O proteassomo é uma protease intracelular multimérica e multicatalítica responsável pela degradação de proteínas envolvidas no controle do ciclo celular, processos de sinalização, apresentação antigênica e no controle de síntese proteica. Ele é constituído por uma unidade catalítica central denominada 20S (20SPT) e por unidades regulatórias (19S) acopladas em uma ou ambas as extremidades para formar o 26S (26SPT). O 26SPT reconhece e direciona os substratos poliubiquitinados para a proteólise por um mecanismo dependente de ATP. Entretanto, o 20SPT também é ativo quando dissociado de unidades regulatórias, degradando proteínas independentemente de poliubiquitinação e consumo de ATP. Proteínas modificadas oxidativamente e outros substratos são degradados desta maneira. O 20SPT é composto por dois anéis heptaméricos centrais (β) onde estão localizados os sítios catalíticos e por dois anéis heptaméricos externos (α) que são responsáveis pela abertura da câmara catalítica. Anteriormente, foi observado pelo grupo que o 20SPT da levedura S.cerevisiae sofre S-glutatiolação na subunidade α5 nos resíduos de Cys76 e Cys221. Assim, foram obtidas neste projeto linhagens de levedura S. cerevisiae portando mutações sítio-específicas na subunidade α5 do 20SPT (α5-C76S ou α5-C221S), e posteriormente, ensaios comparativos quanto às consequências estruturais e funcionais dessas mutações foram realizados. Observamos um aumento na capacidade/velocidade de degradação nas atividades peptidásicas e proteolíticas da mutante C221, e também que, a população de proteassomo isolada dessa linhagem apresenta maior proporção da forma aberta da câmara catalítica, sendo esta imediatamente fechada pela remoção da glutationa do 20SPT na presença de DTT. Resultados opostos foram observados na linhagem mutante C76. Identificamos por espectrometria de massas o resíduo C76 do 20SPT glutationilado na mutante C221. Os ensaios fenotípicos mostraram um aumento da longevidade e resistência ao estresse oxidativo da C221, enquanto que a linhagem C76 mostrou uma dificuldade no crescimento. Contudo, a S-glutationilação do 20SPT é uma modificação química pós-traducional reversível de ocorrência fisiológica dependente do estado redox celular, e, o presente trabalho discute a modulação da câmara catalítica do 20SPT através da S-glutationilação de dois resíduos de cisteína localizados na subunidade α5, e as consequências desta modificação na função proteassomal / The proteasome is a multimeric and multicatalytic intracellular protease responsible for the degradation of proteins involved in cell cycle control, signaling processes, antigen presentation, and control of protein synthesis. It comprises a central catalytic unit called 20S (20SPT) and regulatory units (19S) coupled at one or both ends to form 26S (26SPT). The 26SPT recognizes and directs polyubiquitinylated substrates targeted for proteolysis by an ATP-dependent mechanism. However, 20SPT is also active when dissociated from regulatory units, degrading proteins by a process independent of polyubiquitinylation and ATP consumption. Oxidatively modified proteins and other substrates are degraded in this manner. The 20SPT comprises two central heptamerics rings (β) where are located the catalytic sites and two external heptamerics rings (α) that are responsible for proteasomal gating. Previously, it was observed by our group that the 20SPT of yeast S. cerevisiae is modified by S-glutathionylation in the residues Cys76 and Cys221 of the α5 subunit. Thus, were obtained in this project strains of the S. cerevisiae carrying site-specific mutations in the α5 subunit of the 20SPT (α5-C76S or α5-C221S), and comparative assays as the structural and functional consequences of these mutations were performed. We observed an increase in capacity/speed of degradation in peptidase and proteolytic activity in the C221 strain and also that, the isolated population of the proteasome this strain presents the highest frequency of open catalytic chamber conformation, which is immediately closed by the removal of glutathione of the 20SPT in the presence of DTT. Opposite results were observed in the C76 strain. We identified by mass spectrometry the C76 residue of the 20SPT glutathionyilated the C221 mutant. Phenotypic assays show an increased longevity and resistance to oxidative stress of C221, while the C76 strain showed a difficulty in growth. However, the S-glutathionylation of the 20SPT is a reversible post-translational chemical modification of physiological occurrence dependent on the cellular redox state, and this project discusses the modulation of the catalytic chamber of 20SPT via S-glutationylation of the two cysteine residues located the α5 subunit, and the consequences of this change in proteosomal function

O proteassomo é uma protease intracelular multimérica e multicatalítica responsável pela degradação de proteínas envolvidas no controle do ciclo celular, processos de sinalização, apresentação antigênica e no controle de síntese proteica. Ele é constituído por uma unidade catalítica central denominada 20S (20SPT) e por unidades regulatórias (19S) acopladas em uma ou ambas as extremidades para formar o 26S (26SPT). O 26SPT reconhece e direciona os substratos poliubiquitinados para a proteólise por um mecanismo dependente de ATP. Entretanto, o 20SPT também é ativo quando dissociado de unidades regulatórias, degradando proteínas independentemente de poliubiquitinação e consumo de ATP. Proteínas modificadas oxidativamente e outros substratos são degradados desta maneira. O 20SPT é composto por dois anéis heptaméricos centrais (β) onde estão localizados os sítios catalíticos e por dois anéis heptaméricos externos (α) que são responsáveis pela abertura da câmara catalítica. Anteriormente, foi observado pelo grupo que o 20SPT da levedura S.cerevisiae sofre S-glutatiolação na subunidade α5 nos resíduos de Cys76 e Cys221. Assim, foram obtidas neste projeto linhagens de levedura S. cerevisiae portando mutações sítio-específicas na subunidade α5 do 20SPT (α5-C76S ou α5-C221S), e posteriormente, ensaios comparativos quanto às consequências estruturais e funcionais dessas mutações foram realizados. Observamos um aumento na capacidade/velocidade de degradação nas atividades peptidásicas e proteolíticas da mutante C221, e também que, a população de proteassomo isolada dessa linhagem apresenta maior proporção da forma aberta da câmara catalítica, sendo esta imediatamente fechada pela remoção da glutationa do 20SPT na presença de DTT. Resultados opostos foram observados na linhagem mutante C76. Identificamos por espectrometria de massas o resíduo C76 do 20SPT glutationilado na mutante C221. Os ensaios fenotípicos mostraram um aumento da longevidade e resistência ao estresse oxidativo da C221, enquanto que a linhagem C76 mostrou uma dificuldade no crescimento. Contudo, a S-glutationilação do 20SPT é uma modificação química pós-traducional reversível de ocorrência fisiológica dependente do estado redox celular, e, o presente trabalho discute a modulação da câmara catalítica do 20SPT através da S-glutationilação de dois resíduos de cisteína localizados na subunidade α5, e as consequências desta modificação na função proteassomal / The proteasome is a multimeric and multicatalytic intracellular protease responsible for the degradation of proteins involved in cell cycle control, signaling processes, antigen presentation, and control of protein synthesis. It comprises a central catalytic unit called 20S (20SPT) and regulatory units (19S) coupled at one or both ends to form 26S (26SPT). The 26SPT recognizes and directs polyubiquitinylated substrates targeted for proteolysis by an ATP-dependent mechanism. However, 20SPT is also active when dissociated from regulatory units, degrading proteins by a process independent of polyubiquitinylation and ATP consumption. Oxidatively modified proteins and other substrates are degraded in this manner. The 20SPT comprises two central heptamerics rings (β) where are located the catalytic sites and two external heptamerics rings (α) that are responsible for proteasomal gating. Previously, it was observed by our group that the 20SPT of yeast S. cerevisiae is modified by S-glutathionylation in the residues Cys76 and Cys221 of the α5 subunit. Thus, were obtained in this project strains of the S. cerevisiae carrying site-specific mutations in the α5 subunit of the 20SPT (α5-C76S or α5-C221S), and comparative assays as the structural and functional consequences of these mutations were performed. We observed an increase in capacity/speed of degradation in peptidase and proteolytic activity in the C221 strain and also that, the isolated population of the proteasome this strain presents the highest frequency of open catalytic chamber conformation, which is immediately closed by the removal of glutathione of the 20SPT in the presence of DTT. Opposite results were observed in the C76 strain. We identified by mass spectrometry the C76 residue of the 20SPT glutathionyilated the C221 mutant. Phenotypic assays show an increased longevity and resistance to oxidative stress of C221, while the C76 strain showed a difficulty in growth. However, the S-glutathionylation of the 20SPT is a reversible post-translational chemical modification of physiological occurrence dependent on the cellular redox state, and this project discusses the modulation of the catalytic chamber of 20SPT via S-glutationylation of the two cysteine residues located the α5 subunit, and the consequences of this change in proteosomal function

Cells are constantly exposed to different stresses – one being redox stress, which is induced by metal, reactive oxygen species and reactive nitrogen species. S-nitrosoglutathione reductase (GSNOR) helps modulate redox stress by two different mechanisms – either by reducing S-nitrosoglutathione (GSNO) to oxidized glutathione (GSSG) or by oxidizing hydroxymethyl glutathione (HMGSH), a biproduct of glutathione and formaldehyde, to formic acid. GSNO has the potential to posttranslational modify proteins in two different manners, either by S-nitrosation or by S-glutathionylation. Interestingly, GSNOR can be modified by its substrate GSNO, either by S-nitrosation, which has previously been reported, or, as discussed in this thesis, by S-glutathionylation. As S-glutathionylation has been reported to occur through intermediate species, the S-glutathionylation of GSNOR appears to occur though the S-nitrosated intermediate, instead of the most common route of an oxidation pathway. It is hypothesized that the S-glutathionylation, and the overall presence of glutathione, can act as a buffer to regulate the amount of nitrosation that GSNOR experiences, and thus the enzymatic activity. It is has reported that the S-nitrosation occurs on three different non-structural, non-catalytic, solvent-accessible cysteine residues. Experimentation was conducted using Saccharomyces cerevisiae as a model organism to determine how those three cysteine residues of the GSNOR homolog Sensitive to Formaldehyde 1 (SFA1) participate in the indirect detoxification of formaldehyde, through the hydroxymethyl glutathione pathway. It has been determined that cysteine 370 is not as important as previously thought, but the other one or two cysteines (either cysteine 10 or 271) do indeed play a role in the detoxification, but further analysis needs to be conducted.

While the cardiotoxicity of doxorubicin (DOX) is known to be partly mediated through the generation of reactive oxygen species (ROS), the biochemical mechanisms by which ROS damage cardiomyocytes remain to be determined. This study investigates whether S-glutathionylation of mitochondrial proteins plays a role in DOX-induced myocardial injury using a line of transgenic mice expressing the human mitochondrial glutaredoxin 2 (Glrx2), a thiotransferase catalyzing the reduction as well as formation of protein-glutathione mixed disulfides, in cardiomyocytes. The total glutaredoxin (Glrx) activity was increased by 76% and 53 fold in homogenates of whole heart and isolated heart mitochondria of Glrx2 transgenic mice, respectively, compared to those of nontransgenic mice. The expression of other antioxidant enzymes, with the exception of glutaredoxin 1, was unaltered. Overexpression of Glrx2 completely prevents DOX-induced decreases in NAD- and FAD-linked state 3 respiration and respiratory control ratio (RCR) in heart mitochondria at days 1 and 5 of treatment. The extent of DOX-induced decline in left ventricular function and release of creatine kinase into circulation at day 5 of treatment was also greatly attenuated in Glrx2 transgenic mice. Further studies revealed that heart mitochondria overexpressing Glrx2 released less cytochrome c than did controls in response to treatment with tBid or a peptide encompassing the BH3 domain of Bid. Development of tolerance to DOX toxicity in transgenic mice is also associated with an increase in protein S-glutathionylation in heart mitochondria. Taken together, these results imply that S-glutathionylation of heart mitochondrial proteins plays a role in preventing DOX-induced cardiac injury.

No description available.

Le développement d'organismes photosynthétiques (piégeant le C02 en préservant l'eau douce et les terres cultivables sans ajout d'engrais) capables d'utiliser l'énergie solaire pour produire du dihydrogène (H2) passe par une meilleure compréhension du rôle de l'hydrogénase dans le métabolisme cyanobactérien. Le Laboratoire de Biologie et Biotechnologie des Cyanobatéries où j'ai travaillé durant ma thèse utilise une approche de "Biologie Intégrative" pour analyser le métabolisme qui conduit à la photo-production d’H2 chez la cyanobactérie modèle Synechocystis sp. PCC6803. Mon travail s'est focalisé sur l’analyse des réseaux de régulation amenant à la production d'H2 par l’hydrogénase bidirectionnelle à centre Ni-Fe (composée de 5 sous-unités) codée par l’opéron hox. Lorsque j’ai débuté ce travail, 2 activateurs de l’opéron hox avaient été identifiés: AbrB1 et LexA. Un article dont je suis co-premier auteur est paru (Dutheil et al. 2012 J Bact.), il décrit l'identification par l'utilisation de diverses approches d'un nouveau facteur de transcription de l'opéron hox: AbrB2 (homologue d'AbrB1). J'ai ainsi montré que l'expression de l’opéron hox était régulée négativement par AbrB2 en utilisant des fusions transcriptionnelles au gène rapporteur cat (introduites dans la souche sauvage ou dépourvues d'AbrB2) ainsi que des expériences de qRT-PCR. Par la technique de retard sur gel, nous avons confirmé une interaction directe entre AbrB2 et la région promotrice de l’opéron hox. En collaboration avec deux laboratoires du CEA, nous avons montré qu'un mutant dépourvu d’AbrB2 possède une activité hydrogénase augmentée, confirmant ainsi qu'AbrB2 est un régulateur négatif de la production d'H2.Dans un deuxième temps et en collaboration avec deux post-doc du laboratoire, nous avons mis en évidence le rôle de la cystéine unique d'AbrB2 dans le contrôle redox de son activité de régulation transcriptionnelle.Par la technique du retard sur gel,j’ai montré que cette cystéine n’est pas cruciale pour la fixation d'AbrB2 sur le promoteur hox, mais que par contre, la modification redox de celle-ci l’affecte de manière drastique. Dans le cadre de collaborations, nous avons identifié la modification post-traductionnelle qui peut avoir lieu sur la cysteine d'AbrB2 et il s’agit de la première fois, qu’un tel mécanisme de régulation est identifié pour cette famille de régulateur et chez les cyanobactéries. J’ai construit une souche portant l'allèle muté abrB2 Cys>Ser sur le chromosome et exprimé par le promoteur sauvage d’abrB2. J’ai montré grâce à cette construction et en utilisant diverses techniques (activité hydrogénase, qRT-PCR, Western blot et transcriptome) que la cystéine d'AbrB2 joue un rôle dans son activité de régulation qui est 60% moins bonne sur les 529 gènes cibles (directes ou indirectes) du régulateur muté. L’effet est également visible sur l’activité hydrogénase. Ce résultat a été complété par des tests de surexpression thermoinduite d’AbrB2 qui montrent que la mutation C34S affecte la stabilité de la protéine qui ne s’accumule pas autant que la sauvage dans les même conditions et dont la surexpression est létale. Un manuscrit dont je suis copremier auteur et décrivant ces résultats est en cours de finalisation et sera prochainement soumis à l’Intern. Journ. of Hydrogen Energy.L’ensemble de ces travaux permet de mieux comprendre les mécanismes biologiques liés à l’expression de l’hydrogénase bidirectionnelle et vont dans le sens d’un rôle important de celle-ci dans la détoxification des stress redox. La détermination des relations entre les différents régulateurs de l’hydrogénase et les possibles modifications post-traductionnelles de chacun de ces facteurs que j’ai mises en évidence traduisent une enzyme à la régulation complexe. Ces nouvelles connaissances permettent d’éclairer sous un angle nouveau la photoproduction d’H2 par les cyanobactéries et permettront peut-être d’élaborer des stratégies de production d’H2 efficace. / Developing photosynthetic organism (trapping CO2 while preserving fresh water and arable soils without adding fertilizers) able to use Sun light to produce dihydrogen (H2) is depending on a better understanding of the role of hydrogenase in the cyanobacterial metabolism. The Laboratoire de Biologie et Biotechnologie des Cyanobactéries (LBBC) where I worked during my thesis uses « Integrative Biology » approach to analyze the metabolism leading to H2 photoproduction by the model cyanobacterium Synechocystis sp. PCC6803. My work focused on analyzing the regulation network leading to H2 production by the bidirectionnal hydrogenase with Ni-Fe cluster (composed of 5 subunits) encoded by hox operon. When I started this work, two transcriptionnal activators were identified : AbrB1 and LexA. An article, of which I’m sharing first author position, is published (Dutheil et al. 2012 J Bact.), it describes the identification by different approachs of a new transcriptionnal factor of hox operon : AbrB2 (homologous to AbrB1). I showed that hox expression is negatively regulated by AbrB2 by using transcriptionnal fusion to cat reporter gene (introduced in the wild type background or the AbrB2-deleted one) and qRT-PCR experiments. By the electrophoretic mobility shift assay (EMSA) method, we confirmed a direct interaction between AbrB2 and the promoter region of hox operon. Collaborating with two CEA laboratories, we showed that a mutant lacking AbrB2 harbours an increased hydrogenase activity, validating that AbrB2 is a negative regulator of H2 production.In a second time of my thesis and colaborating with two post-doc of the laboratory, we evidenced the role of the unique cysteine of AbrB2 in redox-controlling the transcriptionnal regulator activity of the protein.Using the EMSA method, I showed that the cysteine is not crucial for AbrB2 fixing on hox promoter, but also that the redox modification occuring on this residue inhibits this same binding activity. Collaborating with other labs, we identified the post-translational modifications that may occur on AbrB2 cysteine and it is the first time that such a regulating mechanism is identified for this family of regulators and in cyanobacteria. I constructed a strand harbouring the abrB2C34S mutant allele on the chromosome and expressed by the abrB2 natural promoter. I showed with this construction and using diverse methods (hydrogenase activity, qRT-PCR, Western blot and transcriptome) that AbrB2 cysteine plays a role in its regulating activity : regulating activity is 60% less efficient towards the 529 target genes (either direct and indirect) of the mutated regulator. The effect is also seen on hydrogenase activity and hox genes. This result was completed by thermoinduced overexpression assays that show that C34S mutation of AbrB2 alters protein stability : the mutated protein accumulates less than wild type allele in the same conditions, which is lethal. A manuscript, of which I’m sharing first author position, and describing those results is being finalised and will be submitted soon to the IJHE (International Journal of Hydrogen Energy).Altogether, my results allow a better understanding of the biological mechanisms linked to bidirectionnal hydrogenase expression and agree with a possible role for hydrogenase in detoxifying redox stresses. The determination of the relationships between the different regulators of hydrogenase, and their possible post-translational modifications that I revealed, highlight an enzyme with complex regulation. This new knowledge brings an original outlook on hydrogen photoproduction by cyanobacteria and shall allow elaboration of efficient H2 production strategies.

To unravel the mysteries of complex biological processes carried out by biomolecules it is necessary to adopt a multifaceted approach, which involves employing a wide variety of tools both computational and experimental. In order to gain a clear understanding of the function of biomolecules their three dimensional structure is required. X-ray crystallography and Nuclear Magnetic Resonance (NMR) spectroscopy are the only two methods capable of providing high-resolution three-dimensional structure of biomolecules. NMR has the advantage of allowing the study of structure of biomolecules in solution and is better equipped to characterize the dynamics of the protein. Protein structure determination by NMR spectroscopy consists of recombinant expression of isotopically labeled proteins, purification, data collection, data processing, resonance assignment, distance restraint and angular restraint generation, structure calculation and structure validation. Apart from 3D structure determination of biomolecules NMR has become the method of choice for studying transient protein-protein interactions, which are notoriously difficult to study at higher resolution by other methods. Mass spectrometry plays an important role in enabling rapid identification of biomolecules and their modifications. The high sensitivity and resolution mass spectrometry offers makes it the method of choice for studying post-transitional modification of proteins. Use of computers in biology has played an essential role in elucidating those structure function relationships in biomolecules that are not possible to study by experimental techniques. The first chapter of this thesis deals with the introduction of methods used in this study. A brief introduction about the theory of Nuclear Magnetic Resonance (NMR) spectroscopy is given. Protein NMR methods used for structure determination of medium sized proteins are discussed. A part of this chapter discusses about the application of mass spectrometry in biochemistry and the use of tandem MS/MS experiments in identification of proteins and peptide fragments. Finally, the last part of this chapter gives an introduction about the theory of molecular dynamics and techniques used in the post processing of MD trajectories to elucidate the dynamics of proteins. The second chapter of this thesis is concerned with NMR characterization of a novel protein-protein interaction between the glycolytic enzyme Triosephosphate isomerase and the redox protein Thioredoxin. Chemical shift perturbation studies have been done to map the binding interfaces of these proteins. The structure of the complex was then modeled using NMR restraints based docking using the known 3D structure of these proteins. The docked complex reveals crucial insights into the glutathione mediated redox regulation of Triosephosphate isomerase and the role of thioredoxin as a deglutathionylating agent. Enzyme activity assays of Triosephosphate isomerase were done to show the inhibitory effects of s-glutathionylation of Cys217 and the role of thioredoxin as a deglutathionylating agent. The third chapter of the thesis is aimed to address some important issues related to the inhibition of Plasmodium falciparum Triosephosphate isomerase by S-glutathionylation. Oxidative stress induces protein glutathionylation which is a reversible post translational modification consisting of the formation of a mixed disulfide between protein cysteines and glutathione. Mass spectrometric analysis of the kilnetics of glutathionylation along with enzyme activity assays clearly show that gluthionylation of either Cys-13 (situated in the dimmer interface) or Cys-217 (situated in Helix G) can render the enzyme inactive. Molecular dynamics simulations provide a mechanistic basis of inhibition and predict that glutathionylation at Cys217 allosterically induces loop 6 disorder. The fourth chapter of this thesis addresses the stabilizing effect of introduction of a cross-strand disulfide bond across a non-hydrogen bonded position of an antiparallel beta sheet. Multidimensional heteronuclear NMR experiments have been used to get the backbone and side-chain resonance assignments, distance and angular restraints. In addition RDC based restraints have been used to calculate the structure of oxidixed form of L79C, T89C thiroedoxin. The observation of predominantly –RH staple conformation among the NMR ensemble in typical of cross-strand disulfides. The fifth chapter of this thesis deals with the dynamics of thioredoxin using computational methods.In this chapter analysis of known complexes of thiroedoxin was done to determine binding hot spot residues using free energy calculations. The physicochemical basis for the multispecificity of thioredoxin is probed using molecular dynamics simulations. In this chapter it has been shown that conformational selection plays a very important role in thioredoxin target recognition.

The toxic metal ion cadmium (Cd2+) is pro-oxidant and specifically disrupts the actin cytoskeleton in renal mesangial cells. This study investigated the role of Cd2+-mediated redox modulation of actin through protein S-glutathionylation and the effects of cytoskeletal changes on focal adhesions (FAs) through a Ca2+/calmodulin dependent-protein kinase II (CaMK-II) pathway. Only at low concentrations of Cd2+ (0.5-2 μM) was there an increase in actin glutathionylation, which was a reactive oxygen species-independent, total glutathione-dependent effect. Immunofluorescence of the cytoskeleton suggests that increases in glutathionylation levels occurring under low [Cd2+] are protective in vivo. Higher concentrations (>= 10 μM) of Cd2+ resulted in loss of vinculin and focal adhesion kinase (FAK) from FAs, concomitant with cytoskeletal disruption. Inhibition of CaMK-II preserved cytoskeletal integrity and focal contacts, while decreasing the migration of FAK-phosphoTyr925 to a membrane-associated compartment. This study adds further insight into the Cd2+-mediated effects on the cytoskeleton and FAs.

The toxic metal ion cadmium (Cd2+) is pro-oxidant and specifically disrupts the actin cytoskeleton in renal mesangial cells. This study investigated the role of Cd2+-mediated redox modulation of actin through protein S-glutathionylation and the effects of cytoskeletal changes on focal adhesions (FAs) through a Ca2+/calmodulin dependent-protein kinase II (CaMK-II) pathway. Only at low concentrations of Cd2+ (0.5-2 μM) was there an increase in actin glutathionylation, which was a reactive oxygen species-independent, total glutathione-dependent effect. Immunofluorescence of the cytoskeleton suggests that increases in glutathionylation levels occurring under low [Cd2+] are protective in vivo. Higher concentrations (>= 10 μM) of Cd2+ resulted in loss of vinculin and focal adhesion kinase (FAK) from FAs, concomitant with cytoskeletal disruption. Inhibition of CaMK-II preserved cytoskeletal integrity and focal contacts, while decreasing the migration of FAK-phosphoTyr925 to a membrane-associated compartment. This study adds further insight into the Cd2+-mediated effects on the cytoskeleton and FAs.

To understand the physiological function of glutaredoxin, a thiotransferase catalyzing the reduction of mixed disulfides of protein and glutathione, we generated a line of knockout mice deficient in the cytosolic glutaredoxin 1 (Grx1). To our surprise, mice deficient in Grx1 were not more susceptible to acute oxidative insults in models of heart and lung injury induced by ischemia/reperfusion and hyperoxia, respectively, suggesting that either changes in S-glutathionylation status of cytosolic proteins are not the major cause of such tissue injury or developmental adaptation in the Glrx1-knockout animals alters the response to oxidative insult. In contrast, mouse embryonic fibroblasts (MEFs) isolated from Grx1-deficient mice displayed an increased vulnerability to diquat and paraquat, but they were not more susceptible to cell death induced by hydrogen peroxide (H2O2) and diamide. A deficiency in Grx1 also sensitized MEFs to protein S-glutathionylation in response to H2O2 treatment and retarded deglutathionylation of the S-glutathionylated proteins, especially for a single prominent protein band. Additional experiments showed that MEFs lacking Grx1 were more tolerant to apoptosis induced by tumor necrosis factor αplus actinomycin D. These findings suggest that various oxidants may damage the cells via distinct mechanisms in which the action of Grx1 may or may not be protective and Grx1 may exert its function on specific target proteins.