Glutathionylation of Pea Chloroplast 2-Cys Prx and Mitochondrial Prx IIF Affects Their Structure and Peroxidase Activity and Sulfiredoxin Deglutathionylates Only the 2-Cys Prx

Together with thioredoxins (Trxs), plant peroxiredoxins (Prxs), and sulfiredoxins (Srxs) are involved in antioxidant defense and redox signaling, while their regulation by post-translational modifications (PTMs) is increasingly regarded as a key component for the transduction of the bioactivity of reactive oxygen and nitrogen species. Among these PTMs, S-glutathionylation is considered a protective mechanism against overoxidation, it also modulates protein activity and allows signaling. This study explores the glutathionylation of recombinant chloroplastic 2-Cys Prx and mitochondrial Prx IIF from Pisum sativum. Glutathionylation of the decameric form of 2-Cys Prx produced a change in the elution volume after FPLC chromatography and converted it to its dimeric glutathionylated form, while Prx IIF in its reduced dimeric form was glutathionylated without changing its oligomeric state. Mass spectrometry demonstrated that oxidized glutathione (GSSG) can glutathionylate resolving cysteine (Cys174), but not the peroxidatic equivalent (Cys52), in 2-Cys Prx. In contrast, GSSG was able to glutathionylate both peroxidatic (Cys59) and resolving (Cys84) cysteine in Prx IIF. Glutathionylation was seen to be dependent on the GSH/GSSG ratio, although the exact effect on the 2-Cys Prx and Prx IIF proteins differed. However, the glutathionylation provoked a similar decrease in the peroxidase activity of both peroxiredoxins. Despite growing evidence of the importance of post-translational modifications, little is known about the enzymatic systems that specifically regulate the reversal of this modification. In the present work, sulfiredoxin from P. sativum was seen to be able to deglutathionylate pea 2-Cys Prx but not pea Prx IIF. Redox changes during plant development and the response to stress influence glutathionylation/deglutathionylation processes, which may represent an important event through the modulation of peroxiredoxin and sulfiredoxin proteins.This research was supported by MICINN, Spain (BFU2014-52452-P co-financed by FEDER) and Seneca Foundation, Murcia, Spain (Excellence Group 19876/GERM/15).Peer reviewedPeer Reviewe

Metabolomic Analysis of Pediatric Patients with Idiosyncratic Drug-Induced Liver Injury According to the Updated RUCAM

Hepatotoxicity, a common adverse drug effect, has been extensively studied in adult patients. However, it is equally important to investigate this condition in pediatric patients to develop personalized treatment strategies for children. This study aimed to identify plasma biomarkers that characterize hepatotoxicity in pediatric patients through an observational case–control study. Metabolomic analysis was conducted on 55 pediatric patients with xenobiotic liver toxicity and 88 healthy controls. The results revealed clear differences between the two groups. Several metabolites, including hydroxydecanoylcarnitine, octanoylcarnitine, lysophosphatidylcholine, glycocholic acid, and taurocholic acid, were identified as potential biomarkers (area under the curve: 0.817; 95% confidence interval: 0.696–0.913). Pathway analysis indicated involvement of primary bile acid biosynthesis and the metabolism of taurine and hypotaurine (p < 0.05). The findings from untargeted metabolomic analysis demonstrated an increase in bile acids in children with hepatotoxicity. The accumulation of cytotoxic bile acids should be further investigated to elucidate the role of these metabolites in drug-induced liver injuryInstitute of Health Carlos III grants (PI17/01989) and Servicio Andaluz de Salud (F2-0071-2015

Genome-Wide Screen of Genes Required for Caffeine Tolerance in Fission Yeast

Isabel A. Calvo et al...Background An excess of caffeine is cytotoxic to all eukaryotic cell types. We aim to study how cells become tolerant to a toxic dose of this drug, and the relationship between caffeine and oxidative stress pathways. Methodology/Principal Findings We searched for Schizosaccharomyces pombe mutants with inhibited growth on caffeine-containing plates. We screened a collection of 2,700 haploid mutant cells, of which 98 were sensitive to caffeine. The genes mutated in these sensitive clones were involved in a number of cellular roles including the H2O2-induced Pap1 and Sty1 stress pathways, the integrity and calcineurin pathways, cell morphology and chromatin remodeling. We have investigated the role of the oxidative stress pathways in sensing and promoting survival to caffeine. The Pap1 and the Sty1 pathways are both required for normal tolerance to caffeine, but only the Sty1 pathway is activated by the drug. Cells lacking Pap1 are sensitive to caffeine due to the decreased expression of the efflux pump Hba2. Indeed, ?hba2 cells are sensitive to caffeine, and constitutive activation of the Pap1 pathway enhances resistance to caffeine in an Hba2-dependent manner. Conclusions/Significance With our caffeine-sensitive, genome-wide screen of an S. pombe deletion collection, we have demonstrated the importance of some oxidative stress pathway components on wild-type tolerance to the drug.This work was supported by Direccion General de Investigacion of Spain Grant BFU2006-02610, and by the Spanish program Consolider-Ingenio 2010 Grant CSD 2007-0020, to E.H.Peer reviewe

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Caracterización bioquímica y molecular del sistema antioxidante peroxirredoxina-sulfirredoxina

Tesis Univ. Granada. Departamento de Bioquímica y Biología Molecular. Leída el 27 de Septiembre del 201

The Dual-Targeted Plant Sulfiredoxin Retroreduces the Sulfinic Form of Atypical Mitochondrial Peroxiredoxin1[W]

Sulfiredoxin (Srx) couples the energy of ATP hydrolysis to the energetically unfavorable process of reducing the inactive sulfinic form of 2-cysteine peroxiredoxins (Prxs) to regenerate its active form. In plants, Srx as well as typical 2-cysteine Prx have been considered as enzymes with exclusive chloroplast localization. This work explores the subcellular localization of Srx in pea (Pisum sativum) and Arabidopsis (Arabidopsis thaliana). Immunocytochemistry, analysis of protein extracts from isolated intact organelles, and cell-free posttranslational import assays demonstrated that plant Srx also localizes to the mitochondrion in addition to plastids. The dual localization was in line with the prediction of a signal peptide for dual targeting. Activity tests and microcalorimetric data proved the interaction between Srx and its mitochondrial targets Prx IIF and thioredoxin. Srx catalyzed the retroreduction of the inactive sulfinic form of atypical Prx IIF using thioredoxin as reducing agent. Arabidopsis Srx also reduced overoxidized human Prx V. These results suggest that plant Srx could play a crucial role in the regulation of Prx IIF activity by controlling the regeneration of its overoxidized form in mitochondria, which are sites of efficient reactive oxygen species production in plants

Dissecting the integrative antioxidant and redox systems in plant mitochondria. Effect of stress and S-nitrosylation

Mitochondrial respiration provides the energy needed to drive metabolic and transport processes in cells. Mitochondria are a significant site of reactive oxygen species (ROS) production in plant cells, and redox-system components obey fine regulation mechanisms that are essential in protecting the mitochondrial integrity. In addition to ROS, there are compelling indications that nitric oxide can be generated in this organelle by both reductive and oxidative pathways. ROS and reactive nitrogen species play a key role in signaling but they can also be deleterious via oxidation of macromolecules. The high production of ROS obligates mitochondria to be provided with a set of ROS scavenging mechanisms. The first line of mitochondrial antioxidants is composed of superoxide dismutase and the enzymes of the ascorbate-glutathione cycle, which are not only able to scavenge ROS but also to repair cell damage and possibly serve as redox sensors. The dithiol-disulfide exchanges form independent signaling nodes and act as antioxidant defense mechanisms as well as sensor proteins modulating redox signaling during development and stress adaptation. The presence of thioredoxin (Trx), peroxiredoxin (Prx) and sulfiredoxin (Srx) in the mitochondria has been recently reported. Cumulative results obtained from studies in salt stress models have demonstrated that these redox proteins play a significant role in the establishment of salt tolerance. The Trx/Prx/Srx system may be subjected to a fine regulated mechanism involving post-translational modifications, among which S-glutathionylation and S-nitrosylation seem to exhibit a critical role that is just beginning to be understood. This review summarizes our current knowledge in antioxidative systems in plant mitochondria, their interrelationships, mechanisms of compensation and some unresolved questions, with special focus on their response to abiotic stress.Peer reviewedPeer Reviewe

Identification of Potential Targets Linked to the Cardiovascular/Alzheimer’s Axis through Bioinformatics Approaches

This research was funded by Instituto de Salud Carlos III grant (PI18-00803), co-funded by the European Regional Development Fund (FEDER) and by Junta de Andalucía grant (PI-0268- 2019). In addition, C.G.-F. and S.G.-S. are funded by postdoctoral and predoctoral fellowships from Instituto de Salud Carlos III with co-funding by FEDER (CD20/00022 and FI19/00118 respectively). R.S.d.l.T. was funded by the University of Granada with co-funding by FEDER, by grant number 8110 (Research investigator call in the framework of the youth guarantee Program)The identification of common targets in Alzheimer's disease (AD) and cardiovascular disease (CVD) in recent years makes the study of the CVD/AD axis a research topic of great interest. Besides aging, other links between CVD and AD have been described, suggesting the existence of common molecular mechanisms. Our study aimed to identify common targets in the CVD/AD axis. For this purpose, genomic data from calcified and healthy femoral artery samples were used to identify differentially expressed genes (DEGs), which were used to generate a protein-protein interaction network, where a module related to AD was identified. This module was enriched with the functionally closest proteins and analyzed using different centrality algorithms to determine the main targets in the CVD/AD axis. Validation was performed by proteomic and data mining analyses. The proteins identified with an important role in both pathologies were apolipoprotein E and haptoglobin as DEGs, with a fold change about +2 and -2, in calcified femoral artery vs healthy artery, respectively, and clusterin and alpha-2-macroglobulin as close interactors that matched in our proteomic analysis. However, further studies are needed to elucidate the specific role of these proteins, and to evaluate its function as biomarkers or therapeutic targets.Instituto de Salud Carlos III grant (PI18-00803) European Regional Development Fund (FEDER) Junta de Andalucía grant (PI-0268- 2019)Instituto de Salud Carlos III FEDER (CD20/00022 and FI19/00118 respectively)University of Granada FEDER 811