Low Molecular Weight Thiols Research Articles

Mechanistic basis for impaired ferroptosis in cells expressing the African-centric S47 variant of p53

A population-restricted single-nucleotide coding region polymorphism (SNP) at codon 47 exists in the human TP53 gene (P47S, hereafter P47 and S47). In studies aimed at identifying functional differences between these variants, we found that the African-specific S47 variant associates with an impaired response to agents that induce the oxidative stress-dependent, nonapoptotic cell death process of ferroptosis. This phenotype is manifested as a greater resistance to glutamate-induced cytotoxicity in cultured cells as well as increased carbon tetrachloride-mediated liver damage in a mouse model. The differential ferroptotic responses associate with intracellular antioxidant differences between P47 and S47 cells, including elevated abundance of the low molecular weight thiols coenzyme A (CoA) and glutathione in S47 cells. Importantly, the disparate ferroptosis phenotypes related to the P47S polymorphism are reversible. Exogenous administration of CoA provides protection against ferroptosis in cultured mouse and human cells, as well as in a mouse model. The combined data support a positive role for p53 in ferroptosis and identify CoA as a regulator of this cell death process. Together, these findings provide mechanistic insight linking redox regulation of p53 to small molecule antioxidants and stress signaling pathways. They also identify potential therapeutic approaches to redox-related pathologies.

Glutathione transferase catalyzes the addition of glutathione to nitro‐conjugated linoleic acid

Nitroalkene fatty acids are formed in vivo and are currently being tested in two Phase 2 clinical trials. They modulate cellular responses through Michael addition reactions with thiol‐containing proteins. Nitro‐conjugated linoleic acid (NO2‐CLA) is formed preferentially both in vivo and in vitro. There are two main positional isomers of NO2‐CLA, 9‐ and 12‐NO2‐CLA. They both react with low molecular weight thiols at two electrophilic sites located on the β‐ and δ‐carbons giving rise to reversible biphasic kinetics. The fast reaction corresponds to the formation of the β‐adduct (the kinetic product), while the slow reaction corresponds to the δ‐adduct (the thermodynamic product) (Turell, Vitturi, J. Biol. Chem., 2017). With the aim of further characterizing the biological chemistry of this bifunctional nitroalkene, we evaluated if its reaction with glutathione (GSH) is catalyzed by glutathione transferases (GST). The reaction was followed by monitoring NO2‐CLA decay at 330 nm using a stopped flow spectrophotometer. A mu‐type glutathione transferase from the parasitic platyhelminth Echinococcus granulosus (EgGST1) was used. Catalysis was observed in both phases when using a mixture of NO2‐CLA isomers. Remarkably, the δ‐addition was accelerated □13 times more than the β‐addition, 4.4 × 106 vs 3.4 × 105 M−1, respectively, at 2 mM GSH and 10 μM NO2‐CLA (25 °C, pH 7.4), suggesting specificity. To evaluate whether there is a preference of the enzyme for the NO2‐CLA isomer, purified 9‐ or 12‐ NO2‐CLA were used. No difference was observed between them. The reaction between GSH and nitro‐oleic acid (NO2‐OA), a nitroalkene fatty acid with only one electrophilic site in β, was also catalyzed by this enzyme. An acceleration of 1 × 105 M−1 was observed (2 mM GSH, 20 μM NO2‐OA), in line with the value obtained for the β‐addition of NO2‐CLA. We are now further characterizing this reaction which is expected to affect nitroalkene fatty acids metabolism and excretion and thus impact their signaling actions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Antimicrobial garlic-derived diallyl polysulfanes: Interactions with biological thiols in Bacillus subtilis

BackgroundDiallylpolysulfanes are the key constituents of garlic oils, known to exhibit broad spectrum anticancer and antimicrobial activity. Studies in vitro, and in mammalian cells, have shown they react, via thiol-polysulfane exchange, with their major low molecular weight thiol, glutathione. However, there are no detailed reports of diallylpolysulfane effects on other common thiol metabolites (cysteine and coenzyme A) or major thiol cofactors (e.g. bacillithiol) that many Gram positive bacteria produce instead of glutathione. MethodsDiallylpolysulfanes were individually purified then screened for antimicrobial activity against Bacillus subtilis. Their impact on thiol metabolites (bacillithiol, cysteine, coenzyme A, protein thiols allyl thiols//persulfides) in B. subtilis cultures were analysed, by HPLC. ResultsDiallylpolysulfane bioactivity increased with increasing chain length up to diallyltetrasulfane, but then plateaued. Within two minutes of treating B. subtilis with diallyltrisulfane or diallyltetrasulfane intracellular bacillithiol levels decreased by ~90%. Cysteine and CoA were also affected but to a lesser degree. This was accompanied by the accumulation of allyl thiol and allyl persulfide. A significant level of protein-S-allylation was also detected. ConclusionsIn addition to the major low molecular weight thiol, diallylpolysulfanes can also have an impact on other thiol metabolites and protein thiols. General significanceThis study shows the rapid parallel impact of polysulfanes on different biological thiols inside Bacillus subtilis alongside the concomitant generation of allyl thiols and persulfides.

Untargeted adductomics of Cys34 modifications to human serum albumin in newborn dried blood spots.

Metabolism of chemicals from the diet, exposures to xenobiotics, the microbiome, and lifestyle factors (e.g., smoking, alcohol intake) produce electrophiles that react with nucleophilic sites in circulating proteins, notably Cys34 of human serum albumin (HSA). To discover potential risk factors resulting from in utero exposures, we are investigating HSA-Cys34 adducts in archived newborn dried blood spots (DBS) that reflect systemic exposures during the last month of gestation. The workflow includes extraction of proteins from DBS, measurement of hemoglobin (Hb) to normalize for blood volume, addition of methanol to enrich HSA by precipitation of Hb and other interfering proteins, digestion with trypsin, and detection of HSA-Cys34 adducts via nanoflow liquid chromatography-high-resolution mass spectrometry. As proof-of-principle, we applied the method to 49 archived DBS collected from newborns whose mothers either actively smoked during pregnancy or were nonsmokers. Twenty-six HSA-Cys34 adducts were detected, including Cys34 oxidation products, mixed disulfides with low molecular weight thiols (e.g., cysteine, homocysteine, glutathione, cysteinylglycine), and other modifications. Data were normalized with a novel method ("scone") to remove unwanted technical variation arising from HSA digestion, blood volume, DBS age, mass spectrometry analysis, and batch effects. Using an ensemble of linear and nonlinear models, the Cys34 adduct of cyanide was found to consistently discriminate between newborns of smoking and nonsmoking mothers with a mean fold change (smoking/nonsmoking) of 1.31. These results indicate that DBS adductomics is suitable for investigating in utero exposures to reactive chemicals and metabolites that may influence disease risks later in life.

YpdA, a putative bacillithiol disulfide reductase, contributes to cellular redox homeostasis and virulence in Staphylococcus aureus.

The intracellular redox environment of Staphylococcus aureus is mainly buffered by bacillithiol (BSH), a low molecular weight thiol. The identity of enzymes responsible for the recycling of oxidized bacillithiol disulfide (BSSB) to the reduced form (BSH) remains elusive. We examined YpdA, a putative bacillithiol reductase, for its role in maintaining intracellular redox homeostasis. The ypdA mutant showed increased levels of BSSB and a lower bacillithiol redox ratio vs. the isogenic parent, indicating a higher level of oxidative stress within the bacterial cytosol. We showed that YpdA consumed NAD(P)H; and YpdA protein levels were augmented in response to stress. Wild type strains overexpressing YpdA showed increased tolerance to oxidants and electrophilic agents. Importantly, YpdA overexpression in the parental strain caused an increase in BSH levels accompanied by a decrease in BSSB concentration in the presence of stress, resulting in an increase in bacillithiol redox ratio vs. the vector control. Additionally, the ypdA mutant exhibited decreased survival in human neutrophils (PMNs) as compared with the parent, while YpdA overexpression protected the resulting strain from oxidative stress in vitro and from killing by human neutrophils ex vivo. Taken together, these data present a new role for YpdA in S. aureus physiology and virulence through the bacillithiol system.

Recent advances in the biosynthesis of lincosamide antibiotics

Lincosamides characterized by an unusual C1 mercapto substituted 8-carbon sugar joined with a proline moiety by an amide linkage, is a small family of antibiotics. The vital role in clinic and the unique structures have attracted much attention for many years. Natural lincosamides contain only four compounds to date: lincomycin A and B, celesticetin and Bu-2545, among which only lincomycin A possesses good in vitro and in vivo potency against a variety of Gram-positive microorganisms, such as Bacillus anthracis , Staphylococcus aureus , Staphylococcus epidermidis , Streptococcus agalactiae , Streptococcus pneumoniae , Streptococcus pyogenes and Streptococcus viridans . Because of the excellent antibiotic activity of lincomycin A, hundreds of synthetic, semisynthetic and biosynthetic fermentation derivatives have been prepared. However, most of them are not suitable for clinical use due to the low biological activity or high toxicity and so on. Only clindamycin, a semisynthetic analog of lincomycin A, is a clinical acceptance and shows better antibiotic activities than lincomycin A. Natural lincosamides have typically been isolated from actinomycetes and are generated from large and complex gene clusters. Despite found and clinical used for nearly half a century, their biosynthetic pathways have not been elucidated completely. During the past few years, great progress was achieved on lincosamides biosynthetic pathway. It is found that the C1 mercapto substituted 8-carbon sugar derived from the condensation of d-ribose 5-phosphate and d-fructose 6-phosphate or d-sedoheptulose 7-phosphate. During the biosynthesis of the octose, two unique low molecular weight thiols: ergothioneine and mycothiol, function through two unusual glycosylations that program lincosamide transfer, activation and modification, providing the first paradigm for ergothioneine associated biochemical processes and for the poorly understood mycothiol dependent biotransformations. The biosynthesis of lincomycin and celesticestin is branched from a pair of homologous proteins, both of which are pyridoxal-5′-phosphate dependent proteins and are highly related in phylogenesis, but cysteine S -conjugated intermediates are processed in different ways and associated with individual downstream enzyme(s) toward distinct S -functionalization of lincomycin and celesticetin. During the formation of the trans 4-propyl- L -proline moiety in lincomycin, two γ-glutamyltranspeptidase homolog proteins (the specific LmbA and a non-specific LmbA2991) catalyze an unusual C−C bond cleavage reaction. Both of them belong to N -terminal nucleophile (Ntn) hydrolase superfamily, and exhibit the activity for post-translational autocatalytic cleavage of a peptide precursor to form a functional heterodimer in which the newly released N -terminal threonine residue acts as a nucleophile to attack a carbonyl group and catalyze the C−C bond cleavage. For the linkage of the octose and the pyrrole rings, the latter are activated by standalone adenylation domains and uploaded to peptidyl carrier proteins, followed by condensation reaction with ergothioneine S -conjugate lincosamine to afford amide bonds. It cannot imagine that such small molecules are biosynthesized via inconceivable process. Although there are still some questions on lincosamides biosynthesis, chances are that more surprise will be unveiled in the near future. Furthermore, on the basis of the biosynthetic mechanism of lincosamides, their biosynthetic pathway could be rationally reconstructed to obtain diverse lincosamide analogues which have distinct physiological activities and are difficult to synthesize, including several unnatural chimeras with antibacterial properties better than lincomycin A, opening the door for combinatorial biosynthesis of hybrid lincosamide antibiotics. But more than that, due to the excellent antibacterial activity of lincomycin A, the lincomycin-producing strain- Streptomyces lincolnensis has been engineered for quite a long time to improve the production of lincomycin A, which may help cut production costs and simplify downstream separation processes. This review mainly covers the most recent advances in the biosynthesis of lincomycin and celesticetin, and on this basis, in the structural diversity expansion and lincomycin-producing strain engineering.

Network Theory and the Resilience of Redox Signaling

The redox status inside and around cells is critically important to control, being used to maintain reduced compounds in the correct state and for cell signaling mechanisms. A myriad of compounds and proteins are involved in a vast network system to regulate the redox state of biological systems. These include reactive molecules such reactive oxygen species (ROS), nitric oxide (NO·) and hydrogen sulfide (H2S) along with systems for their removal, such as antioxidants. Redox buffering involves molecules such as glutathione, low molecular weight thiols and ascorbate. Network Theory attempts to give the mechanisms underlying complex networks a mathematical and model-based underpinning and it has been suggested that metabolic systems can be described as scale-free networks, having a power law degree distribution. Such networks are said to be both robust but vulnerable, suggesting a level of resilience. Redox metabolism also has to be robust, being maintained in what has been described as the Goldilocks Zone, while it is also vulnerable to outside influence, often leading to the phenomenon referred to as oxidative stress. Therefore, it is suggested here that a holistic approach to understand redox networks should embrace Network Theory, which may be able to predict characteristics of the redox network that can be targeted for new therapeutics or agricultural treatments.

Depletion of protein thiols and the accumulation of oxidized thioredoxin in Parkinsonism disclosed by a red-emitting and environment-sensitive probe.

Protein sulfhydryl groups play a vital role in maintaining cellular redox homeostasis and protein functions and have attracted increasing interests for the selective detection of protein thiols over low-molecular-weight thiols (LMWTs). Herein, we reported a red-emitting and environment-sensitive probe (FM-red) for detecting and labeling protein thiols. The probe contains a maleimide unit as a thiol receptor and an environment-sensitive fluorophore as a sensor. The emission signal of the probe was exclusively switched on by binding to protein sulfhydryl groups through the twisted intramolecular charge transfer mechanism, while negligible fluorescence was observed when FM-red reacted with LMWTs. Various experiments verified that FM-red possessed fast responsivity (∼10 min) and high selectivity to sense protein thiols over LMWTs with a red emission (∼655 nm). These favorable properties enable the probe to image protein sulfhydryl groups in live cells and in vivo. In addition, as FM-red has a relatively high molecular weight (MW 688), it is able to separate the labeled proteins from the unlabeled ones after FM-red derivatization via routine protein electrophoresis, which may be applied to determine the redox states of thioredoxin, a small redox protein ubiquitous in all cells. With the aid of the probe, we demonstrated a significant decrease in the protein thiols and the accumulation of oxidized thioredoxin in a cellular model of Parkinson's disease.

Stable integration of the Mrx1-roGFP2 biosensor to monitor dynamic changes of the mycothiol redox potential in Corynebacterium glutamicum

Mycothiol (MSH) functions as major low molecular weight (LMW) thiol in the industrially important Corynebacterium glutamicum. In this study, we genomically integrated an Mrx1-roGFP2 biosensor in C. glutamicum to measure dynamic changes of the MSH redox potential (EMSH) during the growth and under oxidative stress. C. glutamicum maintains a highly reducing intrabacterial EMSH throughout the growth curve with basal EMSH levels of ~− 296 mV. Consistent with its H2O2 resistant phenotype, C. glutamicum responds only weakly to 40 mM H2O2, but is rapidly oxidized by low doses of NaOCl. We further monitored basal EMSH changes and the H2O2 response in various mutants which are compromised in redox-signaling of ROS (OxyR, SigH) and in the antioxidant defense (MSH, Mtr, KatA, Mpx, Tpx). While the probe was constitutively oxidized in the mshC and mtr mutants, a smaller oxidative shift in basal EMSH was observed in the sigH mutant. The catalase KatA was confirmed as major H2O2 detoxification enzyme required for fast biosensor re-equilibration upon return to non-stress conditions. In contrast, the peroxiredoxins Mpx and Tpx had only little impact on EMSH and H2O2 detoxification. Further live imaging experiments using confocal laser scanning microscopy revealed the stable biosensor expression and fluorescence at the single cell level. In conclusion, the stably expressed Mrx1-roGFP2 biosensor was successfully applied to monitor dynamic EMSH changes in C. glutamicum during the growth, under oxidative stress and in different mutants revealing the impact of Mtr and SigH for the basal level EMSH and the role of OxyR and KatA for efficient H2O2 detoxification under oxidative stress.

Mass spectrometric studies of Cu(I)-binding to the N-terminal domains of B. subtilis CopA and influence of bacillithiol

CopA is a Cu(I)-exporting transmembrane P1B-type ATPase from Bacillus subtilis. It contains two N-terminal cytoplasmic domains, CopAab, which bind Cu(I) with high affinity and to form higher-order complexes with multiple Cu(I) ions. To determine the precise nature of these species, electrospray ionisation mass spectrometry (ESI-MS) under non-denaturing conditions was employed. Up to 1 Cu per CopAab resulted in Cu coordination to one or both CopAab domains. At >1 Cu/CopAab, two distinct dimeric charge state envelopes were observed, corresponding to distinct conformations, each with Cu6(CopAab)2 as its major form. The influence of the physiologically relevant low molecular weight thiol bacillithiol (BSH) on Cu(I)-binding to CopAab was assessed. Dimeric CopAab persisted in the presence of BSH, with previously undetected Cu7(CopAab)2 and Cu6(CopAab)2(BSH) forms apparent.

Cold modulated nuclear S-nitrosoproteome analysis indicates redox modulation of novel Brassicaceae specific, myrosinase and napin in Brassica juncea

Stress leads to production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Among the RNS, nitric oxide (NO) is an important signaling molecule that mediates its effect majorly via a reversible post-translational modification (PTM) S-nitrosylation. However, information on the NO mediated cold stress signaling is still missing for nucleus. Therefore, in the present study an attempt was made to analyze NO signaling, particularly, S-nitrosylation in the nuclei of Brassica juncea seedlings. Cold stress (4 °C) induced nitrate reductase (NR) mediated NO production along with the accumulation of available protein thiols (5 fold, at 96 h, 4 °C) and low molecular weight thiols (1.2 fold at 12 h, 4 °C). S-nitrosoglutathione (GSNO, a NO donor) and cold (endogenous, S-nitrosylation without the addition of NO donor) treated, neutravidin-agarose affinity chromatography purified S-nitrosylated proteins were resolved as 55- and 32-spots respectively on the 2-D gels. Functional categorization after MALDI-TOF/TOF identification showed 67% targets to be stress/defense/redox related and 33% to be metabolic, suggesting the potential role(s) of S-nitrosylation in these responses. Cold stress increased S-nitrosylation of Cu/Zn SOD, l-ascorbate oxidase, fructose bisphosphate aldolase (FBA2) and malate dehydrogenase 1 (MDH1), therefore, indicating NO mediated regulation of redox and metabolism related pathways. Identification of targets of glucosinolate hydrolysis pathway (myrosinase and myrosinase-binding protein 2-like) indicated the role of S-nitrosylation in regulating mustard oil bomb. Presence of myrosinase was confirmed by activity measurements and western blotting. Increased S-nitrosylation of myrosinase in cold stress in the nucleus is reported for the first time. Here, we proposed S-nitrosylation mediated nuclear trafficking of non-nuclear targets such as beta-d-xylosidase, daikon cysteine protease, and beta-glucosidase to the nucleus. Similarly, identification of FBA2 and MDH1 in the nucleus, indicated the role of NO in modulating their moonlighting function via S-nitrosylation in stress. Present study provided the first repertoire of cold modulated S-nitrosylated proteins in the nucleus.

Changes in Glutathione Redox Potential Are Linked to Aβ42-Induced Neurotoxicity

Glutathione is the major low-molecular weight thiol ofeukaryotic cells. It is central to one of the two major NADPH-dependent reducing systems and is likely to play a role in combating oxidative stress, a process suggested to play a key role in Alzheimer's disease (AD). However, the nature and relevance of redox changes in the onset and progression of AD are still uncertain. Here, we combine genetically encoded redox sensors with our Drosophila models of amyloid-beta (Aβ) aggregation. We find that changes in glutathione redox potential (EGSH) closely correlate with disease onset and progression. We observe this redox imbalance specifically in neurons, but not in glia cells. EGSH changes and Aβ42 deposition are also accompanied by increased JNK stress signaling. Furthermore, pharmacologic and genetic manipulation of glutathione synthesis modulates Aβ42-mediated neurotoxicity, suggesting a causal relationship between disturbed glutathione redox homeostasis and early AD pathology.

Linking Microbial Activities and Low-Molecular-Weight Thiols to Hg Methylation in Biofilms and Periphyton from High-Altitude Tropical Lakes in the Bolivian Altiplano.

The sources and factors controlling concentrations of monomethylmercury (MMHg) in aquatic ecosystems need to be better understood. Here, we investigated Hg transformations in sediments, periphyton associated with green algae's or aquatic plants, and benthic biofilms from the Lake Titicaca hydrosystem and compared them to the occurrence of active methylating microorganisms and extracellular Hg ligands. Intense Hg methylation was found in benthic biofilms and green algae's periphyton, while it remained low in sediments and aquatic plants' periphyton. Demethylation varied between compartments but remained overall in the same range. Hg methylation was mainly carried out by sulfate reducers, although methanogens also played a role. Its variability between compartments was first explained by the presence or absence of the hgcAB genes. Next, both benthic biofilm and green algae's periphyton exhibited a great diversity of extracellular low-molecular-weight (LMW) thiols (13 or 14 compounds) present at a range of a few nmol L-1 or μmol L-1 but clearly dominated by cysteine and 3-mercaptopropionic acid. Hg methylation was overall positively correlated to the total thiol concentrations, albeit to different extents according to the compartment and conditions. This work is the first examining the interplay between active methylating bacterial communities and extracellular ligands in heterotrophic biofilms and supports the involvement of LMW thiols in Hg methylation in real aquatic systems.

The functional interplay of low molecular weight thiols in Mycobacterium tuberculosis

BackgroundThree low molecular weight thiols are synthesized by Mycobacterium tuberculosis (M.tb), namely ergothioneine (ERG), mycothiol (MSH) and gamma-glutamylcysteine (GGC). They are able to counteract reactive oxygen species (ROS) and/or reactive nitrogen species (RNS). In addition, the production of ERG is elevated in the MSH-deficient M.tb mutant, while the production of MSH is elevated in the ERG-deficient mutants. Furthermore, the production of GGC is elevated in the MSH-deficient mutant and the ERG-deficient mutants. The propensity of one thiol to be elevated in the absence of the other prompted further investigations into their interplay in M.tb.MethodsTo achieve that, we generated two M.tb mutants that are unable to produce ERG nor MSH but are able to produce a moderate (ΔegtD-mshA) or significantly high (ΔegtB-mshA) amount of GGC relative to the wild-type strain. In addition, we generated an M.tb mutant that is unable to produce GGC nor MSH but is able to produce a significantly low level of ERG (ΔegtA-mshA) relative to the wild-type strain. The susceptibilities of these mutants to various in vitro and ex vivo stress conditions were investigated and compared.ResultsThe ΔegtA-mshA mutant was the most susceptible to cellular stress relative to its parent single mutant strains (ΔegtA and ∆mshA) and the other double mutants. In addition, it displayed a growth-defect in vitro, in mouse and human macrophages suggesting; that the complete inhibition of ERG, MSH and GGC biosynthesis is deleterious for the growth of M.tb.ConclusionsThis study indicates that ERG, MSH and GGC are able to compensate for each other to maximize the protection and ensure the fitness of M.tb. This study therefore suggests that the most effective strategy to target thiol biosynthesis for anti-tuberculosis drug development would be the simultaneous inhibition of the biosynthesis of ERG, MSH and GGC.

Paracetamol and Cardiac Congenital Malformations in Prediabetic Pregnancy Women

Hyperglycemia can influence the development of the fetal heart, affecting both its structure and its function. Prospective and retrospective cohort studies have demonstrated an increased risk of congenital abnormalities with gestational diabetes. This observation is probably related to the inclusion of women with unrecognized type 2 diabetes [1, 2]. Substantial literature indicates that diabetes in pregnant rats and mice induces embryo lethality, growth retardation and a variable incidence of birth defects. Then, the maintenance of normal concentrations of metabolites from all nutrient classes may be important for prevention of adverse fetal outcome in diabetic pregnancy. Acetaminophen overdose is the most often cause of acute liver injury and obese women are in particular risk, because is able to induce mitochondrial oxidative stress [3]. Acetaminophen (Paracetamol) over doses decreased embryonic low-molecularweight thiols (glutathione and cysteine), compounds that play a vital role in the detoxication of exogenous and endogenous chemicals [3-5]. The apparent safety of Paracetamol drug, a useful analgesic only (with no anti-inflammatory properties) is compromised by its widespread and extensive chronic use, particularly in Peruvian population, where its analgesic is doing without control [6-9]. In fact, paracetamol though considered safe at a considerable low dose, especially in women could cause kidney derangement and cardiac malformations during pregnant state if the drug is ingested in the first trimester [8]. Major congenital malformations, including those affecting the cardiovascular system, remain the leading cause of mortality and morbidity in infants of diabetic mothers [10]. Thus, there is overcome potential maternal acetaminophen (paracetamol) toxicity [11].

Arabidopsis mutants impaired in glutathione biosynthesis exhibit higher sensitivity towards the glucosinolate hydrolysis product allyl-isothiocyanate

Upon tissue damage the plant secondary metabolites glucosinolates can generate various hydrolysis products, including isothiocyanates (ITCs). Their role in plant defence against insects and pest and their potential health benefits have been well documented, but our knowledge regarding the endogenous molecular mechanisms of their effect in plants is limited. Here we investigated the effect of allyl-isothiocyanate (AITC) on Arabidopsis thaliana mutants impaired in homeostasis of the low-molecular weight thiol glutathione. We show that glutathione is important for the AITC-induced physiological responses, since mutants deficient in glutathione biosynthesis displayed a lower biomass and higher root growth inhibition than WT seedlings. These mutants were also more susceptible than WT to another ITC, sulforaphane. Sulforaphane was however more potent in inhibiting root growth than AITC. Combining AITC with the glutathione biosynthesis inhibitor L-buthionine-sulfoximine (BSO) led to an even stronger phenotype than observed for the single treatments. Furthermore, transgenic plants expressing the redox-sensitive fluorescent biomarker roGFP2 indicated more oxidative conditions during AITC treatment. Taken together, we provide genetic evidence that glutathione plays an important role in AITC-induced growth inhibition, although further studies need to be conducted to reveal the underlying mechanisms.

Inhibition of thioredoxin A1 from Corynebacterium pseudotuberculosis by polyanions and flavonoids

In pathogens, the thioredoxin system forms part of the defense against oxidative stress and ensures the formation of the proper disulfide bonds to ensure protein function. In Corynebacterium pseudotuberculosis, the role and mechanism of TrxA1 has not been elucidated, but, the significant homology among different Trxs and the conservation of the residues that form their active sites underline the importance of the Trx systems. Proteins involved in redox metabolism and low molecular weight thiols, which might interact with them, become attractive targets to modulate the activity of pathogens.The activity of the protein was investigated using a turbidimetric assay system. The influence of different pH and low molecular weight thiols were tested. Additionally, this assay was used to investigate the inhibitory potential of ligands from different molecular families, such as, polyanions (suramin and heparin) and flavonoids (hesperetin and hesperidin). All four compounds showed inhibition of the protein activity by approximately 80%. The interactions between these compounds and Cp-TrxA1 were investigated using CD spectroscopy, NMR, molecular docking and dynamics. Our results demonstrate that suramin and hesperetin can serve as lead molecules for the development of specific inhibitors for the C. pseudotuberculosis TrxA1.

Hydrogen Sulfide Sensing through Reactive Sulfur Species (RSS) and Nitroxyl (HNO) in Enterococcus faecalis

Recent studies of hydrogen sulfide (H2S) signaling implicate low molecular weight (LMW) thiol persulfides and other reactive sulfur species (RSS) as signaling effectors. Here, we show that a CstR protein from the human pathogen Enterococcus faecalis ( E. faecalis), previously identified in Staphylococcus aureus ( S. aureus), is an RSS-sensing repressor that transcriptionally regulates a cst-like operon in response to both exogenous sulfide stress and Angeli's salt, a precursor of nitroxyl (HNO). E. faecalis CstR reacts with coenzyme A persulfide (CoASSH) to form interprotomer disulfide and trisulfide bridges between C32 and C61', which negatively regulate DNA binding to a consensus CstR DNA operator. A Δ cstR strain exhibits deficiency in catheter colonization in a catheter-associated urinary tract infection (CAUTI) mouse model, suggesting sulfide regulation and homeostasis is critical for pathogenicity. Cellular polysulfide metabolite profiling of sodium sulfide-stressed E. faecalis confirms an increase in both inorganic polysulfides and LMW thiols and persulfides sensed by CstR. The cst-like operon encodes two authentic thiosulfate sulfurtransferases and an enzyme we characterize here as an NADH and FAD-dependent coenzyme A (CoA) persulfide reductase (CoAPR) that harbors an N-terminal CoA disulfide reductase (CDR) domain and a C-terminal rhodanese homology domain (RHD). Both cysteines in the CDR (C42) and RHD (C508) domains are required for CoAPR activity and complementation of a sulfide-induced growth phenotype of a S. aureus strain lacking cstB, encoding a nonheme FeII persulfide dioxygenase. We propose that S. aureus CstB and E. faecalis CoAPR employ orthogonal chemistries to lower CoASSH that accumulates under conditions of cellular sulfide toxicity and signaling.

Structure and function of the bacillithiol-S-transferase BstA from Staphylococcus aureus.

Bacillithiol is a low-molecular weight thiol produced by many gram-positive organisms, including Staphylococcus aureus and Bacillus anthracis. It is the major thiol responsible for maintaining redox homeostasis and cellular detoxification, including inactivation of the antibiotic fosfomycin. The metal-dependent bacillithiol transferase BstA is likely involved in these sorts of detoxification processes, but the exact substrates and enzyme mechanism have not been identified. Here we report the 1.34 Å resolution X-ray crystallographic structure of BstA from S. aureus. Our structure confirms that BstA belongs to the YfiT-like metal-dependent hydrolase superfamily. Like YfiT, our structure contains nickel within its active site, but our functional data suggest that BstA utilizes zinc for activity. Although BstA and YfiT both contain a core four helix bundle and coordinate their metal ions in the same fashion, significant differences between the protein structures are described here.

Identification of the S-transferase like superfamily bacillithiol transferases encoded by Bacillus subtilis.

Bacillithiol is a low molecular weight thiol found in Firmicutes that is analogous to glutathione, which is absent in these bacteria. Bacillithiol transferases catalyze the transfer of bacillithiol to various substrates. The S-transferase-like (STL) superfamily contains over 30,000 putative members, including bacillithiol transferases. Proteins in this family are extremely divergent and are related by structural rather than sequence similarity, leaving it unclear if all share the same biochemical activity. Bacillus subtilis encodes eight predicted STL superfamily members, only one of which has been shown to be a bacillithiol transferase. Here we find that the seven remaining proteins show varying levels of metal dependent bacillithiol transferase activity. We have renamed the eight enzymes BstA-H. Mass spectrometry and gene expression studies revealed that all of the enzymes are produced to varying levels during growth and sporulation, with BstB and BstE being the most abundant and BstF and BstH being the least abundant. Interestingly, several bacillithiol transferases are induced in the mother cell during sporulation. A strain lacking all eight bacillithiol transferases showed normal growth in the presence of stressors that adversely affect growth of bacillithiol-deficient strains, such as paraquat and CdCl2. Thus, the STL bacillithiol transferases represent a new group of proteins that play currently unknown, but potentially significant roles in bacillithiol-dependent reactions. We conclude that these enzymes are highly divergent, perhaps to cope with an equally diverse array of endogenous or exogenous toxic metabolites and oxidants.