based on chitosan/poly(vinyl alcohol) (PVA)/ bovine bone powder (BBP)) for the treatment of AD-like skin lesions was evaluated. 2,4-Dinitrochlorobenzene (DNCB) was applied to the dorsal skin on days 1-3 for sensitization. Mice were challenged with DNCB on the ear (on days 14-29) and dorsal skin (on days 14, 17, 20, 23, 26, and 29) and treated with 4-PSQ, dexamethasone, biofilm (biofilm sample without 4-PSQ) or 4-PSQ-loaded biofilms. On the day 30, skin severity scores and scratching behavior were determined. After that, animals were sacrificed, and ears and dorsal skin were removed for determination of inflammatory and oxidative parameters. DNCB induced the skin lesions, scratching behavior and ear swelling, increased myeloperoxidase (MPO) activity (ear and back) and reactive species (RS) levels (back). 4-PSQ, 4-PSQ-loaded biofilms and biofilm treatments ameliorated skin severity scores, scratching behavior and inflammatory response induced by DNCB. 4-PSQ and 4-PSQ-loaded biofilm treatments partially protected against the increase in the RS levels induced by DNCB. Our results revealed that the incorporation of 4-PSQ improved the therapeutic effect of the biofilm. The efficacy of 4-PSQ in treating AD-like lesions was similar or better than dexamethasone. In summary, 4-PSQ has a potential therapeutic advantage in the treatment and management of AD.

Identification and characterization of the zebrafish glutathione S-transferase Pi-1.

Zebrafish has in recent years emerged as a popular vertebrate model for use in pharmacological and toxicological studies. While there have been sporadic studies on the zebrafish glutathione S-transferases (GSTs), the zebrafish GST gene superfamily still awaits to be fully elucidated. We report here the identification of 15 zebrafish cytosolic GST genes in NCBI GenBank database and the expression, purification, and enzymatic characterization of the zebrafish cytosolic GST Pi-1 (GSTP1). The cDNA encoding the zebrafish GSTP1 was cloned from a 3-month-old female zebrafish, expressed in Eschelichia coli host cells, and purified. Purified GSTP1 displayed glutathione-conjugating activity toward 1-chloro-2,4-dinitrobenzene as a representative substrate. The enzymatic characteristics of the zebrafish GSTP1, including pH-dependency, effects of metal cations, and kinetic parameters, were studied. Moreover, the expression of zebrafish GSTP1 at different developmental stages during embryogenesis, throughout larval development, onto maturity was examined.

SeGSTo, a novel glutathione S-transferase from the beet armyworm (Spodoptera exigua), involved in detoxification and oxidative stress.

Members of the glutathione S-transferase superfamily can protect organisms against oxidative stress. In this study, we characterized an omega glutathione S-transferase from Spodoptera exigua (SeGSTo). The SeGSTo gene contains an open reading frame (ORF) of 744 nucleotides encoding a 248-amino acid polypeptide. The predicted molecular mass and isoelectric point of SeGSTo are 29007 Da and 7.74, respectively. Multiple amino acid sequence alignment analysis shows that the SeGSTo sequence is closely related to the class 4 GSTo of Bombyx mori BmGSTo4 (77 % protein sequence similarity). Homologous modeling and molecular docking reveal that Cys35 may play an essential role in the catalytic process. Additionally, the phylogenetic tree indicates that SeGSTo belongs to the omega group of the GST superfamily. During S. exigua development, SeGSTo is expressed in the midgut of the fifth instar larval stage, but not in the epidermis or fat body. Identification of recombinant SeGSTo via SDS-PAGE and Western blot shows that its molecular mass is 30 kDa. The recombinant SeGSTo was able to protect super-coiled DNA from damage in a metal-catalyzed oxidation (MCO) system and catalyze the 1-chloro-2,4-dinitrobenzene (CDNB), but not 1,2-dichloro-4-nitrobenzene (DCNB), 4-nitrophenethyl bromide (4-NPB), or 4-nitrobenzyl chloride (4-NBC). The optimal reaction pH and temperature were 8 and 50 °C, respectively, in the catalysis of CDNB by recombinant SeGSTo. The mRNA expression of SeGSTo was up-regulated by various oxidative stresses, such as CdCl2, CuSO4, and isoprocarb, and the catalytic activity of recombinant SeGSTo was noticeably inhibited by heavy metals (Cu(2+) and Cd(2+)) and various pesticides. Taken together, these results indicate that SeGSTo plays an important role in the antioxidation and detoxification of pesticides.

Efficient nitro reduction and dechlorination of 2,4-dinitrochlorobenzene through the integration of bioelectrochemical system into upflow anaerobic sludge blanket: A comprehensive study.

Bioelectrochemical system (BES) coupled upflow anaerobic sludge blanket (UASB) was developed for the removal of recalcitrant pollutants but lack of a comprehensive study. Thus in this study an integrated UASB-BES system was operated continuously for 240 d to systematically investigate the feasibility of the enhanced reduction of 2,4-dinitrochlorobenzene (DNCB), with the key operation parameters, the system stability as well as the microbial biodiversity emphasized. The results indicate that high voltage supplied had a positive effect on DNCB reduction but a negative impact for the overhigh voltage (>1.6 V). The ability to resist shock loading was strengthened in the UASB-BES system in comparison with the control UASB system. High-throughput sequencing analysis suggested that the enhanced reduction of DNCB in UASB-BES could be attributed to higher diversity and the enrichment of reduction-related species, potential electroactive species and fermentative species. Both DNCB removal and dechlorination gradually increased with the increase of operation time, indicating the improved performance of the coupled UASB-BES system. The heatmap visualized only slight differences in the microbial community during long-term operation, indicating the stability of the microbial community. The observed efficient and stable performance highlights the potential for long-term operation and full-scale application of the UASB-BES coupled system particularly for highly recalcitrant pollutants removal.

Trimeric microsomal glutathione transferase 2 displays one third of the sites reactivity.

Human microsomal glutathione transferase 2 (MGST2) is a trimeric integral membrane protein that belongs to the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family. The mammalian MAPEG family consists of six members where four have been structurally determined. MGST2 activates glutathione to form a thiolate that is crucial for GSH peroxidase activity and GSH conjugation reactions with electrophilic substrates, such as 1-chloro-2,4-dinitrobenzene (CDNB). Several studies have shown that MGST2 is able to catalyze a GSH conjugation reaction with the epoxide LTA4 forming the pro-inflammatory LTC4. Unlike its closest homologue leukotriene C4 synthase (LTC4S), MGST2 appears to activate its substrate GSH using only one of the three potential active sites [Ahmad S, et al. (2013) Biochemistry. 52, 1755-1764]. In order to demonstrate and detail the mechanism of one-third of the sites reactivity of MGST2, we have determined the enzyme oligomeric state, by Blue native PAGE and Differential Scanning Calorimetry, as well as the stoichiometry of substrate and substrate analog inhibitor binding to MGST2, using equilibrium dialysis and Isothermal Titration Calorimetry, respectively. Global simulations were used to fit kinetic data to determine the catalytic mechanism of MGST2 with GSH and CDNB (1-chloro-2,4-dinitrobenzene) as substrates. The best fit was observed with 1/3 of the sites catalysis as compared with a simulation where all three sites were active. In contrast to LTC4S, MGST2 displays a 1/3 the sites reactivity, a mechanism shared with the more distant family member MGST1 and recently suggested also for microsomal prostaglandin E synthase-1.

Electrochemical evaluation of glutathione S-transferase kinetic parameters.

Glutathione S-transferases (GSTs), are a family of enzymes belonging to the phase II metabolism that catalyse the formation of thioether conjugates between the endogenous tripeptide glutathione and xenobiotic compounds. The voltammetric behaviour of glutathione (GSH), 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione S-transferase (GST), as well as the catalytic conjugation reaction of GSH to CDNB by GST was investigated at room temperature, T=298.15K (25°C), at pH6.5, for low concentration of substrates and enzyme, using differential pulse (DP) voltammetry at a glassy carbon electrode. Only GSH can be oxidized; a sensitivity of 0.14nA/µM and a LOD of 6.4µM were obtained. The GST kinetic parameter electrochemical evaluation, in relation to its substrates, GSH and CDNB, using reciprocal Michaelis-Menten and Lineweaver-Burk double reciprocal plots, was determined. A value of KM~100µM was obtained for either GSH or CDNB, and Vmax varied between 40 and 60µmol/min per mg of GST.

Stable isotope labeling method for the investigation of protein haptenation by electrophilic skin sensitizers.

The risk of contact sensitization is a major consideration in the development of new formulations for personal care products. However, developing a mechanistic approach for non-animal risk assessment requires further understanding of haptenation of skin proteins by sensitizing chemicals, which is the molecular initiating event causative of skin sensitization. The non-stoichiometric nature of protein haptenation results in relatively low levels of modification, often of low abundant proteins, presenting a major challenge for their assignment in complex biological matrices such as skin. Instrumental advances over the last few years have led to a considerable increase in sensitivity of mass spectrometry (MS) techniques. We have combined these advancements with a novel dual-labeling/LC-MS(E) approach to provide an in-depth direct comparison of human serum albumin (HSA), 2,4-dinitro-1-chlorobenzene (DNCB), 5-chloro-2-methyl-4-isothiazolin-3-one (MCI), trans-cinnamaldehyde, and 6-methyl coumarin. These data have revealed novel insights into the differences in protein haptenation between sensitizers with different reaction mechanisms and sensitizing potency; the extreme sensitizers DNCB and MCI were shown to modify a greater number of nucleophilic sites than the moderate sensitizer cinnamaldehyde; and the weak/non-sensitizer 6-methyl coumarin was restricted to only a single nucleophilic residue within HSA. The evaluation of this dual labeling/LC-MS(E) approach using HSA as a model protein has also demonstrated that this strategy could be applied to studying global haptenation in complex mixtures of skin-related proteins by different chemicals.

Purification and characterization of the Staphylococcus aureus bacillithiol transferase BstA.

BACKGROUND: Gram-positive bacteria in the phylum Firmicutes synthesize the low molecular weight thiol bacillithiol rather than glutathione or mycothiol. The bacillithiol transferase YfiT from Bacillus subtilis was identified as a new member of the recently discovered DinB/YfiT-like Superfamily. Based on structural similarity using the Superfamily program, we have determined 30 of 31 Staphylococcus aureus strains encode a single bacillithiol transferase from the DinB/YfiT-like Superfamily, while the remaining strain encodes two proteins. METHODS: We have cloned, purified, and confirmed the activity of a recombinant bacillithiol transferase (henceforth called BstA) encoded by the S. aureus Newman ORF NWMN_2591. Moreover, we have studied the saturation kinetics and substrate specificity of this enzyme using in vitro biochemical assays. RESULTS: BstA was found to be active with the co-substrate bacillithiol, but not with other low molecular weight thiols tested. BstA catalyzed bacillithiol conjugation to the model substrates monochlorobimane, 1-chloro-2,4-dinitrobenzene, and the antibiotic cerulenin. Several other molecules, including the antibiotic rifamycin S, were found to react directly with bacillithiol, but the addition of BstA did not enhance the rate of reaction. Furthermore, cells growing in nutrient rich medium exhibited low BstA activity. CONCLUSIONS: BstA is a bacillithiol transferase from S. aureus that catalyzes the detoxification of cerulenin. Additionally, we have determined that bacillithiol itself might be capable of directly detoxifying electrophilic molecules. GENERAL SIGNIFICANCE: BstA is an active bacillithiol transferase from S. aureus Newman and is the first DinB/YfiT-like Superfamily member identified from this organism. Interestingly, BstA is highly divergent from B. subtilis YfiT.