Requirements for the biogenesis of [2Fe-2S] proteins in the human and yeast cytosol.

The biogenesis of iron-sulfur (Fe/S) proteins entails the synthesis and trafficking of Fe/S clusters, followed by their insertion into target apoproteins. In eukaryotes, the multiple steps of biogenesis are accomplished by complex protein machineries in both mitochondria and cytosol. The underlying biochemical pathways have been elucidated over the past decades, yet the mechanisms of cytosolic [2Fe-2S] protein assembly have remained ill-defined. Similarly, the precise site of glutathione (GSH) requirement in cytosolic and nuclear Fe/S protein biogenesis is unclear, as is the molecular role of the GSH-dependent cytosolic monothiol glutaredoxins (cGrxs). Here, we investigated these questions in human and yeast cells by various in vivo approaches. [2Fe-2S] cluster assembly of cytosolic target apoproteins required the mitochondrial ISC machinery, the mitochondrial transporter Atm1/ABCB7 and GSH, yet occurred independently of both the CIA system and cGrxs. This mechanism was strikingly different from the ISC-, Atm1/ABCB7-, GSH-, and CIA-dependent assembly of cytosolic-nuclear [4Fe-4S] proteins. One notable exception to this cytosolic [2Fe-2S] protein maturation pathway defined here was yeast Apd1 which used the CIA system via binding to the CIA targeting complex through its C-terminal tryptophan. cGrxs, although attributed as [2Fe-2S] cluster chaperones or trafficking proteins, were not essential in vivo for delivering [2Fe-2S] clusters to either CIA components or target apoproteins. Finally, the most critical GSH requirement was assigned to Atm1-dependent export, i.e. a step before GSH-dependent cGrxs function. Our findings extend the general model of eukaryotic Fe/S protein biogenesis by adding the molecular requirements for cytosolic [2Fe-2S] protein maturation.

Identification and characterization of multidomain monothiol glutaredoxin 3 from diploblastic Hydra.

Intracellular antioxidant glutaredoxin controls cell proliferation and survival. Based on the active site, structure, and conserved domain motifs, it is classified into two classes. Class I contains dithiol Grxs with two cysteines in the consensus active site sequence CXXC, while class II has monothiol Grxs with one cysteine residue in the active site. Monothiol Grxs can also have an additional N-terminal thioredoxin (Trx)-like domain. Previously, we reported the characterization of Grx1 from Hydra vulgaris (HvGrx1), which is a dithiol isoform. Here, we report the molecular cloning, expression, analysis, and characterization of another isoform of Grx, which is the multidomain monothiol glutaredoxin-3 from Hydra vulgaris (HvGrx3). It encodes a protein with 303 amino acids and is significantly larger and more divergent than HvGrx1. In-silico analysis revealed that Grx1 and Grx3 have 22.5% and 9.9% identical nucleotide and amino acid sequences, respectively. HvGrx3 has two glutaredoxin domains and a thioredoxin-like domain at its amino terminus, unlike HvGrx1, which has a single glutaredoxin domain. Like other monothiol glutaredoxins, HvGrx3 failed to reduce glutathione-hydroxyethyl disulfide. In the whole Hydra, HvGrx3 was found to be expressed all over the body column, and treatment with H2O2 led to a significant upregulation of HvGrx3. When transfected in HCT116 (human colon cancer cells) cells, HvGrx3 enhanced cell proliferation and migration, indicating that this isoform could be involved in these cellular functions. These transfected cells also tolerate oxidative stress better.

Fmp40 ampylase regulates cell survival upon oxidative stress by controlling Prx1 and Trx3 oxidation.

Reactive oxygen species (ROS), play important roles in cellular signaling, nonetheless are toxic at higher concentrations. Cells have many interconnected, overlapped or backup systems to neutralize ROS, but their regulatory mechanisms remain poorly understood. Here, we reveal an essential role for mitochondrial AMPylase Fmp40 from budding yeast in regulating the redox states of the mitochondrial 1-Cys peroxiredoxin Prx1, which is the only protein shown to neutralize H2O2 with the oxidation of the mitochondrial glutathione and the thioredoxin Trx3, directly involved in the reduction of Prx1. Deletion of FMP40 impacts a cellular response to H2O2 treatment that leads to programmed cell death (PCD) induction and an adaptive response involving up or down regulation of genes encoding, among others the catalase Cta1, PCD inducing factor Aif1, and mitochondrial redoxins Trx3 and Grx2. This ultimately perturbs the reduced glutathione and NADPH cellular pools. We further demonstrated that Fmp40 AMPylates Prx1, Trx3, and Grx2 in vitro and interacts with Trx3 in vivo. AMPylation of the threonine residue 66 in Trx3 is essential for this protein's proper endogenous level and its precursor forms' maturation under oxidative stress conditions. Additionally, we showed the Grx2 involvement in the reduction of Trx3 in vivo. Taken together, Fmp40, through control of the reduction of mitochondrial redoxins, regulates the hydrogen peroxide, GSH and NADPH signaling influencing the yeast cell survival.

Specificity and dynamics of H2O2 detoxification by the cytosolic redox regulatory network as revealed by in vitro reconstitution.

The thiol redox state is a decisive functional characteristic of proteins in cell biology. Plasmatic cell compartments maintain a thiol-based redox regulatory network linked to the glutathione/glutathione disulfide couple (GSH/GSSG) and the NAD(P)H system. The basic network constituents are known and in vivo cell imaging with gene-encoded probes have revealed insight into the dynamics of the [GSH]2/[GSSG] redox potential, cellular H2O2 and NAD(P)H+H+ amounts in dependence on metabolic and environmental cues. Less understood is the contribution and interaction of the network components, also because of compensatory reactions in genetic approaches. Reconstituting the cytosolic network of Arabidopsis thaliana in vitro from fifteen recombinant proteins at in vivo concentrations, namely glutathione peroxidase-like (GPXL), peroxiredoxins (PRX), glutaredoxins (GRX), thioredoxins, NADPH-dependent thioredoxin reductase A and glutathione reductase and applying Grx1-roGFP2 or roGFP2-Orp1 as dynamic sensors, allowed for monitoring the response to a single H2O2 pulse. The major change in thiol oxidation as quantified by mass spectrometry-based proteomics occurred in relevant peptides of GPXL, and to a lesser extent of PRX, while other Cys-containing peptides only showed small changes in their redox state and protection. Titration of ascorbate peroxidase (APX) into the system together with dehydroascorbate reductase lowered the oxidation of the fluorescent sensors in the network but was unable to suppress it. The results demonstrate the power of the network to detoxify H2O2, the partially independent branches of electron flow with significance for specific cell signaling and the importance of APX to modulate the signaling without suppressing it and shifting the burden to glutathione oxidation.

Identification and Functional Analysis of Lysine 2-Hydroxyisobutyrylation in Cyanobacteria.

Cyanobacteria are the oldest prokaryotic photoautotrophic microorganisms and have evolved complicated post-translational modification (PTM) machinery to respond to environmental stress. Lysine 2-hydroxyisobutyrylation (Khib) is a newly identified PTM that is reported to play important roles in diverse biological processes, however, its distribution and function in cyanobacteria have not been reported. Here, we performed the first systematic studies of Khib in a model cyanobacterium Synechococcus sp. strain PCC 7002 (Syn7002) using peptide prefractionation, pan-Khib antibody enrichment, and high-accuracy mass spectrometry (MS) analysis. A total of 1875 high-confidence Khib sites on 618 proteins were identified, and a large proportion of Khib sites are present on proteins in the cellular metabolism, protein synthesis, and photosynthesis pathways. Using site-directed mutagenesis and functional studies, we showed that Khib of glutaredoxin (Grx) affects the efficiency of the PS II reaction center and H2O2 resistance in Syn7002. Together, this study provides novel insights into the functions of Khib in cyanobacteria and suggests that reversible Khib may influence the stress response and photosynthesis in both cyanobacteria and plants.

Salvia miltiorrhiza Bge. processed with porcine cardiac blood inhibited GLRX5-mediated ferroptosis alleviating cerebral ischemia-reperfusion injury.

BACKGROUND: Cerebral ischemia-reperfusion injury (CIRI) is a destructive adverse reaction of ischemic stroke, leading to high disability and mortality rates. Salvia miltiorrhiza Bge. (Danshen, DS) processed with porcine cardiac blood (PCB-DS), a characteristic processed product, has promising anti-ischemic effects. However, the underlying mechanism of PCB-DS against CIRI remains unclear. PURPOSE: Ferroptosis is demonstrated to be involved in CIRI. The aim of this study was to explore the molecular mechanism underlying PCB-DS inhibited GLRX5-mediated ferroptosis alleviating CIRI, which was different from DS. METHODS: Quality evaluation of PCB-DS and DS was conducted by UPLC. Pharmacological activities of PCB-DS and DS against CIRI were compared using neurobehavioral scores, infarct volume, proinflammatory factors, and pathological examinations. Proteomics was employed to explore the potential specific mechanism of PCB-DS against CIRI, which was different from DS. Based on the differential protein GLRX5, ferroptosis-related iron, GSH, MDA, SOD, ROS, liperfluo, and mitochondrial morphology were analyzed. Then, the proteins of GLRX5-mediated iron-starvation response and SLC7A11/GPX4 were analyzed. Finally, OGD/R-induced SH-SY5Y cells upon GLRX5 silencing were constructed to demonstrate that PCB-DS improved CIRI by GLRX5-mediated ferroptosis. RESULTS: PCB-DS better alleviated CIRI through decreasing neurological score, reducing the infarct volume, and suppressing the release of inflammatory cytokines than DS. Proteomics suggested that PCB-DS may ameliorate CIRI by inhibiting GLRX5-mediated ferroptosis, which was different from DS. PCB-DS reversed the abnormal mitochondrial morphology, iron, GSH, MDA, SOD, ROS, and liperfluo to inhibit ferroptosis in vitro and in vivo. PCB-DS directly activated GLRX5 suppressing the iron-starvation response and downregulated the SLC7A11/GPX4 signaling pathway to inhibit ferroptosis. Finally, silencing GLRX5 activated the iron-starvation response in SH-SY5Y cells and PCB-DS unimproved OGD/R injury upon GLRX5 silencing. CONCLUSION: Different from DS, PCB-DS suppressed ferroptosis to alleviate CIRI through inhibiting GLRX5-mediated iron-starvation response. These findings give a comprehensive understanding of the molecular mechanism underlying the effect of PCB-DS against CIRI and provide evidence to assess the product in clinical studies.

Immune targeting of filarial glutaredoxin through a multi-epitope peptide-based vaccine: A reverse vaccinology approach.

Despite the efforts of global programme to eliminate lymphatic filariasis (GPELF), the threat of lymphatic filariasis (LF) still looms over humanity in terms of long-term disabilities, and morbidities across the globe. In light of this situation, investigators have chosen to focus on the development of immunotherapeutics targeting the physiologically important filarial-specific proteins. Glutaredoxin (16.43 kDa) plays a pivotal role in filarial redox biology, serving as a vital contributor. In the context of the intra-host survival of filarial parasites, this antioxidant helps in mitigating the oxidative stress imposed by the host immune system. Given its significant contribution, the development of a vaccine targeting glutaredoxin holds promise as a new avenue for achieving a filaria-free world. Herein, multi-epitope-based vaccine was designed using advanced immunoinformatics approach. Initially, 4B-cell epitopes and 6 T-cell epitopes (4 MHC I and 2 MHC II) were identified from the 146 amino acid long sequence of glutaredoxin of the human filarid, Wuchereria bancrofti. Subsequent clustering of these epitopes with linker peptides finalized the vaccine structure. To boost TLR-mediated innate immunity, TLR-specific adjuvants were incorporated into the designed vaccine. After that, experimental analyses confirm the designed vaccine, Vac4 as anefficient ligand of human TLR5 to elicit protective innate immunity against filarial glutaredoxin. Immune simulation further demonstrated abundant levels of IgG and IgM as crucial contributors in triggering vaccine-induced adaptive responses in the recipients. Hence, to facilitate the validation of immunogenicity of the designed vaccine, Vac4 was cloned in silico in pET28a(+) expression vector for recombinant production. Taken together, our findings suggest that vaccine-mediated targeting of filarial glutaredoxin could be a future option for intervening LF on a global scale.

Capsicum chinense Jacq.-derived glutaredoxin (CcGRXS12) alters redox status of the cells to confer resistance against pepper mild mottle virus (PMMoV-I).

KEY MESSAGE: The CcGRXS12 gene protects plants from cellular oxidative damage that are caused by both biotic and abiotic stresses. The protein possesses GSH-disulphide oxidoreductase property but lacks Fe-S cluster assembly mechanism. Glutaredoxins (Grxs) are small, ubiquitous and multi-functional proteins. They are present in different compartments of plant cells. A chloroplast targeted Class I GRX (CcGRXS12) gene was isolated from Capsicum chinense during the pepper mild mottle virus (PMMoV) infection. Functional characterization of the gene was performed in Nicotiana benthamiana transgenic plants transformed with native C. chinense GRX (Nb:GRX), GRX-fused with GFP (Nb:GRX-GFP) and GRX-truncated for chloroplast sequences fused with GFP (Nb:Δ2MGRX-GFP). Overexpression of CcGRXS12 inhibited the PMMoV-I accumulation at the later stage of infection, accompanied with the activation of salicylic acid (SA) pathway pathogenesis-related (PR) transcripts and suppression of JA/ET pathway transcripts. Further, the reduced accumulation of auxin-induced Glutathione-S-Transferase (pCNT103) in CcGRXS12 overexpressing lines indicated that the protein could protect the plants from the oxidative stress caused by the virus. PMMoV-I infection increased the accumulation of pyridine nucleotides (PNs) mainly due to the reduced form of PNs (NAD(P)H), and it was high in Nb:GRX-GFP lines compared to other transgenic lines. Apart from biotic stress, CcGRXS12 protects the plants from abiotic stress conditions caused by H2O2 and herbicide paraquat. CcGRXS12 exhibited GSH-disulphide oxidoreductase activity in vitro; however, it was devoid of complementary Fe-S cluster assembly mechanism found in yeast. Overall, this study proves that CcGRXS12 plays a crucial role during biotic and abiotic stress in plants.

Exploration of glutaredoxin-1 oxidative modification in carbon nanomaterial-induced hepatotoxicity.

Herein, we present toxicological assessments of carbon nanomaterials in HL-7702 cells, and it was found that reactive oxygen species (ROS) levels were elevated. Mass spectrometry results indicated that cysteine sulfhydryl of glutaredoxin-1 (GLRX1) was oxidized to sulfenic acids and sulfonic acids by excessive ROS, which broke the binding of GLRX1 to apoptosis signal-regulating kinase 1, causing the activation of the JNK/p38 signaling pathway and ultimately hepatocyte apoptosis. However, a lower level of ROS upregulated GLRX1 instead of sulfonation modification of its active sites. Highly expressed GLRX1 in turn enabled the removal of intracellular ROS, thereby exerting inconspicuous toxic effects on cells. Taken together, these findings emphasized that CNM-induced hepatotoxicity is attributable to oxidative modifications of GLRX1 arising from redox imbalance.

The significance of glutaredoxins for diabetes mellitus and its complications.

Diabetes mellitus is a non-communicable metabolic disease hallmarked by chronic hyperglycemia caused by beta-cell failure. Diabetic complications affect the vasculature and result in macro- and microangiopathies, which account for a significantly increased morbidity and mortality. The rising incidence and prevalence of diabetes is a major global health burden. There are no feasible strategies for beta-cell preservation available in daily clinical practice. Therefore, patients rely on antidiabetic drugs or the application of exogenous insulin. Glutaredoxins (Grxs) are ubiquitously expressed and highly conserved members of the thioredoxin family of proteins. They have specific functions in redox-mediated signal transduction, iron homeostasis and biosynthesis of iron-sulfur (FeS) proteins, and the regulation of cell proliferation, survival, and function. The involvement of Grxs in chronic diseases has been a topic of research for several decades, suggesting them as therapeutic targets. Little is known about their role in diabetes and its complications. Therefore, this review summarizes the available literature on the significance of Grxs in diabetes and its complications. In conclusion, Grxs are differentially expressed in the endocrine pancreas and in tissues affected by diabetic complications, such as the heart, the kidneys, the eye, and the vasculature. They are involved in several pathways essential for insulin signaling, metabolic inflammation, glucose and fatty acid uptake and processing, cell survival, and iron and mitochondrial metabolism. Most studies describe significant changes in glutaredoxin expression and/or activity in response to the diabetic metabolism. In general, mitigated levels of Grxs are associated with oxidative distress, cell damage, and even cell death. The induced overexpression is considered a potential part of the cellular stress-response, counteracting oxidative distress and exerting beneficial impact on cell function such as insulin secretion, cytokine expression, and enzyme activity.

Localization of four class I glutaredoxins in the cytosol and the secretory pathway and characterization of their biochemical diversification.

Class I glutaredoxins (GRXs) are catalytically active oxidoreductases and considered key proteins mediating reversible glutathionylation and deglutathionylation of protein thiols during development and stress responses. To narrow in on putative target proteins, it is mandatory to know the subcellular localization of the respective GRXs and to understand their catalytic activities and putative redundancy between isoforms in the same compartment. We show that in Arabidopsis thaliana, GRXC1 and GRXC2 are cytosolic proteins with GRXC1 being attached to membranes through myristoylation. GRXC3 and GRXC4 are identified as type II membrane proteins along the early secretory pathway with their enzymatic function on the luminal side. Unexpectedly, neither single nor double mutants lacking both GRXs isoforms in the cytosol or the ER show phenotypes that differ from wild-type controls. Analysis of electrostatic surface potentials and clustering of GRXs based on their electrostatic interaction with roGFP2 mirrors the phylogenetic classification of class I GRXs, which clearly separates the cytosolic GRXC1 and GRXC2 from the luminal GRXC3 and GRXC4. Comparison of all four studied GRXs for their oxidoreductase function highlights biochemical diversification with GRXC3 and GRXC4 being better catalysts than GRXC1 and GRXC2 for the reduction of bis(2-hydroxyethyl) disulfide. With oxidized roGFP2 as an alternative substrate, GRXC1 and GRXC2 catalyze the reduction faster than GRXC3 and GRXC4, which suggests that catalytic efficiency of GRXs in reductive reactions depends on the respective substrate. Vice versa, GRXC3 and GRXC4 are faster than GRXC1 and GRXC2 in catalyzing the oxidation of pre-reduced roGFP2 in the reverse reaction.

Computational models as catalysts for investigating redoxin systems.

Thioredoxin, glutaredoxin and peroxiredoxin systems play central roles in redox regulation, signaling and metabolism in cells. In these systems, reducing equivalents from NAD(P)H are transferred by coupled thiol-disulfide exchange reactions to redoxins which then reduce a wide array of targets. However, the characterization of redoxin activity has been unclear, with redoxins regarded as enzymes in some studies and redox metabolites in others. Consequently, redoxin activities have been quantified by enzyme kinetic parameters in vitro, and redox potentials or redox ratios within cells. By analyzing all the reactions within these systems, computational models showed that many kinetic properties attributed to redoxins were due to system-level effects. Models of cellular redoxin networks have also been used to estimate intracellular hydrogen peroxide levels, analyze redox signaling and couple omic and kinetic data to understand the regulation of these networks in disease. Computational modeling has emerged as a powerful complementary tool to traditional redoxin enzyme kinetic and cellular assays that integrates data from a number of sources into a single quantitative framework to accelerate the analysis of redoxin systems.

Deciphering the mechanism of glutaredoxin-catalyzed roGFP2 redox sensing reveals a ternary complex with glutathione for protein disulfide reduction.

Glutaredoxins catalyze the reduction of disulfides and are key players in redox metabolism and regulation. While important insights were gained regarding the reduction of glutathione disulfide substrates, the mechanism of non-glutathione disulfide reduction remains highly debated. Here we determined the rate constants for the individual redox reactions between PfGrx, a model glutaredoxin from Plasmodium falciparum, and redox-sensitive green fluorescent protein 2 (roGFP2), a model substrate and versatile tool for intracellular redox measurements. We show that the PfGrx-catalyzed oxidation of roGFP2 occurs via a monothiol mechanism and is up to three orders of magnitude faster when roGFP2 and PfGrx are fused. The oxidation kinetics of roGFP2-PfGrx fusion constructs reflect at physiological GSSG concentrations the glutathionylation kinetics of the glutaredoxin moiety, thus allowing intracellular structure-function analysis. Reduction of the roGFP2 disulfide occurs via a monothiol mechanism and involves a ternary complex with GSH and PfGrx. Our study provides the mechanistic basis for understanding roGFP2 redox sensing and challenges previous mechanisms for protein disulfide reduction.

Chloroplasts lacking class I glutaredoxins are functional but show a delayed recovery of protein cysteinyl redox state after oxidative challenge.

Redox status of protein cysteinyl residues is mediated via glutathione (GSH)/glutaredoxin (GRX) and thioredoxin (TRX)-dependent redox cascades. An oxidative challenge can induce post-translational protein modifications on thiols, such as protein S-glutathionylation. Class I GRX are small thiol-disulfide oxidoreductases that reversibly catalyse S-glutathionylation and protein disulfide formation. TRX and GSH/GRX redox systems can provide partial backup for each other in several subcellular compartments, but not in the plastid stroma where TRX/light-dependent redox regulation of primary metabolism takes place. While the stromal TRX system has been studied at detail, the role of class I GRX on plastid redox processes is still unknown. We generate knockout lines of GRXC5 as the only chloroplast class I GRX of the moss Physcomitrium patens. While we find that PpGRXC5 has high activities in GSH-dependent oxidoreductase assays using hydroxyethyl disulfide or redox-sensitive GFP2 as substrates in vitro, Δgrxc5 plants show no detectable growth defect or stress sensitivity, in contrast to mutants with a less negative stromal EGSH (Δgr1). Using stroma-targeted roGFP2, we show increased protein Cys steady state oxidation and decreased reduction rates after oxidative challenge in Δgrxc5 plants in vivo, indicating kinetic uncoupling of the protein Cys redox state from EGSH. Compared to wildtype, protein Cys disulfide formation rates and S-glutathionylation levels after H2O2 treatment remained unchanged. Lack of class I GRX function in the stroma did not result in impaired carbon fixation. Our observations suggest specific roles for GRXC5 in the efficient transfer of electrons from GSH to target protein Cys as well as negligible cross-talk with metabolic regulation via the TRX system. We propose a model for stromal class I GRX function in efficient catalysis of protein dithiol/disulfide equilibria upon redox steady state alterations affecting stromal EGSH and highlight the importance of identifying in vivo target proteins of GRXC5.

A Prognostic Activity of Glutaredoxin 1 Protein (Grx1) in Colon Cancer.

Glutaredoxin 1 (Grx1) is an essential enzyme that regulates redox signal transduction and repairs protein oxidation by reversing S-glutathionylation, an oxidative modification of protein cysteine residues. Grx1 removes glutathione from proteins to restore their reduced state (protein-SH) and regulate protein-SSG levels in redox signaling networks. Thus, it can exert an influence on the development of cancer. To further investigate this problem, we performed an analysis of Grx1 expression in colon adenocarcinoma samples from the Polish population of patients with primary colon adenocarcinoma (stages I and II of colon cancer) and those with regional lymph node metastasis (stage III of colon cancer). Our study revealed a significant correlation between the expression of Grx1 protein through immunohistochemical analysis and various clinical characteristics of patients, such as histological grade, depth of invasion, angioinvasion, staging, regional lymph node invasion, and PCNA expression. It was found that almost 88% of patients with stage I had high levels of Grx1 expression, while only 1% of patients with stage III exhibited high levels of Grx1 protein expression. Furthermore, the study discovered that high levels of Grx1 expression were present in samples of colon mucosa without any pathological changes. These results were supported by in vitro analysis conducted on colorectal cancer cell lines that corresponded to stages I, II, and III of colorectal cancer, using qRT-PCR and Western blot.

Teaching NeuroImage: Glutaredoxin-5-Associated Variant Nonketotic Hyperglycinemia.

A 5-year-old boy presented with subacute motor regression since age 2.5 years. Examination revealed spasticity of bilateral lower extremities, generalized dystonia, and pseudobulbar palsy. Investigations revealed raised plasma lactate (2.5 mmol/L, normal range 0.8-1.5 mmol/L) and no evidence of sideroblastic anemia. Neuroimaging showed cavitating leukoencephalopathy with involvement of long tracts (corticospinal, spinothalamic tracts) and dorsolateral columns of cervicothoracic cord (Figures 1 and 2). A next-generation sequencing test identified a novel homozygous missense variant (c.171C > A, p.Phe57Leu) in exon 1 of the Glutaredoxin-5 (GLRX5) gene.

Glutaredoxin 2 Protein (Grx2) as an Independent Prognostic Factor Associated with the Survival of Colon Adenocarcinoma Patients.

Glutaredoxin 2 (Grx2; Glrx2) is a glutathione-dependent oxidoreductase located in mitochondria, which is central to the regulation of glutathione homeostasis and mitochondrial redox, and plays a crucial role in highly metabolic tissues. In response to mitochondrial redox signals and oxidative stress, Grx2 can catalyze the oxidation and S-glutathionylation of membrane-bound thiol proteins in mitochondria. Therefore, it can have a significant impact on cancer development. To investigate this further, we performed an immunohistochemical analysis of Grx2 protein expression in colon adenocarcinoma samples collected from patients with primary colon adenocarcinoma (stage I and II) and patients with metastasis to regional lymph nodes (stage III). The results of our study revealed a significant relationship between the immunohistochemical expression of Grx2 and tumor histological grade, depth of invasion, regional lymph node involvement, angioinvasion, staging, and PCNA immunohistochemical expression. It was found that 87% of patients with stage I had high levels of Grx2 expression. In contrast, only 33% of patients with stage II and 1% of patients with stage III had high levels of Grx2 expression. Moreover, the multivariate analysis revealed that the immunohistochemical expression of Grx2 protein apart from the grade of tumor differentiation was an independent prognostic factors for the survival of patients with colon adenocarcinoma. Studies analyzing Grx2 levels in patients' blood confirmed that the highest levels of serum Grx2 protein was also found in stage I patients, which was reflected in the survival curves. A higher level of Grx2 in the serum has been associated with a more favorable outcome. These results were supported by in vitro analysis conducted on colorectal cancer cell lines that corresponded to stages I, II, and III of colorectal cancer, using qRT-PCR and Western Blot.

Glutaredoxin regulation of primary root growth is associated with early drought stress tolerance in pearl millet.

Seedling root traits impact plant establishment under challenging environments. Pearl millet is one of the most heat and drought tolerant cereal crops that provides a vital food source across the sub-Saharan Sahel region. Pearl millet's early root system features a single fast-growing primary root which we hypothesize is an adaptation to the Sahelian climate. Using crop modeling, we demonstrate that early drought stress is an important constraint in agrosystems in the Sahel where pearl millet was domesticated. Furthermore, we show that increased pearl millet primary root growth is correlated with increased early water stress tolerance in field conditions. Genetics including genome-wide association study and quantitative trait loci (QTL) approaches identify genomic regions controlling this key root trait. Combining gene expression data, re-sequencing and re-annotation of one of these genomic regions identified a glutaredoxin-encoding gene PgGRXC9 as the candidate stress resilience root growth regulator. Functional characterization of its closest Arabidopsis homolog AtROXY19 revealed a novel role for this glutaredoxin (GRX) gene clade in regulating cell elongation. In summary, our study suggests a conserved function for GRX genes in conferring root cell elongation and enhancing resilience of pearl millet to its Sahelian environment.

Pearl millet is a staple food for over 90 million people living in regions of Africa and India that typically experience high temperatures and little rainfall. It was domesticated about 4,500 years ago in the Sahel region of West Africa and is one of the most heat and drought tolerant cereal crops worldwide. In most plants, organs known as roots absorb water and essential nutrients from the soil. Young pearl millet plants develop a fast-growing primary root, but it is unclear how this unique feature helps the crop to grow in hot and dry conditions. Using weather data collected from the Sahel over a 20-year period, Fuente, Grondin et al. predicted by modelling that early drought stress is the major factor limiting pearl millet growth and yield in this region. Field experiments found that plants with primary roots that grow faster within soil were better at tolerating early drought than those with slower growing roots. Further work using genetic approaches revealed that a gene known as PgGRXC9 promotes the growth of the primary root. To better understand how this gene works, the team examined a very similar gene in a well-studied model plant known as Arabidopsis. This suggested that PgGRXC9 helps the primary root to grow by stimulating cell elongation within the root. Since it is well adapted to dry conditions, pearl millet is expected to play an important role in helping agriculture adjust to climate change. The findings of Fuente, Grondin et al. may be used by plant breeders to create more resilient and productive varieties of pearl millet.

The integration of TRX/GRX systems and phytohormonal signalling pathways in plant stress and development.

Plant acclimation to changing environmental conditions involves the interaction of different signalling molecules, including reactive oxygen species and hormones. Redox regulation exerted by thioredoxin (TRX) and glutaredoxin (GRX), two oxidoreductases, is emerging as a specific point of control mediating signal transduction pathways associated with plant growth and stress response. Phytohormones are messengers that coordinate plant cell activities to regulate growth, defence, and productivity, although their cross-talk with components of the redox system is less known. The present review focuses on our current knowledge of the interplay that occurs between TRX and GRX systems and phytohormonal signalling pathways in connection with the control of plant development and stress responses. Here, we consider the regulation that phytohormones exert on TRX and GRX systems, as well as the involvement of these redox proteins in the control of phytohormone-mediated signalling pathways.

Molecular identification and functional characterization of an environmental stress responsive glutaredoxin gene ROXY1 in Quercus glauca.

Quercus glauca is a valuable natural resource with both economic and ecological values. It is one of the dominant forest tree species widely distributed in Southern China. As a perennial broadleaf plant, Q. glauca inevitably encounters numerous stresses from environment. Glutaredoxins (GRXs) are a kind of small oxidoreductases that play an important role in response to oxidative stress. CC-type GRXs also known as ROXYs are specific to land plants. In this study, we isolated a CC-type GRX gene, QgROXY1, from Q. glauca. Expression of QgROXY1 is induced by a variety of environmental stimuli. QgROXY1 protein localizes to both cytoplasm and nucleus; whereas the nucleus localized QgROXY1 could physically interact with the basic region/leucine zipper motif (bZIP) transcription factor AtTGA2 from Arabidopsis thaliana. Transgenic A. thaliana ectopically expressing QgROXY1 is hypersensitive to exogenously applied salicylic acid. Induction of plant defense gene is significantly impaired in QgROXY1 transgenic plants that results in enhanced susceptibility to infection of Botrytis cinerea pathogen, indicating the evolutionary conserved function among ROXY homologs in weedy and woody plants. This is the first described function for the ROXYs in tree plants. Through this case study, we demonstrated the feasibility and efficacy of molecular technology applied to characterization of gene function in tree species.