RESUMEN
Genetically-encoded redox biosensors have become invaluable tools for monitoring cellular redox processes with high spatiotemporal resolution, coupling the presence of the redox-active analyte with a change in fluorescence signal that can be easily recorded. This review summarizes the available fluorescence recording methods and presents an in-depth classification of the redox biosensors, organized by the analytes they respond to. In addition to the fluorescent protein-based architectures, this review also describes the recent advances on fluorescent, chemigenetic-based redox biosensors and other emerging chemigenetic strategies. This review examines how these biosensors are designed, the biosensors sensing mechanism, and their practical advantages and disadvantages.
Asunto(s)
Técnicas Biosensibles , Oxidación-Reducción , Técnicas Biosensibles/métodos , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas Luminiscentes/química , AnimalesRESUMEN
Reversibly switchable monomeric Cherry (rsCherry) is a photoswitchable variant of the red fluorescent protein mCherry. We report that this protein gradually and irreversibly loses its red fluorescence in the dark over a period of months at 4 °C and a few days at 37 °C. We also find that its ancestor, mCherry, undergoes a similar fluorescence loss but at a slower rate. X-ray crystallography and mass spectrometry reveal that this is caused by the cleavage of the p-hydroxyphenyl ring from the chromophore and the formation of two novel types of cyclic structures at the remaining chromophore moiety. Overall, our work sheds light on a new process occurring within fluorescent proteins, further adding to the chemical diversity and versatility of these molecules.
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Oxígeno , Conformación Proteica , Modelos Moleculares , Proteínas Luminiscentes/química , Cristalografía por Rayos X , Proteínas Fluorescentes Verdes/química , Proteína Fluorescente RojaRESUMEN
Protein S-persulfidation (P-SSH) is recognized as a common posttranslational modification. It occurs under basal conditions and is often observed to be elevated under stress conditions. However, the mechanism(s) by which proteins are persulfidated inside cells have remained unclear. Here we report that 3-mercaptopyruvate sulfur transferase (MPST) engages in direct protein-to-protein transpersulfidation reactions beyond its previously known protein substrates thioredoxin and MOCS3/Uba4, associated with H2S generation and transfer RNA thiolation, respectively. We observe that depletion of MPST in human cells lowers overall intracellular protein persulfidation levels and identify a subset of proteins whose persulfidation depends on MPST. The predicted involvement of these proteins in the adaptation to stress responses supports the notion that MPST-dependent protein persulfidation promotes cytoprotective functions. The observation of MPST-independent protein persulfidation suggests that other protein persulfidases remain to be identified.
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Sulfurtransferasas , Humanos , Cisteína , Sulfuro de Hidrógeno/metabolismo , Azufre/metabolismoRESUMEN
Antibiotic-producing microorganisms usually require one or more self-resistance determinants to survive antibiotic production. The effectors of these mechanisms are proteins that inactivate the antibiotic, facilitate its transport, or modify the target to render it insensitive to the molecule. Streptomyces bacteria biosynthesize various bioactive natural products and possess resistance systems for most metabolites, which are coregulated with antibiotic biosynthesis genes. Streptomyces olindensis strain DAUFPE 5622 produces the antitumor antibiotic cosmomycin D (COSD), a member of the anthracycline family. In this study, we propose three self-resistance mechanisms, anchored or based in the COSD biosynthetic gene cluster. These include cosIJ (an ABC transporter), cosU (a UvrA class IIa protein), and a new self-resistance mechanism encoded by cosP, which shows response against peroxides by the enzyme mycothiol peroxidase (MPx). Activity-based investigations of MPx and its mutant enzyme confirmed peroxidation during the production of COSD. Overexpression of the ABC transporter, the UvrA class IIa protein, and the MPx led to an effective response against toxic anthracyclines, such as cosmomycins. Our findings help to understand how thiol peroxidases play an antioxidant role in the anthracycline producer S. olindensis DAUFPE 5622, a mechanism which has been reported for neoplastic cells that are resistant to doxorubicin (DOX). IMPORTANCE Anthracycline compounds are DNA intercalating agents widely used in cancer chemotherapeutic protocols. This work focused on the self-resistance mechanisms developed by the cosmomycin-producing bacterium Streptomyces olindensis. Our findings showed that cysteine peroxidases, such as mycothiol peroxidase, encoded by the gene cosP, protected S. olindensis against peroxidation during cosmomycin production. This observation can contribute to much better understanding of resistance both in the producers, eventually enhancing production, and in some tumoral cell lines.
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Antioxidantes , Cisteína , Transportadoras de Casetes de Unión a ATP , Antraciclinas/metabolismo , Antibacterianos/farmacología , Cisteína/metabolismo , Glicopéptidos , Inositol , Oxidorreductasas/metabolismo , Peroxidasa/metabolismo , Peroxidasas/metabolismo , StreptomycesRESUMEN
Initially adopted as a mucolytic about 60 years ago, the cysteine prodrug N-acetylcysteine (NAC) is the standard of care to treat paracetamol intoxication, and is included on the World Health Organization's list of essential medicines. Additionally, NAC increasingly became the epitome of an "antioxidant". Arguably, it is the most widely used "antioxidant" in experimental cell and animal biology, as well as clinical studies. Most investigators use and test NAC with the idea that it prevents or attenuates oxidative stress. Conventionally, it is assumed that NAC acts as (i) a reductant of disulfide bonds, (ii) a scavenger of reactive oxygen species and/or (iii) a precursor for glutathione biosynthesis. While these mechanisms may apply under specific circumstances, they cannot be generalized to explain the effects of NAC in a majority of settings and situations. In most cases the mechanism of action has remained unclear and untested. In this review, we discuss the validity of conventional assumptions and the scope of a newly discovered mechanism of action, namely the conversion of NAC into hydrogen sulfide and sulfane sulfur species. The antioxidative and cytoprotective activities of per- and polysulfides may explain many of the effects that have previously been ascribed to NAC or NAC-derived glutathione.
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Acetilcisteína , Acetilcisteína/farmacología , Animales , Antioxidantes/farmacología , Humanos , Sulfuro de Hidrógeno , AzufreRESUMEN
3-Mercaptopyruvate sulfurtransferase (MPST) catalyzes the desulfuration of 3-mercaptopyruvate to generate an enzyme-bound hydropersulfide. Subsequently, MPST transfers the persulfide's outer sulfur atom to proteins or small molecule acceptors. MPST activity is known to be involved in hydrogen sulfide generation, tRNA thiolation, protein urmylation and cyanide detoxification. Tissue-specific changes in MPST expression correlate with ageing and the development of metabolic disease. Deletion and overexpression experiments suggest that MPST contributes to oxidative stress resistance, mitochondrial respiratory function and the regulation of fatty acid metabolism. However, the role and regulation of MPST in the larger physiological context remain to be understood.
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Azufre/metabolismo , Sulfurtransferasas/metabolismo , Animales , Cisteína/análogos & derivados , Cisteína/química , Cisteína/metabolismo , Humanos , Estructura Molecular , Azufre/química , Sulfurtransferasas/químicaRESUMEN
Peroxiredoxins are central to cellular redox homeostasis and signaling. They serve as peroxide scavengers, sensors, signal transducers, and chaperones, depending on conditions and context. Typical 2-Cys peroxiredoxins are known to switch between different oligomeric states, depending on redox state, pH, posttranslational modifications, and other factors. Quaternary states and their changes are closely connected to peroxiredoxin activity and function but so far have been studied, almost exclusively, outside the context of the living cell. Here we introduce the use of homo-FRET (Förster resonance energy transfer between identical fluorophores) fluorescence polarization to monitor dynamic changes in peroxiredoxin quaternary structure inside the crowded environment of living cells. Using the approach, we confirm peroxide- and thioredoxin-related quaternary transitions to take place in cellulo and observe that the relationship between dimer-decamer transitions and intersubunit disulfide bond formation is more complex than previously thought. Furthermore, we demonstrate the use of the approach to compare different peroxiredoxin isoforms and to identify mutations and small molecules affecting the oligomeric state inside cells. Mutagenesis experiments reveal that the dimer-decamer equilibrium is delicately balanced and can be shifted by single-atom structural changes. We show how to use this insight to improve the design of peroxiredoxin-based redox biosensors.
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Peroxirredoxinas/química , Línea Celular , Transferencia Resonante de Energía de Fluorescencia , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Mutación , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Multimerización de Proteína/efectos de los fármacos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Hydrogen peroxide (H2O2) is a key redox intermediate generated within cells. Existing probes for H2O2 have not solved the problem of detection of the ultra-low concentrations of the oxidant: these reporters are not sensitive enough, or pH-dependent, or insufficiently bright, or not functional in mammalian cells, or have poor dynamic range. Here we present HyPer7, the first bright, pH-stable, ultrafast, and ultrasensitive ratiometric H2O2 probe. HyPer7 is fully functional in mammalian cells and in other higher eukaryotes. The probe consists of a circularly permuted GFP integrated into the ultrasensitive OxyR domain from Neisseria meningitidis. Using HyPer7, we were able to uncover the details of H2O2 diffusion from the mitochondrial matrix, to find a functional output of H2O2 gradients in polarized cells, and to prove the existence of H2O2 gradients in wounded tissue in vivo. Overall, HyPer7 is a probe of choice for real-time H2O2 imaging in various biological contexts.
Asunto(s)
Movimiento Celular , Peróxido de Hidrógeno/metabolismo , Mitocondrias/metabolismo , Oxidantes/metabolismo , Animales , Transporte Biológico , Extensiones de la Superficie Celular/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Células HeLa , Humanos , Imagenología Tridimensional , Larva/metabolismo , Membranas Mitocondriales/metabolismo , Pez CebraRESUMEN
SIGNIFICANCE: Decrypting the cellular response to oxidative stress relies on a comprehensive understanding of the redox signaling pathways stimulated under oxidizing conditions. Redox signaling events can be divided into upstream sensing of oxidants, midstream redox signaling of protein function, and downstream transcriptional redox regulation. Recent Advances: A more and more accepted theory of hydrogen peroxide (H2O2) signaling is that of a thiol peroxidase redox relay, whereby protein thiols with low reactivity toward H2O2 are instead oxidized through an oxidative relay with thiol peroxidases. CRITICAL ISSUES: These ultrareactive thiol peroxidases are the upstream redox sensors, which form the first cellular port of call for H2O2. Not all redox-regulated interactions between thiol peroxidases and cellular proteins involve a transfer of oxidative equivalents, and the nature of redox signaling is further complicated through promiscuous functions of redox-regulated "moonlighting" proteins, of which the precise cellular role under oxidative stress can frequently be obscured by "polygamous" interactions. An ultimate goal of redox signaling is to initiate a rapid response, and in contrast to prokaryotic oxidant-responsive transcription factors, mammalian systems have developed redox signaling pathways, which intersect both with kinase-dependent activation of transcription factors, as well as direct oxidative regulation of transcription factors through peroxiredoxin (Prx) redox relays. FUTURE DIRECTIONS: We highlight that both transcriptional regulation and cell fate can be modulated either through oxidative regulation of kinase pathways, or through distinct redox-dependent associations involving either Prxs or redox-responsive moonlighting proteins with functional promiscuity. These protein associations form systems of crossregulatory networks with multiple nodes of potential oxidative regulation for H2O2-mediated signaling.
Asunto(s)
Redes Reguladoras de Genes , Peróxido de Hidrógeno/farmacología , Peroxidasas/metabolismo , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Estrés Oxidativo , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
Hydrogen peroxide (H2O2) is a strong oxidant capable of oxidizing cysteinyl thiolates, yet only a few cysteine-containing proteins have exceptional reactivity toward H2O2 One such example is the prokaryotic transcription factor OxyR, which controls the antioxidant response in bacteria, and which specifically and rapidly reduces H2O2 In this study, we present crystallographic evidence for the H2O2-sensing mechanism and H2O2-dependent structural transition of Corynebacterium glutamicum OxyR by capturing the reduced and H2O2-bound structures of a serine mutant of the peroxidatic cysteine, and the full-length crystal structure of disulfide-bonded oxidized OxyR. In the H2O2-bound structure, we pinpoint the key residues for the peroxidatic reduction of H2O2, and relate this to mutational assays showing that the conserved active-site residues T107 and R278 are critical for effective H2O2 reduction. Furthermore, we propose an allosteric mode of structural change, whereby a localized conformational change arising from H2O2-induced intramolecular disulfide formation drives a structural shift at the dimerization interface of OxyR, leading to overall changes in quaternary structure and an altered DNA-binding topology and affinity at the catalase promoter region. This study provides molecular insights into the overall OxyR transcription mechanism regulated by H2O2.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Corynebacterium glutamicum/metabolismo , Peróxido de Hidrógeno/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Sitios de Unión/genética , Catalasa/química , Catalasa/genética , Catalasa/metabolismo , Corynebacterium glutamicum/genética , Cristalografía por Rayos X , Genes Bacterianos , Cinética , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Estructura Cuaternaria de Proteína , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
SIGNIFICANCE: Mycothiol (MSH, AcCys-GlcN-Ins) is the main low-molecular weight (LMW) thiol of most Actinomycetes, including the human pathogen Mycobacterium tuberculosis that affects millions of people worldwide. Strains with decreased MSH content show increased susceptibilities to hydroperoxides and electrophilic compounds. In M. tuberculosis, MSH modulates the response to several antituberculosis drugs. Enzymatic routes involving MSH could provide clues for specific drug design. Recent Advances: Physicochemical data argue against a rapid, nonenzymatic reaction of MSH with oxidants, disulfides, or electrophiles. Moreover, exposure of the bacteria to high concentrations of two-electron oxidants resulted in protein mycothiolation. The recently described glutaredoxin-like protein mycoredoxin-1 (Mrx-1) provides a route for catalytic reduction of mycothiolated proteins, protecting critical cysteines from irreversible oxidation. The description of MSH/Mrx-1-dependent activities of peroxidases helped to explain the higher susceptibility to oxidants observed in Actinomycetes lacking MSH. Moreover, the first mycothiol-S-transferase, member of the DinB superfamily of proteins, was described. In Corynebacterium, both the MSH/Mrx-1 and the thioredoxin pathways reduce methionine sulfoxide reductase A. A novel tool for in vivo imaging of the MSH/mycothiol disulfide (MSSM) status allows following changes in the mycothiol redox state during macrophage infection and its relationship with antibiotic sensitivity. CRITICAL ISSUES: Redundancy of MSH with other LMW thiols is starting to be unraveled and could help to rationalize the differences in the reported importance of MSH synthesis observed in vitro versus in animal infection models. FUTURE DIRECTIONS: Future work should be directed to establish the structural bases of the specificity of MSH-dependent enzymes, thus facilitating drug developments. Antioxid. Redox Signal. 28, 487-504.
Asunto(s)
Cisteína/química , Glicopéptidos/química , Inositol/química , Mycobacterium tuberculosis/metabolismo , Oxidantes/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Cisteína/metabolismo , Glicopéptidos/metabolismo , Inositol/metabolismo , Mycobacterium tuberculosis/patogenicidad , Oxidantes/química , Oxidación-Reducción , Estrés Oxidativo , Peroxidasas/química , Peroxidasas/metabolismo , Compuestos de Sulfhidrilo/químicaRESUMEN
Mycothiol (MSH) is the major low molecular weight (LMW) thiol in Actinomycetes and functions in post-translational thiol-modification by protein S-mycothiolation as emerging thiol-protection and redox-regulatory mechanism. Here, we have used shotgun-proteomics to identify 26 S-mycothiolated proteins in the pathogen Corynebacterium diphtheriae DSM43989 under hypochlorite stress that are involved in energy metabolism, amino acid and nucleotide biosynthesis, antioxidant functions and translation. The glyceraldehyde-3-phosphate dehydrogenase (GapDH) represents the most abundant S-mycothiolated protein that was modified at its active site Cys153 in vivo. Exposure of purified GapDH to H2O2 and NaOCl resulted in irreversible inactivation due to overoxidation of the active site in vitro. Treatment of GapDH with H2O2 or NaOCl in the presence of MSH resulted in S-mycothiolation and reversible GapDH inactivation in vitro which was faster compared to the overoxidation pathway. Reactivation of S-mycothiolated GapDH could be catalyzed by both, the Trx and the Mrx1 pathways in vitro, but demycothiolation by Mrx1 was faster compared to Trx. In summary, we show here that S-mycothiolation can function in redox-regulation and protection of the GapDH active site against overoxidation in C. diphtheriae which can be reversed by both, the Mrx1 and Trx pathways.
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Corynebacterium diphtheriae/enzimología , Cisteína/química , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Glicopéptidos/química , Inositol/química , Proteómica/métodos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Dominio Catalítico/efectos de los fármacos , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Peróxido de Hidrógeno/farmacología , Oxidación-Reducción , Estrés Oxidativo , Procesamiento Proteico-Postraduccional , Hipoclorito de Sodio/farmacologíaRESUMEN
The Mycobacterium tuberculosis rv2466c gene encodes an oxidoreductase enzyme annotated as DsbA. It has a CPWC active-site motif embedded within its thioredoxin fold domain and mediates the activation of the prodrug TP053, a thienopyrimidine derivative that kills both replicating and nonreplicating bacilli. However, its mode of action and actual enzymatic function in M. tuberculosis have remained enigmatic. In this study, we report that Rv2466c is essential for bacterial survival under H2O2 stress. Further, we discovered that Rv2466c lacks oxidase activity; rather, it receives electrons through the mycothiol/mycothione reductase/NADPH pathway to activate TP053, preferentially via a dithiol-disulfide mechanism. We also found that Rv2466c uses a monothiol-disulfide exchange mechanism to reduce S-mycothiolated mixed disulfides and intramolecular disulfides. Genetic, phylogenetic, bioinformatics, structural, and biochemical analyses revealed that Rv2466c is a novel mycothiol-dependent reductase, which represents a mycoredoxin cluster of enzymes within the DsbA family different from the glutaredoxin cluster to which mycoredoxin-1 (Mrx1 or Rv3198A) belongs. To validate this DsbA-mycoredoxin cluster, we also characterized a homologous enzyme of Corynebacterium glutamicum (NCgl2339) and observed that it demycothiolates and reduces a mycothiol arsenate adduct with kinetic properties different from those of Mrx1. In conclusion, our work has uncovered a DsbA-like mycoredoxin that promotes mycobacterial resistance to oxidative stress and reacts with free mycothiol and mycothiolated targets. The characterization of the DsbA-like mycoredoxin cluster reported here now paves the way for correctly classifying similar enzymes from other organisms.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Profármacos/farmacología , Proteína Disulfuro Isomerasas/metabolismo , Pirimidinas/farmacología , Activación Metabólica , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Cisteína/metabolismo , Pruebas Antimicrobianas de Difusión por Disco , Drogas en Investigación/química , Drogas en Investigación/metabolismo , Drogas en Investigación/farmacología , Eliminación de Gen , Conformación Molecular , Simulación del Acoplamiento Molecular , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/crecimiento & desarrollo , Oxidación-Reducción , Filogenia , Profármacos/química , Profármacos/metabolismo , Conformación Proteica , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/genética , Pirimidinas/química , Pirimidinas/metabolismo , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Arsenic (As) is widespread in the environment and highly toxic. It has been released by volcanic and anthropogenic activities and causes serious health problems worldwide. To survive arsenic-rich environments, soil and saprophytic microorganisms have developed molecular detoxification mechanisms to survive arsenic-rich environments, mainly by the enzymatic conversion of inorganic arsenate (AsV) to arsenite (AsIII) by arsenate reductases, which is then extruded by arsenite permeases. One of these Gram-positive bacteria, Corynebacterium glutamicum, the workhorse of biotechnological research, is also resistant to arsenic. To sanitize contaminated soils and waters, C. glutamicum strains were modified to work as arsenic "biocontainers." Two chromosomally encoded ars operons (ars1 and ars2) are responsible for As resistance. The genes within these operons encode for metalloregulatory proteins (ArsR1/R2), arsenite permeases (Acr3-1/-2), and arsenate reductases (ArsC1/C2/C1'). ArsC1/C2 arsenate reductases are coupled to the low molecular weight thiol mycothiol (MSH) and to the recently discovered mycoredoxin-1 (Mrx-1) present in most Actinobacteria. This MSH/Mrx-1 redox system protects cells against different forms of stress, including reactive oxygen species (ROS), metals, and antibiotics. ROS can modify functional sulfur cysteines by oxidizing the thiol (-SH) to a sulfenic acid (-SOH). These oxidation-sensitive protein cysteine thiols are redox regulated by the MSH/Mrx-1 couple in Corynebacterium and Mycobacterium. In summary, the molecular mechanisms involved in arsenic resistance system in C. glutamicum have paved the way for understanding the cellular response against oxidative stress in Actinobacteria.
Asunto(s)
Arsénico/metabolismo , Corynebacterium glutamicum/metabolismo , Arsénico/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biodegradación Ambiental , Corynebacterium glutamicum/genética , Regulación Bacteriana de la Expresión Génica , Operón , Oxidación-ReducciónRESUMEN
Peroxiredoxins catalyze the reduction of peroxides, a process of vital importance to survive oxidative stress. A nucleophilic cysteine, also known as the peroxidatic cysteine, is responsible for this catalytic process. We used the Mycobacterium tuberculosis alkyl hydroperoxide reductase E (MtAhpE) as a model to investigate the effect of the chemical environment on the specificity of the reaction. Using an integrative structural (R116A - PDB ; F37H - PDB ), kinetic and computational approach, we explain the mutational effects of key residues in its environment. This study shows that the active site residues are specifically oriented to create an environment which selectively favours a reaction with peroxides.
Asunto(s)
Mycobacterium tuberculosis/enzimología , Peroxirredoxinas/metabolismo , Dominio Catalítico , Cinética , Simulación de Dinámica Molecular , Oxidación-Reducción , Peróxidos/química , Peróxidos/metabolismo , Peroxirredoxinas/química , Peroxirredoxinas/genética , Conformación ProteicaRESUMEN
Cysteine glutathione peroxidases (CysGPxs) control oxidative stress levels by reducing hydroperoxides at the expense of cysteine thiol (-SH) oxidation, and the recovery of their peroxidatic activity is generally accomplished by thioredoxin (Trx). Corynebacterium glutamicum mycothiol peroxidase (Mpx) is a member of the CysGPx family. We discovered that its recycling is controlled by both the Trx and the mycothiol (MSH) pathway. After H2 O2 reduction, a sulfenic acid (-SOH) is formed on the peroxidatic cysteine (Cys36), which then reacts with the resolving cysteine (Cys79), forming an intramolecular disulfide (S-S), which is reduced by Trx. Alternatively, the sulfenic acid reacts with MSH and forms a mixed disulfide. Mycoredoxin 1 (Mrx1) reduces the mixed disulfide, in which Mrx1 acts in combination with MSH and mycothiol disulfide reductase as a biological relevant monothiol reducing system. Remarkably, Trx can also take over the role of Mrx1 and reduce the Mpx-MSH mixed disulfide using a dithiol mechanism. Furthermore, Mpx is important for cellular survival under H2 O2 stress, and its gene expression is clearly induced upon H2 O2 challenge. These findings add a new dimension to the redox control and the functioning of CysGPxs in general.
Asunto(s)
Proteínas Bacterianas/metabolismo , Corynebacterium glutamicum/enzimología , Cisteína/metabolismo , Glicopéptidos/metabolismo , Inositol/metabolismo , Peroxidasas/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Corynebacterium glutamicum/genética , Disulfuros/metabolismo , Peróxido de Hidrógeno/farmacología , Cinética , Datos de Secuencia Molecular , Oxidación-Reducción , Estrés Oxidativo , Oxidorreductasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Análisis de Secuencia de Proteína , Tiorredoxinas/metabolismoRESUMEN
Methionine sulfoxide reductases are conserved enzymes that reduce oxidized methionines in proteins and play a pivotal role in cellular redox signaling. We have unraveled the redox relay mechanisms of methionine sulfoxide reductase A of the pathogen Corynebacterium diphtheriae (Cd-MsrA) and shown that this enzyme is coupled to two independent redox relay pathways. Steady-state kinetics combined with mass spectrometry of Cd-MsrA mutants give a view of the essential cysteine residues for catalysis. Cd-MsrA combines a nucleophilic cysteine sulfenylation reaction with an intramolecular disulfide bond cascade linked to the thioredoxin pathway. Within this cascade, the oxidative equivalents are transferred to the surface of the protein while releasing the reduced substrate. Alternatively, MsrA catalyzes methionine sulfoxide reduction linked to the mycothiol/mycoredoxin-1 pathway. After the nucleophilic cysteine sulfenylation reaction, MsrA forms a mixed disulfide with mycothiol, which is transferred via a thiol disulfide relay mechanism to a second cysteine for reduction by mycoredoxin-1. With x-ray crystallography, we visualize two essential intermediates of the thioredoxin relay mechanism and a cacodylate molecule mimicking the substrate interactions in the active site. The interplay of both redox pathways in redox signaling regulation forms the basis for further research into the oxidative stress response of this pathogen.
Asunto(s)
Corynebacterium diphtheriae/enzimología , Cisteína/metabolismo , Glicopéptidos/metabolismo , Inositol/metabolismo , Metionina Sulfóxido Reductasas/química , Metionina Sulfóxido Reductasas/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Disulfuros/metabolismo , Metionina/análogos & derivados , Metionina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Oxidorreductasas/metabolismo , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Tiorredoxinas/metabolismoRESUMEN
Despite current remediation efforts, arsenic contamination in water sources is still a major health problem, highlighting the need for new approaches. In this work, strains of the nonpathogenic and highly arsenic-resistant bacterium Corynebacterium glutamicum were used as inexpensive tools to accumulate inorganic arsenic, either as arsenate (As(V)) or arsenite (As(III)) species. The assays made use of "resting cells" from these strains, which were assessed under well-established conditions and compared with C. glutamicum background controls. The two mutant As(V)-accumulating strains were those used in a previously published study: (i) ArsC1/C2, in which the gene/s encoding the mycothiol-dependent arsenate reductases is/are disrupted, and (ii) MshA/C mutants unable to produce mycothiol, the low molecular weight thiol essential for arsenate reduction. The As(III)-accumulating strains were either those lacking the arsenite permease activities (Acr3-1 and Acr3-2) needed in As(III) release or recombinant strains overexpressing the aquaglyceroporin genes (glpF) from Corynebacterium diphtheriae or Streptomyces coelicolor, to improve As(III) uptake. Both genetically modified strains accumulated 30-fold more As(V) and 15-fold more As(III) than the controls. The arsenic resistance of the modified strains was inversely proportional to their metal accumulation ability. Our results provide the basis for investigations into the use of these modified C. glutamicum strains as a new bio-tool in arsenic remediation efforts.