Role In Oxidative Stress Response Research Articles

Screening and functional identification of antioxidant microRNA-size sRNAs from Spirulina platensis using high-throughput sequencing.

MiRNA-size small RNAs, abbreviated as sRNAs, are increasingly being discovered as research progresses and omics technologies development in prokaryotes. However, there is a paucity of data concerning whether or not sRNAs exist in cyanobacteria and regulate the resistance to oxidative stress. In this investigation, small RNA libraries were constructed from the control, 50-nM and 100-nM H2O2 treatments of Spirulina platensis. By high-throughput sequencing, 23 candidate sRNAs showed significantly differential expression under oxidative stress, among which eight sRNAs were identified with the similar expression patterns as the sequencing results by real-time qPCR. By nucleic acid hybridisation, the corresponding expression changes also demonstrated that sequencing results of sRNAs were feasible and credible. By bioinformatics prediction and structure identification, 43 target genes were predicted for 8 sRNAs in plant miRNA database, among which 29 were annotated into the genome and related metabolic pathways of S. platensis. By COG functional classification and KEGG pathway analysis, 31 target genes were predicted to be directly or indirectly involved in the defence mechanism of H2O2 stress. Thirteen target genes displayed reversely changing patterns compared with those of their sRNAs under H2O2 treatment. These findings provide compelling evidence that these sRNAs in S. platensis play a crucial role in oxidative stress responses, and thus provide a theoretical reference for improving the stress-triggering physiological regulation.

Heme oxygenase 1 plays a crucial role in swamp eel response to oxidative stress induced by cadmium exposure or Aeromonas hydrophila infection.

Oxidative stress contributes a lot to initiation and progression of pathological conditions. Heme oxygenase 1 (HO1), a cytoprotective enzyme, is usually upregulated to alleviate oxidative stress in vivo. The function of teleost HO1 in the response to oxidative stress induced by heavy metal exposure and in pathogenic bacterial infection remains uncertain. In the present study, both complementary DNA and genomic sequence of a HO1-like gene cloned from the liver of swamp eel (Monopterus albus) are reported. Sequence analysis showed that the putative amino acid sequence contained a conserved heme oxygenase signature and displayed higher similarity to HO1 genes of other teleosts. Expression profile of swamp eel HO1 was investigated in healthy tissues and in tissues following stimulation with pathogenic bacteria (Aeromonas hydrophila) or cadmium chloride (CdCl2) exposure. Results demonstrated that HO1 messenger RNA (mRNA) was highly expressed in the liver and relatively less in other tissues. Bacterial infection with A. hydrophila significantly changed HO1 mRNA expression in the liver, spleen, and kidney, and the mRNA expression of HO1 and Nrf2 in the liver was elevated after the fish were exposed to CdCl2. Subsequently, the swamp eel HO1 was subcloned into a pET28a expression vector and transformed into Escherichia coli BL21 (DE3). Recombinant HO1 (rHO1) was successfully induced by 0.1mmol/l IPTG and purified by Ni-NTA His Bind Resin purification system. To determine whether the rHO1 could confer stress tolerance in vitro, the viability of control and HO1-expressing E. coli under CdCl2 stress was compared by spot assay. The rHO1 protein significantly increased survival rates of the bacterial hosts. To evaluate whether intraperitoneal injection with rHO1 protected the liver of swamp eel against A. hydrophila-induced oxidative stress, mRNA expression of HO1, Nrf2, hepcidin, and IL-1β as well as the oxidative stress-related parameters (ROS and total antioxidant capacity (T-AOC)) in the liver were examined. The results showed that exogenous rHO1 could significantly upgrade the mRNA expression of HO1 and hepcidin, coupled with increased ROS and T-AOC levels. However, Nrf2 and IL-1β expression levels were significantly downregulated and upregulated, respectively. These results suggested that HO1 should not only play a protective role in oxidative stress response and its adverse effects deserved further investigation.

Abstract B05: Tumor evolution and oxidative stress in recurrent human papillomavirus-mediated oropharyngeal squamous cell carcinoma

Abstract Background: Human papillomavirus (HPV)-mediated head and neck squamous cell carcinomas (HPVmHNSCC) are more responsive to platinum-based concurrent chemoradiation treatment than non-HPVmHNSCC. Previously, our group demonstrated that recurrent HPVmHNSCC are genomically more characteristic of HPV-negative than non-recurrent HPVmHNSCC. Moreover, we observed a propensity for NFE2L2 hotspot mutations among HPVmHNSCC that recurred. Since NFE2L2 is a transcription factor regulating oxidative stress, we hypothesized that genes involved with the management of oxidative stress would be upregulated in recurrent HPVmHNSCC. Methods: We performed whole-exome sequencing (WES) and RNA-Seq on matched primary and metachronous HPVmHNSCC from the Universities of Pittsburgh (UPitt) and Washington (UW). Somatic nucleotide (SNVs) and copy number variants (CNVs) were quantified. Clonal evolution analyses were performed. Differential expression and gene set enrichment (GSEA) were quantified. Results: RNA-Seq was available for 10 tumor pairs and WES for 7/10. Consistent with prior WES data, KMT2D was the most frequently mutated gene. We found two unique NFE2L2 hotspot mutations in one paired set. The primary tumor had an NFE2L2H150Y mutation while the recurrent tumor had an NFE2L2E79K mutation. Metachronous tumors possessed significantly more deleterious somatic alterations in genes involved with regulation of oxidative stress and NFE2L2 networks compared to the primary tumors. Clonality analyses revealed a propensity for subclonal mutations in oxidative-stress related genes. RNA-Seq analysis revealed differential expression of genes involved in mitochondrial oxidative stress (MAP3K9) and oxidoreductase activity (CYP24A1, HEPHL1). Moreover, 6/8 enriched gene sets among the metachronous recurrent tumors by GSEA were involved with oxidative stress response, mitochondrial genes, and electron transport. Conclusions: Here, using WES and RNA-Seq of pairs of primary and metachronous HPVmHNSCC we identified increased expression of and somatic alterations in oxidative stress regulatory pathways in recurrent HPVmHNSCC that appear to occur later in tumor evolution. One possible explanation for this is that treatment selects for tumor clones that can withstand the oxidative environment induced by chemoradiation. Ultimately, understanding the role of oxidative stress response under therapy may be critical to mitigating recurrence and treating recurrences with a favorable genomic profile. Citation Format: Daniel Faden, Richard A. Harbison, Qing Zhang, Jeffrey Delrow, Umamaheswar Duvvuri. Tumor evolution and oxidative stress in recurrent human papillomavirus-mediated oropharyngeal squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B05.

Nrf2 suppresses allergic lung inflammation by attenuating the type2-innate lymphoid cell response

Abstract The Keap1-Nrf2 system has an essential role in oxidative-stress responses by inducing a number of cytoprotective genes. Under stress, damaged epithelial cells release cytokines that activate Type 2 Innate lymphoid cells (ILC2s), which mediate the allergic immune response. Here we investigated the role of the Keap1-Nrf2 pathway in ILC2 homeostasis and allergic inflammation, using Nrf-2-knockout mice. ILC2s from Nrf2-deficient mice showed a transient, upregulated IL-33 response, and underwent hyper-proliferation in response to combined stimulation by IL-33 with IL-2, IL-7, or TSLP. This enhanced proliferation was correlated with an increased activation of downstream signals, including JAK1, Akt, and Erk1/2. In contrast, activating Nrf2 with a chemical inducer (CDDO-Im) decreased the viability of wild-type, but not of the Nrf2-deficient ILC2s. The reduced ILC2 viability resembled that exerted by the corticosteroid dexamethasone; however, unlike the latter, the Nrf2-dependent cell death was mediated by neither caspase 3-dependent apoptosis nor necroptosis. Using a mouse intratracheal IL-33-administration allergy model, we found that the activation of Nrf2 by CDDO-Im in vivo decreased the numbers of pulmonary ILC2s and eosinophils. These findings indicated that Nrf2 is an important regulator of the allergic response, by determining the survival and death of ILC2s, and suggest that Nrf2 activation is a potential therapeutic strategy for steroid-resistant allergy alleviation.

Protein‐DNA Interactome of the OxyR Oxidative‐Stress Transcription Factors in Aliivibrio fischeri

Transcription factors (TFs) are sequence‐specific DNA‐binding proteins that dictate cell function by controlling selective usage of genomic information. One of the largest prokaryotic family of TFs is the LysR type transcriptional regulators (LTTRs). These TFs tend to regulate a diverse set of genes involved in virulence, metabolism, quorum sensing, and oxidative stress. Hydrogen peroxide (H2O2) is a well‐known cell damaging agent, which can lead to oxidative stress, disease, and important beneficial signaling roles. OxyR is a transcription factor with an N‐terminal DNA‐binding helix‐turn‐helix motif and a C‐terminal co‐inducer‐binding domain, that is involved in the defense mechanism against hydrogen peroxide‐induced oxidative stress. Although, different OxyR proteins share functional and sequence similarities across different bacterial species, they respond differently to hydrogen peroxide. The symbiotic relationship between Euprymna scolopes and Aliivibrio fischeri is dependent on the initial colonization survival of the bacteria in an environment rich reactive oxygen species. It has been documented that A. fischeri’s genome encodes two OxyR genes, OxyR1 and OxyR2. Studying OxyR1 and OxyR2 protein‐DNA interactomes will allow us to identify key differences and their possible roles in oxidative stress responses. Currently, we have over‐expressed and purified the full‐length OxyR1 and OxyR2 and their DNA binding domain. Protein functionality has been tested by Electric Mobility Shift Assay (EMSA), and High‐Throughput Systematic Evolution of Ligands by Exponential Enrichment (HT‐SELEX) will be used to determine the protein‐DNA interactomes of the OxyR1 and OxyR2. Genomic targets of OxyR1 and OxyR2 will be bioinformatically predicted once the DNA‐binding specificities are established.Support or Funding InformationNational Institutes of Health SC1GM127231.

Biological functions, pathways of glutathione peroxidases and their expression in osteoarthritis

Purpose: To determine the biological functions of selenoprotein glutathione peroxidase (GPXs) and thier expressions in osteoarthritis (OA), and to fully explore the possible association between GPXs and OA in order to provide new insights to the study of OA pathogenesis and novel prophylactic and therapeutic strategies for OA. Methods: The biological functions of the five GPX-genes were analyzed using bioinformatics and data mining techniques. A protein-protein interaction network (PPI) was constructed to identify the core genes associated with the biological functions of GPXs. Furthermore, the expressions of the five GPX-genes in OA patients and healthy individuals were determined using gene chip datasets from public databases based on meta-analysis methods. Results: KEGG pathway enrichment analysis showed that the signalling pathways of GPXs were mainly enriched in glutathione metabolism, thyroid hormone synthesis, and amyotrophic lateral sclerosis. The PPI network of the five GPXs indicated that SOD2 was the core protein of this network (Figure 1). The microRNA interaction network of GPXs revealed 16 microRNAs for GPX1, 12 microRNAs for GPX6, and 2 microRNAs each for GPX2 and GPX4 (Figure 2). Quercetin, acetaminophen and aflatoxin B1 play important roles in the interaction between GPXs and environmental factors. Meta-analysis showed that GPX2, GPX3, GPX6 expressions showed a decreasing trend in the OA group compared with the control group (P<0.05). However, meta-analysis showed that the GPX1 and GPX4 expressions were not statistically different from those in the control group (P>0.05) (Figure 3). Conclusions: GPXs may play an important role in oxidative stress response and anti-oxidation in OA. Glutathione metabolism disorder and thyroid hormone synthesis abnormalities may increase the risk of OA. SOD2 may affect the development and progression of OA. The interaction between GPXs and MicroRNA or environmental factors provide some insights into the molecular mechanisms underlying OA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Influence of gene position on the expression divergence of oxidative response genes in intraspecific yeast.

Phenotypic variation can arise from differences in the protein coding sequence and in the regulatory elements. However, little is known about the contribution of regulatory difference to the expression divergence, especially the cis and trans regulatory variation to the expression divergence in intraspecific populations. In this study, we used two different yeast strains, BY4743 and RM11-1a/α, to study the regulatory variation to the expression divergence between BY and RM under oxidative stress condition. Our results indicated that the expression divergence of BY and RM is mainly due to trans regulatory variations under both normal and oxidative stress conditions. However, cis regulatory variation seems to play a very important role in oxidative stress response in yeast because 36% of genes showed an increase in cis regulatory variation effect compared with 13% of genes that showed an increase in trans regulatory variation effect after oxidative stress. Our data also indicated that genes located on the longer arm of the chromosomes are more susceptible to cis variation effect under oxidative stress than genes on the shorter arm of the chromosomes.

Spatiotemporal regulation of NADP(H) phosphatase Nocturnin and its role in oxidative stress response

An intimate link exists between circadian clocks and metabolism with nearly every metabolic pathway in the mammalian liver under circadian control. Circadian regulation of metabolism is largely driven by rhythmic transcriptional activation of clock-controlled genes. Among these output genes, Nocturnin (Noct) has one of the highest amplitude rhythms at the mRNA level. The Noct gene encodes a protein (NOC) that is highly conserved with the endonuclease/exonuclease/phosphatase (EEP) domain-containing CCR4 family of deadenylases, but highly purified NOC possesses little or no ribonuclease activity. Here, we show that NOC utilizes the dinucleotide NADP(H) as a substrate, removing the 2' phosphate to generate NAD(H), and is a direct regulator of oxidative stress response through its NADPH 2' phosphatase activity. Furthermore, we describe two isoforms of NOC in the mouse liver. The cytoplasmic form of NOC is constitutively expressed and associates externally with membranes of other organelles, including the endoplasmic reticulum, via N-terminal glycine myristoylation. In contrast, the mitochondrial form of NOC possesses high-amplitude circadian rhythmicity with peak expression level during the early dark phase. These findings suggest that NOC regulates local intracellular concentrations of NADP(H) in a manner that changes over the course of the day.

Analysis of Functional Domains in Rho5, the Yeast Homolog of Human Rac1 GTPase, in Oxidative Stress Response.

The small GTPase Rho5 of Saccharomyces cerevisiae is required for proper regulation of different signaling pathways, which includes the response to cell wall, osmotic, nutrient, and oxidative stress. We here show that proper in vivo function and intracellular distribution of Rho5 depends on its hypervariable region at the carboxyterminal end, which includes the CAAX box for lipid modification, a preceding polybasic region (PBR) carrying a serine residue, and a 98 amino acid–specific insertion only present in Rho5 of S. cerevisiae but not in its human homolog Rac1. Results from trapping GFP-Rho5 variants to the mitochondrial surface suggest that the GTPase needs to be activated at the plasma membrane prior to its translocation to mitochondria in order to fulfil its role in oxidative stress response. These findings are supported by heterologous expression of a codon-optimized human RAC1 gene, which can only complement a yeast rho5 deletion in a chimeric fusion with RHO5 sequences that restore the correct spatiotemporal distribution of the encoded protein.

NADPH Oxidase Is Crucial for the Cellular Redox Homeostasis in Fungal Pathogen Botrytis cinerea.

During interactions, both plants and pathogens produce reactive oxygen species (ROS). Plants generate ROS for defense induction, while pathogens synthesize ROS for growth, sporulation, and virulence. NADPH oxidase (NOX) complex in the plasma membrane represents a main protein complex for ROS production in pathogens. Although NOX plays a crucial role in pathogenicity of pathogens, the underlying molecular mechanisms of NOX, especially the proteins regulated by NOX, remain largely unknown. Here, we applied an iodoacetyl tandem mass tag-based redox proteomic assay to investigate the protein redox dynamics in deletion mutant of bcnoxR, which encodes a regulatory subunit of NOX in the fungal pathogen Botrytis cinerea. In total, 214 unique peptidyl cysteine (Cys) thiols from 168 proteins were identified and quantified in both the wild type and ∆bcnoxR mutant. The Cys thiols in the ∆bcnoxR mutant were generally more oxidized than those in the wild type, suggesting that BcNoxR is essential for maintaining the equilibrium of the redox state in B. cinerea. Site-specific thiol oxidation analysis indicated that 142 peptides containing the oxidized thiols changed abundance significantly in the ∆bcnoxR mutant. Proteins containing these differential peptides are classified into various functional categories. Functional analysis revealed that one of these proteins, 6-phosphate dehydrogenase, played roles in oxidative stress response and pathogenesis of B. cinerea. These results provide insight into the potential target proteins and the ROS signal transduction pathway regulated by NOX.

Role of AccMGST1 in oxidative stress resistance in Apis cerana cerana.

As detoxification enzymes, proteins in the glutathione S-transferase (GST) superfamily are reported to participate in oxidative stress resistance. Nevertheless, microsomal GSTs (MGSTs), a unique subclass of the GST superfamily associated with membranes, are rarely studied in insects. Here, we isolated an MGST gene in Apis cerana cerana (AccMGST1) and verified its role in oxidative stress response. We found higher expression of AccMGST1 in protective or defensive tissue, that is, the epidermis, which indicated its role in stress resistance. Real-time quantitative PCR (qRT-PCR) analysis indicated that AccMGST1 was upregulated by oxidative stresses at the transcriptional level. In contrast, AccMGST1 expression was inhibited when the antioxidant vitamin C (VC) was fed to experimental bees. Through western blotting, we found that the protein level of AccMGST1 under oxidative stress corresponded to the transcript level. Disc diffusion and mixed-function oxidation (MFO) assays suggested that AccMGST1 can protect not only cells but also DNA against oxidative damage. Furthermore, we discovered that the expression patterns of known antioxidant genes were changed in A. cerana cerana after AccMGST1 was silenced by RNA interference (RNAi). Thus, we concluded that the gene AccMGST1 exerts a significant role in the antioxidant mechanism.

Stress-Free Evolution: The Nrf-Coordinated Oxidative Stress Response in Early Diverging Metazoans

Environmental stress from ultraviolet radiation, elevated temperatures or metal toxicity can lead to reactive oxygen species in cells, leading to oxidative DNA damage, premature aging, neurodegenerative diseases, and cancer. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activates many cytoprotective proteins within the nucleus to maintain homeostasis during oxidative stress. In vertebrates, Nrf2 levels are regulated by the Kelch-family protein Keap1 (Kelch-like ECH-associated protein 1) in the absence of stress according to a canonical redox control pathway. Little, however, is known about the redox control pathway used in early diverging metazoans. Our study examines the presence of known oxidative stress regulatory elements within non-bilaterian metazoans including free living and parasitic cnidarians, ctenophores, placozoans, and sponges. Cnidarians, with their pivotal position as the sister phylum to bilaterians, play an important role in understanding the evolutionary history of response to oxidative stress. Through comparative genomic and transcriptomic analysis our results show that Nrf homologs evolved early in metazoans, whereas Keap1 appeared later in the last common ancestor of cnidarians and bilaterians. However, key Nrf-Keap1 interacting domains are not conserved within the cnidarian lineage, suggesting this important pathway evolved with the radiation of bilaterians. Several known downstream Nrf targets are present in cnidarians suggesting that cnidarian Nrf plays an important role in oxidative stress response even in the absence of Keap1. Comparative analyses of key oxidative stress sensing and response proteins in early diverging metazoans thus provide important insights into the molecular basis of how these lineages interact with their environment and suggest a shared evolutionary history of regulatory pathways. Exploration of these pathways may prove important for the study of cancer therapeutics and broader research in oxidative stress, senescence, and the functional responses of early diverging metazoans to environmental change.

Identification of an adaptor protein-2 mu gene (AccAP2m) in Apis cerana cerana and its role in oxidative stress responses.

The adaptor protein-2 (AP-2) complex refers to a class of proteins that play an important role in cargo recognition and are involved in clathrin-mediated endocytosis of cargo proteins from the plasma membrane in animal cells. However, there have been few studies of AP-2 in insects. Here, we isolated an AP-2 gene from Apis cerana cerana (A. cerana cerana) and explored the function of this gene, named AccAP2m. The obtained amino acid sequence of AccAP2m is highly conserved across species and contains conserved features of the AP-2 family. Quantitative real-time PCR analysis showed that AccAP2m mRNA levels were high in newly emerged workers, and the expression levels were higher in muscle than in other tissues. The expression of AccAP2m was altered in response to various environmental stress factors. Moreover, overexpression of recombinant AccAP2m protein enhanced the resistance of the bacteria to HgCl2 , CdCl2 , and cumyl hydroperoxide. In addition, after knockdown of AccAP2m in A. cerana cerana by RNA interference (RNAi), the transcription of some antioxidant genes was downregulated, and the activities of some antioxidant enzymes decreased. Taken together, these results indicate that AccAP2m may play an indispensable role in response to oxidative stresses.

Bacterial lipocalins protect bacteria from antibiotic-induced membrane lipid peroxidation

Burkholderia cenocepacia, an opportunistic bacterium causing chronic respiratory infections in patients with cystic fibrosis, produces the bacterial lipocalin protein BcnA upon exposure to sublethal concentrations of bactericidal antibiotics. BcnA captures antibiotics outside bacterial cells, providing a novel extracellular mechanism of antimicrobial resistance. Sublethal concentrations of bactericidal antibiotics (e.g. polymyxin B, norfloxacin) and superoxide ion stress inducers (e.g. paraquat) stimulate transcription of bcnA, suggesting BcnA may also play a role in oxidative stress responses. bcnA gene homologues can be genetically linked to an upstream gene encoding a predicted membrane cytochrome b-561 protein, which we have named bcoA (bcnA cytochrome oxidase-associated gene). RT-PCR showed that both bcnA and bcoA are co-transcribed in B. cenocepacia. We hypothesize that BcnA and BcoA may be components of an unrecognized pathway to protect B. cenocepacia and other bacteria from membrane lipid-derived peroxidation damage. Here, we show that bcnA and bcoA deletion mutants display enhanced membrane lipid peroxidation and fail to survive under conditions that stimulate peroxidative stress (e.g. cold stress and salt stress). Compared to wild type, the levels of the lipid peroxidation biomarker malondialdehyde are also increased in the mutants upon exposure to near-lethal concentrations of polymyxin B and norfloxacin. No differences in bacterial survival were detected in parental and mutants under anaerobiosis. These findings indicate that BcnA and BcoA participate in a previously unrecognised peroxidation detoxification pathway that likely protects the outer membrane lipid bilayer under extreme stress conditions such as antibiotic killing.

Elucidation of the mechanism of disulfide exchange between staphylococcal thioredoxin2 and thioredoxin reductase2: A structural insight

The redox homeostasis of cytoplasm is maintained by a series of disulfide exchange reactions mediated by proteins belonging to the thioredoxin superfamily. Thioredoxin and thioredoxin reductase, being the major members of the family, play a key role in oxidative stress response of Staphylococcus aureus. In this report, we have identified and characterised an active thioredoxin system of the mentioned pathogen. Crystal structure of thioredoxin2 (SaTrx2) in its reduced form reveals that it contains the conserved redox active WCXXC motif and a thioredoxin fold. Thioredoxin reductase2 (SaTR2) is a flavoprotein and consists of two Rossmann folds as the binding sites for FAD and NADPH. Crystal structure of the SaTR2 holoenzyme shows that the protein consists of two domains and the catalytic site comprises of an intramolecular disulfide bond formed between two sequentially distal cysteine residues. Biophysical and biochemical studies unveil that SaTrx2 and SaTR2 can physically interact in solution and in the course of sustaining the redox equilibrium, the latter reduces the former. Molecular docking has been performed to illustrate the interface formed between SaTrx2 and SaTR2 during the disulfide exchange reaction.

Novel interactions of Selenium Binding Protein family with the PICOT containing proteins AtGRXS14 and AtGRXS16 in Arabidopsis thaliana

During abiotic stress the primary symptom of phytotoxicity can be ROS production which is strictly regulated by ROS scavenging pathways involving enzymatic and non-enzymatic antioxidants. Furthermore, ROS are well-described secondary messengers of cellular processes, while during the course of evolution, plants have accomplished high degree of control over ROS and used them as signalling molecules. Glutaredoxins (GRXs) are small and ubiquitous glutathione (GSH) -or thioredoxin reductase (TR)-dependent oxidoreductases belonging to the thioredoxin (TRX) superfamily which are conserved in most eukaryotes and prokaryotes. In Arabidopsis thaliana GRXs are subdivided into four classes playing a central role in oxidative stress responses and physiological functions. In this work, we describe a novel interaction of AtGRXS14 with the Selenium Binding Protein 1 (AtSBP1), a protein proposed to be integrated in a regulatory network that senses alterations in cellular redox state and acts towards its restoration. We further show that SBP protein family interacts with AtGRXS16 that also contains a PICOT domain, like AtGRXS14.

Nrf2 represses the onset of type 1 diabetes in non-obese diabetic mice.

The transcription factor Nrf2 (NF-E2-related factor 2) plays a critical role in oxidative stress responses. While activation of Nrf2 signaling is known to exert anti-inflammatory effects, Nrf2 function in inflammation-mediated autoimmune disorders, such as type 1 diabetes, is not well established. To address the roles of Nrf2 in protection against autoreactive T-cell-induced type 1 diabetes, we used non-obese diabetic (NOD) mice, a polygenic model of human type 1 diabetes, to generate a genetic model that allowed us to assess the contribution of Nrf2 activation to preventing and/or treating type 1 diabetes. As Keap1 negatively regulates Nrf2, we used Keap1 gene knockdown driven by either hypomorphic or knockout alleles of Keap1,which enhances Nrf2 signaling to moderate and excess levels, respectively. We found that Nrf2 activation in NOD::Keap1FA/- mice inhibited T-cell infiltration within or near the islets, ameliorated impairment of insulin secretion, and prevented development of diabetes mellitus in the NOD mice. Notably, Nrf2 activation decreased both plasma interferon-γ (IFN-γ) levels and IFN-γ-positive cell numbers in the pancreatic islets. These findings were also observed in mice with two hypomorphic Keap1 alleles (Keap1FA/FA). Both NOD::Keap1FA/- and NOD::Keap1FA/FA mice had decreased incidence of diabetes mellitus, demonstrating that the activation of Nrf2 signaling prevents the onset of type 1 diabetes mellitus in NOD mice. Thus, Nrf2 appears to be a potential target for preventing and treating type 1 diabetes.

The Zinc-Metallothionein Redox System Reduces Oxidative Stress in Retinal Pigment Epithelial Cells.

Oxidative stress affects all the structures of the human eye, particularly the retina and its retinal pigment epithelium (RPE). The RPE limits oxidative damage by several protective mechanisms, including the non-enzymatic antioxidant system zinc-metallothionein (Zn-MT). This work aimed to investigate the role of Zn-MT in the protection of RPE from the oxidative damage of reactive oxygen intermediates by analytical and biochemical-based techniques. The Zn-MT system was induced in an in vitro model of RPE cells and determined by elemental mass spectrometry with enriched isotopes and mathematical calculations. Induced-oxidative stress was quantified using fluorescent probes. We observed that 25, 50 or 100 μM of zinc induced Zn-MT synthesis (1.6-, 3.6- and 11.9-fold, respectively), while pre-treated cells with zinc (25, 50, and 100 μM) and subsequent 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) treatment increased Zn-MT levels in a lesser extent (0.8-, 2.1-, 6.1-fold, respectively), exerting a stoichiometric transition in the Zn-MT complex. Moreover, AAPH treatment decreased MT levels (0.4-fold), while the stoichiometry remained constant or slightly higher when compared to non-treated cells. Convincingly, induction of Zn-MT significantly attenuated oxidative stress produced by free radicals’ generators. We conclude that the stoichiometry of Zn-MT plays an important role in oxidative stress response, related with cellular metal homeostasis.

The reduced activity of PP-1\u03b1 under redox stress condition is a consequence of GSH-mediated transient disulfide formation

Heart failure is the most common cause of morbidity and hospitalization in the western civilization. Protein phosphatases play a key role in the basal cardiac contractility and in the responses to β-adrenergic stimulation with type-1 phosphatase (PP-1) being major contributor. We propose here that formation of transient disulfide bridges in PP-1α might play a leading role in oxidative stress response. First, we established an optimized workflow, the so-called “cross-over-read” search method, for the identification of disulfide-linked species using permutated databases. By applying this method, we demonstrate the formation of unexpected transient disulfides in PP-1α to shelter against over-oxidation. This protection mechanism strongly depends on the fast response in the presence of reduced glutathione. Our work points out that the dimerization of PP-1α involving Cys39 and Cys127 is presumably important for the protection of PP-1α active surface in the absence of a substrate. We finally give insight into the electron transport from the PP-1α catalytic core to the surface. Our data suggest that the formation of transient disulfides might be a general mechanism of proteins to escape from irreversible cysteine oxidation and to prevent their complete inactivation.

The roles of Arabidopsis proteins of Lhcb4, Lhcb5 and Lhcb6 in oxidative stress under natural light conditions

Under light conditions, highly reactive oxygen species (ROS) can be generated in the antenna systems and the reaction center of photosystems (PS). The protective roles of Lhcb4 (CP29), Lhcb5 (CP26) and Lhcb6 (CP24), three minor chlorophyll binding antenna proteins during photoinhibition have been well studied. However, their regulatory mechanisms against oxidative damages under natural light conditions remain unknown. Here we investigated their specific roles in oxidative stress responses and photosynthetic adaptation by using the Arabidopsis thaliana knockout lines grown in the field condition. All three mutant lines exhibited decreased energy-transfer efficiency from the LHCII (light-harvesting complex II) to the PSII reaction center. Oxygen evolution capacity decreased slightly in the plants lacking Lhcb4 (koLHCB4) and Lhcb6 (koLHCB6). Photosynthetic rates and fitness for the plants lacking Lhcb5 (koLHCB5) or koLHCB6 grown in the field were affected, but not in the plants lacking Lhcb4. Antioxidant analysis indicated the lowest antioxidant enzyme activities and the lowest levels of non-enzymatic antioxidants in koLHCB6 plants. In addition, koLHCB6 plants accumulated much higher levels of superoxide and hydrogen, and suffered more severe oxidative-damages in the field. Our results clearly demonstrate that Lhcb6 may be involved in alleviating oxidative stress and photoprotection under natural conditions.