Cellular Compartmentalization, Glutathione Transport and Its Relevance in Some Pathologies.

Reduced glutathione (GSH) is the most abundant non-protein endogenous thiol. It is a ubiquitous molecule produced in most organs, but its synthesis is predominantly in the liver, the tissue in charge of storing and distributing it. GSH is involved in the detoxification of free radicals, peroxides and xenobiotics (drugs, pollutants, carcinogens, etc.), protects biological membranes from lipid peroxidation, and is an important regulator of cell homeostasis, since it participates in signaling redox, regulation of the synthesis and degradation of proteins (S-glutathionylation), signal transduction, various apoptotic processes, gene expression, cell proliferation, DNA and RNA synthesis, etc. GSH transport is a vital step in cellular homeostasis supported by the liver through providing extrahepatic organs (such as the kidney, lung, intestine, and brain, among others) with the said antioxidant. The wide range of functions within the cell in which glutathione is involved shows that glutathione's role in cellular homeostasis goes beyond being a simple antioxidant agent; therefore, the importance of this tripeptide needs to be reassessed from a broader metabolic perspective.

Glutaredoxin: Discovery, redox defense and much more.

Glutaredoxin, Grx, is a small protein containing an active site cysteine pair and was discovered in 1976 by Arne Holmgren. The Grx system, comprised of Grx, glutathione, glutathione reductase, and NADPH, was first described as an electron donor for Ribonucleotide Reductase but, from the first discovery in E.coli, the Grx family has impressively grown, particularly in the last two decades. Several isoforms have been described in different organisms (from bacteria to humans) and with different functions. The unique characteristic of Grxs is their ability to catalyse glutathione-dependent redox regulation via glutathionylation, the conjugation of glutathione to a substrate, and its reverse reaction, deglutathionylation. Grxs have also recently been enrolled in iron sulphur cluster formation. These functions have been implied in various physiological and pathological conditions, from immune defense to neurodegeneration and cancer development thus making Grx a possible drug target. This review aims to give an overview on Grxs, starting by a phylogenetic analysis of vertebrate Grxs, followed by an analysis of the mechanisms of action, the specific characteristics of the different human isoforms and a discussion on aspects related to human physiology and diseases.

Protective effects of N-acetyl-cysteine in mitochondria bioenergetics, oxidative stress, dynamics and S-glutathionylation alterations in acute kidney damage induced by folic acid.

Folic acid (FA)-induced acute kidney injury (AKI) is a widely used model for studies of the renal damage and its progression to chronic state. However, the molecular mechanisms by which FA induces AKI remain poorly understood. Since renal function depends on mitochondrial homeostasis, it has been suggested that mitochondrial alterations contribute to AKI development. Additionally, N-acetyl-cysteine (NAC) can be a protective agent to prevent mitochondrial and renal dysfunction in this model, given its ability to increase mitochondrial glutathione (GSH) and to control the S-glutathionylation levels, a reversible post-translational modification that has emerged as a mechanism able to link mitochondrial energy metabolism and redox homeostasis. However, this hypothesis has not been explored. The present study demonstrates for the first time that, at 24 h, FA induced mitochondrial bioenergetics, redox state, dynamics and mitophagy alterations, which are involved in the mechanisms responsible for the AKI development. On the other hand, NAC preadministration was able to prevent mitochondrial bioenergetics, redox state and dynamics alterations as well as renal damage. The protective effects of NAC on mitochondria and renal function could be related to its observed capacity to preserve the S-glutathionylation process and GSH levels in mitochondria. Taken together, our results support the idea that these mitochondrial processes can be targets for the prevention of the renal damage and its progression in FA-induced AKI model.

Kinetic studies reveal a key role of a redox-active glutaredoxin in the evolution of the thiol-redox metabolism of trypanosomatid parasites.

Trypanosomes are flagellated protozoan parasites (kinetoplastids) that have a unique redox metabolism based on the small dithiol trypanothione (T(SH)2). Although GSH may still play a biological role in trypanosomatid parasites beyond being a building block of T(SH)2, most of its functions are replaced by T(SH)2 in these organisms. Consequently, trypanosomes have several enzymes adapted to using T(SH)2 instead of GSH, including the glutaredoxins (Grxs). However, the mechanistic basis of Grx specificity for T(SH)2 is unknown. Here, we combined fast-kinetic and biophysical approaches, including NMR, MS, and fluorescent tagging, to study the redox function of Grx1, the only cytosolic redox-active Grx in trypanosomes. We observed that Grx1 reduces GSH-containing disulfides (including oxidized trypanothione) in very fast reactions (k > 5 × 105 m-1 s-1). We also noted that disulfides without a GSH are much slower oxidants, suggesting a strongly selective binding of the GSH molecule. Not surprisingly, oxidized Grx1 was also reduced very fast by T(SH)2 (4.8 × 106 m-1 s-1); however, GSH-mediated reduction was extremely slow (39 m-1 s-1). This kinetic selectivity in the reduction step of the catalytic cycle suggests that Grx1 uses preferentially a dithiol mechanism, forming a disulfide on the active site during the oxidative half of the catalytic cycle and then being rapidly reduced by T(SH)2 in the reductive half. Thus, the reduction of glutathionylated substrates avoids GSSG accumulation in an organism lacking GSH reductase. These findings suggest that Grx1 has played an important adaptive role during the rewiring of the thiol-redox metabolism of kinetoplastids.

Structural Basis for Redox Regulation of Cytoplasmic and Chloroplastic Triosephosphate Isomerases from Arabidopsis thaliana.

In plants triosephosphate isomerase (TPI) interconverts glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) during glycolysis, gluconeogenesis, and the Calvin-Benson cycle. The nuclear genome of land plants encodes two tpi genes, one gene product is located in the cytoplasm and the other is imported into the chloroplast. Herein we report the crystal structures of the TPIs from the vascular plant Arabidopsis thaliana (AtTPIs) and address their enzymatic modulation by redox agents. Cytoplasmic TPI (cTPI) and chloroplast TPI (pdTPI) share more than 60% amino acid identity and assemble as (ß-α)8 dimers with high structural homology. cTPI and pdTPI harbor two and one accessible thiol groups per monomer respectively. cTPI and pdTPI present a cysteine at an equivalent structural position (C13 and C15 respectively) and cTPI also contains a specific solvent accessible cysteine at residue 218 (cTPI-C218). Site directed mutagenesis of residues pdTPI-C15, cTPI-C13, and cTPI-C218 to serine substantially decreases enzymatic activity, indicating that the structural integrity of these cysteines is necessary for catalysis. AtTPIs exhibit differential responses to oxidative agents, cTPI is susceptible to oxidative agents such as diamide and H2O2, whereas pdTPI is resistant to inhibition. Incubation of AtTPIs with the sulfhydryl conjugating reagents methylmethane thiosulfonate (MMTS) and glutathione inhibits enzymatic activity. However, the concentration necessary to inhibit pdTPI is at least two orders of magnitude higher than the concentration needed to inhibit cTPI. Western-blot analysis indicates that residues cTPI-C13, cTPI-C218, and pdTPI-C15 conjugate with glutathione. In summary, our data indicate that AtTPIs could be redox regulated by the derivatization of specific AtTPI cysteines (cTPI-C13 and pdTPI-C15 and cTPI-C218). Since AtTPIs have evolved by gene duplication, the higher resistance of pdTPI to redox agents may be an adaptive consequence to the redox environment in the chloroplast.

When Bad Guys Become Good Ones: The Key Role of Reactive Oxygen Species and Nitric Oxide in the Plant Responses to Abiotic Stress.

The natural environment of plants is composed of a complex set of abiotic stresses and their ability to respond to these stresses is highly flexible and finely balanced through the interaction between signaling molecules. In this review, we highlight the integrated action between reactive oxygen species (ROS) and reactive nitrogen species (RNS), particularly nitric oxide (NO), involved in the acclimation to different abiotic stresses. Under stressful conditions, the biosynthesis transport and the metabolism of ROS and NO influence plant response mechanisms. The enzymes involved in ROS and NO synthesis and scavenging can be found in different cells compartments and their temporal and spatial locations are determinant for signaling mechanisms. Both ROS and NO are involved in long distances signaling (ROS wave and GSNO transport), promoting an acquired systemic acclimation to abiotic stresses. The mechanisms of abiotic stresses response triggered by ROS and NO involve some general steps, as the enhancement of antioxidant systems, but also stress-specific mechanisms, according to the stress type (drought, hypoxia, heavy metals, etc.), and demand the interaction with other signaling molecules, such as MAPK, plant hormones, and calcium. The transduction of ROS and NO bioactivity involves post-translational modifications of proteins, particularly S-glutathionylation for ROS, and S-nitrosylation for NO. These changes may alter the activity, stability, and interaction with other molecules or subcellular location of proteins, changing the entire cell dynamics and contributing to the maintenance of homeostasis. However, despite the recent advances about the roles of ROS and NO in signaling cascades, many challenges remain, and future studies focusing on the signaling of these molecules in planta are still necessary.

S-nitrosoglutathione inhibits inducible nitric oxide synthase upregulation by redox posttranslational modification in experimental diabetic retinopathy.

PURPOSE: Diabetic retinopathy (DR) is associated with nitrosative stress. The purpose of this study was to evaluate the beneficial effects of S-nitrosoglutathione (GSNO) eye drop treatment on an experimental model of DR. METHODS: Diabetes (DM) was induced in spontaneously hypertensive rats (SHR). Treated animals received GSNO eye drop (900 nM or 10 µM) twice daily in both eyes for 20 days. The mechanisms of GSNO effects were evaluated in human RPE cell line (ARPE-19). RESULTS: In animals with DM, GSNO decreased inducible nitric oxide synthase (iNOS) expression and prevented tyrosine nitration formation, ameliorating glial dysfunction measured with glial fibrillary acidic protein, resulting in improved retinal function. In contrast, in nondiabetic animals, GSNO induced oxidative/nitrosative stress in tissue resulting in impaired retinal function. Nitrosative stress was present markedly in the RPE layer accompanied by c-wave dysfunction. In vitro study showed that treatment with GSNO under high glucose condition counteracted nitrosative stress due to iNOS downregulation by S-glutathionylation, and not by prevention of decreased GSNO and reduced glutathione levels. This posttranslational modification probably was promoted by the release of oxidized glutathione through GSNO denitrosylation via GSNO-R. In contrast, in the normal glucose condition, GSNO treatment promoted nitrosative stress by NO formation. CONCLUSIONS: In this study, a new therapeutic modality (GSNO eye drop) targeting nitrosative stress by redox posttranslational modification of iNOS was efficient against early damage in the retina due to experimental DR. The present work showed the potential clinical implications of balancing the S-nitrosoglutathione/glutathione system in treating DR.

Investigação de potenciais biomarcadores redox - um enfoque em aldeídos e seus produtos / Potential redox biomarkers investigation - focus on aldehydes and their products

As espécies reativas são associadas a processos toxicológicos e fisiopatológicos, agindo como importantes mediadores, por exemplo, na sinalização celular. Diversas classes de compostos têm sido utilizadas como possíveis biomarcadores de estresse redox, destacando-se os aldeídos α,ß-insaturados, capazes de alquilar biomoléculas como o DNA. Para evitar efeitos deletérios, estes aldeídos são detoxificados por glutationilação e posterior metabolização a derivados mercaptúricos. Contudo, avaliar o estado redox em sistemas biológicos ainda é tarefa bastante complexa, sendo a dificuldade em quantificar de forma prática e acurada os efeitos de sinalização e/ou dano molecular o maior problema dos estudos redox. Assim, o objetivo deste trabalho foi desenvolver métodos acurados e sensíveis de análise de potenciais biomarcadores de estresse redox, isto é: nucleosídeos modificados, aldeídos endógenos e exógenos, glutationa e produtos de glutationilação, e avaliá-los em sistemas modelos, celular e animal, e em humanos. A avaliação dos níveis urinários de três nucleosídeos modificados por metodologia de HPLC-MS/MS desenvolvida pelo grupo em moradores da cidade de São Paulo - região com poluição atmosférica - demonstrou aumento significativo de 1,N2-propanodGuo comparado aos moradores de região não poluída. Ademais, comprova-se pela primeira vez que células deficientes em reparo de ligações cruzadas apresentam níveis basais elevados de 1,N2-propanodGuo, em duas linhagens independentes, colocando este aduto como potencial mediador de carcinogênese em pacientes portadores de Anemia de Fanconi. Utilizando cérebro de ratos SOD1G93A (modelo de Esclerose Lateral Amiotrófica - ELA), verificou-se aumento de 50% nos níveis de 1,N2-propanodGuo e de 100% nos de 1,N6-εdAdo em fase sintomática, sugerindo influência do conteúdo lipídico cerebral, levando a comprometimento do metabolismo neuronal e morte celular. O perfil de aldeídos determinado em cérebro de ratos SOD1G93A demonstrou aumento de trans-hexa-2-enal e trans,trans-hexa-2,4-dienal em fase assintomática e de trans,trans-deca-2,4-dienal em fase sintomática, não sendo observada nenhuma alteração na medula. Conhecer estas variações permite direcionar estudos de modificações em biomoléculas, além de a metodologia per se corroborar com as áreas de análises lipidômicas. Técnicas distintas e o preparo de amostras refletiram nos níveis de glutationa reduzida (GSH) e oxidada (GSSG) relatados. A técnica de espectrometria de massas mostrou-se mais precisa que a detecção eletroquímica; e a alquilação do grupo tiol minimizou interferências de matriz. Por análise de HPLC-UV/Vis-ESI-MS/MS, a quantificação de trans-4-hidroxi-2-nonenal (HNE) e crotonaldeido conjugados com GSH demonstrou não haver alterações em cérebro e medula de ratos SOD1G93A. Contudo, há formação esteroespecífica dos adutos de HNE in vivo. Ressalta-se que a metodologia desenvolvida é extremamente sensível e específica e permite análise simultânea de GSH, GSSG, cisteína, cistina e dos adutos supracitados, servindo para análise de outros adutos de glutationilação de aldeídos que possam ser importantes em doenças associadas a estresse redox

Free radicais and oxidant species are associated with toxicological and pathophysiological processes. It has been demonstrated that production of reactive oxygen species may be involved in cell signaling and regulation. Several biomarkers of redox processes have been used, including adducts formed through the reaction of α,ß-unsaturated aldehydes with biomolecules such as DNA. In order to avoid these deleterious effects, aldehydes are detoxified through glutathionylation and further metabolized to mercapturic derivatives. However, assessing the redox status in biological systems is still a very complex task, and the difficulty in practical and accurate quantification of signaling effects and/or molecular damage is a major problem in redox studies. The objective of this work was to develop accurate and sensitive methods for analysis of potential biomarkers of redox stress, i.e., modified nucleosides, endogenous and exogenous aldehydes, glutathione and glutathionylation products, and their evaluation in cell, animal model and humans. Evaluation of urinary levels of 1,N2-propano-2'-deoxyguanosine (1,N2-propanodGuo), 1,N2-etheno-2'-deoxyguanosine and 8-oxo-7,8-dihydro-2'-deoxyguanosine in residents of São Paulo City - polluted region - showed a significant increase (p<0.05) in 1,N2-propanodGuo levels compared to residents of an unpolluted region by a HPLC-MS/MS methodology developed by the group. Moreover, it was proven, for the first time, that repair deficient cells have basal levels of 1,N2-propanodGuo higher than proficient cells in two independent strains, placing 1,N2-propanodGuo as a potential mediator of carcinogenesis in Fanconi Anemia patients. In an Amyotrophic Lateral Sclerosis (ALS) animal model (SOD1G93A rat) , a 50% increase in the levels of 1,N2-propanodGuo and 100% in the 1,N6-etheno-2'-deoxyadenosine in brain tissue in the symptomatic phase was observed, suggesting that the high brain lipid content may play a role, leading to impairment of cell metabolism and neuronal cell death. There is an increase of trans-hex-2-enal and trans,trans-hexa-2,4-dienal in asymptomatic SOD1G93A rats brain and of trans,trans-deca-2,4-dienal in symptomatic ones. However, no alteration was observed in spinal cord. Our approach contributes to a better understanding of the aldehyde status in vivo and allows us to predict biomolecule modifications. The developed methodology can contribute to lipidomic studies. The use of different techniques and sample preparation reflected in the reported levels of reduced (GSH) and oxidized glutathione (GSSG). The mass spectrometry technique proved to be more accurate than the electrochemical one, and the use of thiol alkylating agent minimizes matrix interference. No changes were observed in the levels of the GSH conjugates of trans-4-hydroxy-2-nonenal (HNE) and crotonaldehyde in brain and spinal cord of SOD1G93A rats quantified by HPLC-UV/Vis-ESI-MS/MS compared to controls. However, it was observed stereospecific HNE adducts formation in vivo. Note that this methodology is extremely sensitive and specific and allows simultaneous analysis of GSH, GSSG, Cys, cystine and the aforementioned adducts, serving for analysis of other aldehyde-glutathionylation adducts that may be important in pathologies associated with stress redox

Redox regulation of the proteasome via S-glutathionylation.

The proteasome is a multimeric and multicatalytic intracellular protease responsible for the degradation of proteins involved in cell cycle control, various signaling processes, antigen presentation, and control of protein synthesis. The central catalytic complex of the proteasome is called the 20S core particle. The majority of these are flanked on one or both sides by regulatory units. Most common among these units is the 19S regulatory unit. When coupled to the 19S unit, the complex is termed the asymmetric or symmetric 26S proteasome depending on whether one or both sides are coupled to the 19S unit, respectively. The 26S proteasome recognizes poly-ubiquitinylated substrates targeted for proteolysis. Targeted proteins interact with the 19S unit where they are deubiquitinylated, unfolded, and translocated to the 20S catalytic chamber for degradation. The 26S proteasome is responsible for the degradation of major proteins involved in the regulation of the cellular cycle, antigen presentation and control of protein synthesis. Alternatively, the proteasome is also active when dissociated from regulatory units. This free pool of 20S proteasome is described in yeast to mammalian cells. The free 20S proteasome degrades proteins by a process independent of poly-ubiquitinylation and ATP consumption. Oxidatively modified proteins and other substrates are degraded in this manner. The 20S proteasome comprises two central heptamers (ß-rings) where the catalytic sites are located and two external heptamers (α-rings) that are responsible for proteasomal gating. Because the 20S proteasome lacks regulatory units, it is unclear what mechanisms regulate the gating of α-rings between open and closed forms. In the present review, we discuss 20S proteasomal gating modulation through a redox mechanism, namely, S-glutathionylation of cysteine residues located in the α-rings, and the consequence of this post-translational modification on 20S proteasomal function.