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.

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.

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.

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.

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.

Structural conservation in the glutathione binding in Sphingomonas sp. glutaredoxin Grx3 and variations for cold adaptation.

Glutaredoxin 3 (Grx3), a redox protein with a thioredoxin-fold structure, maintains structural integrity and glutathione (GSH) binding capabilities across varying habitat temperatures. The cis-Pro loop, essential for GSH binding, relies on the Arg-Asp salt bridge (α2-α3) and Gln-His hydrogen bond (ß3-ß4) for its conformation. In some psychrophilic Grx3 variants, Arg in α2 is replaced with Tyr, and His in ß4 is replaced with Phe. This study examines the roles of these bonds in Grx3's structure, function, and cold adaptation, using SpGrx3 from the Arctic bacterium Sphingomonas sp. Despite its cold habitat, SpGrx3 maintains the Arg51-Asp69 salt bridge and Gln56-His63 hydrogen bond. The R51Y substitution disrupts the α2-α3 salt bridge, while the H63F and H63Y substitutions hinder the salt bridge through cation-π interactions with Arg51, involving Phe63/Tyr63, thereby enhancing flexibility. Conversely, mutations that disrupt the hydrogen bond (Q56A, H63A, and H63F) reduce thermal stability. In the psychrophilic Grx3 configuration A48T/R51Y/H63F, a Thr48-Gln56 hydrogen bond stabilizes the cis-Pro loop, enhancing flexibility by disrupting both bonds. Furthermore, all mutants exhibit reduced α-helical content and catalytic efficiency. In summary, the highly conserved Arg51-Asp69 salt bridge and Gln56-His63 hydrogen bond are crucial for stabilizing the cis-Pro loop and catalytic activity in SpGrx3. His63 is favored as it avoids cation-π interactions with Arg51, unlike Phe63/Tyr63. Psychrophilic Grx3 variants have adapted to cold environments by reducing GSH binding and increasing structural flexibility. These findings deepen our understanding of the structural conservation in Grx3 for GSH binding and the critical alterations required for cold adaptation.

Macrophage-restricted overexpression of glutaredoxin 1 protects against atherosclerosis by preventing nutrient stress-induced macrophage dysfunction and reprogramming.

BACKGROUND AND AIMS: Deficiency in the thiol transferase glutaredoxin 1 (Grx1) in aging mice promotes, in a sexually dimorphic manner, dysregulation of macrophages and atherogenesis. However, the underlying mechanisms are not known. Here we tested the hypothesis that macrophage-restricted overexpression of Grx1 protects atherosclerosis-prone mice against macrophage reprogramming and dysfunction induced by a high-calorie diet (HCD) and thereby reduces the severity of atherosclerosis. METHODS: We generated lentiviral vectors carrying cluster of differentiation 68 (CD68) promoter-driven enhanced green fluorescent protein (EGFP) or Grx1 constructs and conducted bone marrow (BM) transplantation studies to overexpress Grx1 in a macrophage-specific manner in male and female atherosclerosis-prone LDLR-/- mice, and fed these mice a HCD to induce atherogenesis. Atherosclerotic lesion size was determined in both the aortic root and the aorta. We isolated BM-derived macrophages (BMDM) to assess protein S-glutathionylation levels and loss of mitogen-activated protein kinase phosphatase 1 (MKP-1) activity as measures of HCD-induced thiol oxidative stress. We also conducted gene profiling on these BMDM to determine the impact of Grx1 activity on HCD-induced macrophage reprogramming. RESULTS: Overexpression of Grx1 protected macrophages against HCD-induced protein S-glutathionylation, reduced monocyte chemotaxis in vivo, limited macrophage recruitment into atherosclerotic lesions, and was sufficient to reduce the severity of atherogenesis in both male and female mice. Gene profiling revealed major sex differences in the transcriptional reprogramming of macrophages induced by HCD feeding, but Grx1 overexpression only partially reversed HCD-induced transcriptional reprogramming of macrophages. CONCLUSIONS: Macrophage Grx1 plays a major role in protecting mice atherosclerosis mainly by maintaining the thiol redox state of the macrophage proteome and preventing macrophage dysfunction.

Glutaredoxin in Rice Growth, Development, and Stress Resistance: Mechanisms and Research Advances.

Rice (Oryza sativa L.) is a staple food for more than half of the global population. Various abiotic and biotic stresses lead to accumulation of reactive oxygen species in rice, which damage macromolecules and signaling pathways. Rice has evolved a variety of antioxidant systems, including glutaredoxin (GRX), that protect against various stressors. A total of 48 GRX gene loci have been identified on 11 of the 12 chromosomes of the rice genome; none were found on chromosome 9. GRX proteins were classified into four categories according to their active sites: CPYC, CGFS, CC, and GRL. In this paper, we summarized the recent research advances regarding the roles of GRX in rice development regulation and response to stresses, and discussed future research perspectives related to rice production. This review could provide information for rice researchers on the current status of the GRX and serve as guidance for breeding superior varieties.

Iron homeostasis proteins Grx4 and Fra2 control activity of the Schizosaccharomyces pombe iron repressor Fep1 by facilitating [2Fe-2S] cluster removal.

The Bol2 homolog Fra2 and monothiol glutaredoxin Grx4 together play essential roles in regulating iron homeostasis in Schizosaccharomyces pombe. In vivo studies indicate that Grx4 and Fra2 act as coinhibitory partners that inactivate the transcriptional repressor Fep1 in response to iron deficiency. In Saccharomyces cerevisiae, Bol2 is known to form a [2Fe-2S]-bridged heterodimer with the monothiol Grxs Grx3 and Grx4, with the cluster ligands provided by conserved residues in Grx3/4 and Bol2 as well as GSH. In this study, we characterized this analogous [2Fe-2S]-bridged Grx4-Fra2 complex in S. pombe by identifying the specific residues in Fra2 that act as ligands for the Fe-S cluster and are required to regulate Fep1 activity. We present spectroscopic and biochemical evidence confirming the formation of a [2Fe-2S]-bridged Grx4-Fra2 heterodimer with His66 and Cys29 from Fra2 serving as Fe-S cluster ligands in S. pombe. In vivo transcription and growth assays confirm that both His66 and Cys29 are required to fully mediate the response of Fep1 to low iron conditions. Furthermore, we analyzed the interaction between Fep1 and Grx4-Fra2 using CD spectroscopy to monitor changes in Fe-S cluster coordination chemistry. These experiments demonstrate unidirectional [2Fe-2S] cluster transfer from Fep1 to Grx4-Fra2 in the presence of GSH, revealing the Fe-S cluster dependent mechanism of Fep1 inactivation mediated by Grx4 and Fra2 in response to iron deficiency.

On the occurrence of a glutaredoxin-like small protein in the anaerobic protozoan parasite Entamoeba histolytica.

BACKGROUND: Entamoeba histolytica, an intestinal parasitic protozoan that usually lives and multiplies within the human gut, is the causative agent of amoebiasis. To date, de novo glutathione biosynthesis and its associated enzymes have not been identified in the parasite. Cysteine has been proposed to be the main intracellular thiol. METHODS: Using bioinformatics tools to search for glutaredoxin homologs in the E. histolytica genome database, we identified a coding sequence for a putative Grx-like small protein (EhGLSP) in the E. histolytica HM-1:IMSS genome. We produced the recombinant protein and performed its biochemical characterization. RESULTS: Through in vitro experiments, we observed that recombinant EhGLSP could bind GSH and L-Cys as ligands. However, the protein exhibited very low GSH-dependent disulfide reductase activity. Interestingly, via UV-Vis spectroscopy and chemical analysis, we detected that recombinant EhGLSP (freshly purified from Escherichia coli cells by IMAC) was isolated together with a redox-labile [FeS] bio-inorganic complex, suggesting that this protein could have some function linked to the metabolism of this cofactor. Western blotting showed that EhGLSP protein levels were modulated in E. histolytica cells exposed to exogenous oxidative species and metronidazole, suggesting that this protein cooperates with the antioxidant mechanisms of this parasite. CONCLUSIONS AND GENERAL SIGNIFICANCE: Our findings support the existence of a new metabolic actor in this pathogen. To the best of our knowledge, this is the first report on this protein class in E. histolytica.

Identification and adaptive evolution analysis of glutaredoxin genes in Populus spp.

Glutaredoxin (GRX) is a class of small redox proteins widely involved in cellular redox homeostasis and the regulation of various cellular processes. The role of GRX gene in the differentiation of Populus spp. is rarely reported. We compared the similarities and differences of GRX genes among four sections of poplar using bioinformatics, corrected the annotations of some GRX genes, and focused on analysing their transcript profiling and adaptive evolution in Populus spp. A total of 219 GRX genes were identified in four sections of poplar, among which annotations for 13 genes were corrected. Differences in GRX genes were found between sect. Turanga, represented by P. euphratica, and other poplar sections. Most notably, P. euphratica had the smallest number of duplication events for GRX genes (n = 9) and no tandem duplications, whereas there were >25 duplication events for all other poplars. Furthermore, we detected 18 pairs of GRX genes under positive selection pressure in various sections of poplar, and identified two groups of GRX genes in the Salicaceae that potentially underwent positive selection. Expression profiling results showed that the PtrGRX34 and its orthologous genes were upregulated under stress treatments. In summary, the GRX gene family underwent expansion during poplar differentiation, and some genes underwent rapid evolution during this process, which may be beneficial for Populus spp. to adapt to environmental changes. This study may provide more insights into the molecular mechanisms of Populus spp. adaptation to environmental changes and the adaptive evolution of GRX genes.

Glutaredoxin 2 protects lens epithelial cells from epithelial-mesenchymal transition by suppressing mitochondrial oxidative stress-related upregulation of integrin-linked kinase.

Glutaredoxin 2 (Grx2), a mitochondrial glutathione-dependent oxidoreductase, is crucial for maintaining redox homeostasis and cellular functions in the lens. The oxidative stress-induced epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) is related to posterior capsule opacification. In this study, we investigated the effects of Grx2 on oxidative stress-induced EMT in LECs during posterior capsule opacification. We found that Grx2 expression was substantially decreased during the EMT of LECs and in a mouse model of cataract surgery. Deletion of Grx2 aggravated the generation of reactive oxygen species, including those that are mitochondria-derived, and promoted the proliferation and EMT of the LECs. This was reversed by Grx2 overexpression. In vivo, proteomic liquid chromatography-mass spectrometry analysis showed that integrin-linked kinase (ILK) was significantly upregulated in the lens posterior capsule of a Grx2 knockout (KO) mouse model. Compared with that of the wild-type group, the expression of ILK and EMT markers was increased in the Grx2 KO group which was reversed in the Grx2 knock-in group. Inhibition of ILK partially blocked Grx2 knockdown-induced EMT and prevented the increased phosphorylation of Akt and GSK-3ß and the nuclear translocation of ß-catenin in the Grx2 KO group. Finally, inhibition of the Wnt/ß-catenin pathway partially blocked the Grx2 knockdown-induced EMT. In conclusion, we demonstrated that Grx2 protects LECs from oxidative stress-related EMT by regulating the ILK/Akt/GSK-3ß axis.

Structural and functional fine mapping of cysteines in mammalian glutaredoxin reveal their differential oxidation susceptibility.

Protein-S-glutathionylation is a post-translational modification involving the conjugation of glutathione to protein thiols, which can modulate the activity and structure of key cellular proteins. Glutaredoxins (GLRX) are oxidoreductases that regulate this process by performing deglutathionylation. However, GLRX has five cysteines that are potentially vulnerable to oxidative modification, which is associated with GLRX aggregation and loss of activity. To date, GLRX cysteines that are oxidatively modified and their relative susceptibilities remain unknown. We utilized molecular modeling approaches, activity assays using recombinant GLRX, coupled with site-directed mutagenesis of each cysteine both individually and in combination to address the oxidizibility of GLRX cysteines. These approaches reveal that C8 and C83 are targets for S-glutathionylation and oxidation by hydrogen peroxide in vitro. In silico modeling and experimental validation confirm a prominent role of C8 for dimer formation and aggregation. Lastly, combinatorial mutation of C8, C26, and C83 results in increased activity of GLRX and resistance to oxidative inactivation and aggregation. Results from these integrated computational and experimental studies provide insights into the relative oxidizability of GLRX's cysteines and have implications for the use of GLRX as a therapeutic in settings of dysregulated protein glutathionylation.

Glutaredoxin-1 promotes lymphangioleiomyomatosis progression through inhibiting Bim-mediated apoptosis via COX2/PGE2/ERK pathway.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is a female-predominant interstitial lung disease, characterized by progressive cyst formation and respiratory failure. Clinical treatment with the mTORC1 inhibitor rapamycin could relieve partially the respiratory symptoms, but not curative. It is urgent to illustrate the fundamental mechanisms of TSC2 deficiency to the development of LAM, especially mTORC1-independent mechanisms. Glutaredoxin-1 (Glrx), an essential glutathione (GSH)-dependent thiol-oxidoreductase, maintains redox homeostasis and participates in various processes via controlling protein GSH adducts. Redox signalling through protein GSH adducts in LAM remains largely elusive. Here, we demonstrate the underlying mechanism of Glrx in the pathogenesis of LAM. METHODS: 1. Abnormal Glrx expression in various kinds of human malignancies was identified by the GEPIA tumour database, and the expression of Glrx in LAM-derived cells was detected by real-time quantitative reverse transcription (RT-qPCR) and immunoblot. 2. Stable Glrx knockdown cell line was established to evaluate cellular impact. 3. Cell viability was determined by CCK8 assay. 4. Apoptotic cell number and intracellular reactive oxygen species (ROS) level were quantified by flow cytometry. 5. Cox2 expression and PGE2 production were detected to clarify the mechanism of Bim expression modulated by Glrx. 6. S-glutathionylated p65 was enriched and detected by immunoprecipitation and the direct regulation of Glrx on p65 was determined. 7. The xenograft animal model was established and photon flux was analyzed using IVIS Spectrum. RESULTS: In LAM, TSC2 negatively regulated abnormal Glrx expression and activation in a mTORC1-independent manner. Knockdown of Glrx increased the expression of Bim and the accumulation of ROS, together with elevated S-glutathionylated proteins, contributing to the induction of apoptotic cell death and inhibited cell proliferation. Knockdown of Glrx in TSC2-deficient LAM cells increased GSH adducts on nuclear factor-kappa B p65, which contributed to a decrease in the expression of Cox2 and the biosynthesis of PGE2. Inhibition of PGE2 metabolism attenuated phosphorylation of ERK, which led to the accumulation of Bim, due to the imbalance of its phosphorylation and proteasome degradation. In xenograft tumour models, knockdown of Glrx in TSC2-deficient LAM cells inhibited tumour growth and increased tumour cell apoptosis. CONCLUSIONS: Collectively, we provide a novel redox-dependent mechanism in the pathogenesis of LAM and propose that Glrx may be a beneficial strategy for the treatment of LAM or other TSC-related diseases.

Redox regulation, thioredoxins, and glutaredoxins in retrograde signalling and gene transcription.

Integration of reactive oxygen species (ROS)-mediated signal transduction pathways via redox sensors and the thiol-dependent signalling network is of increasing interest in cell biology for their implications in plant growth and productivity. Redox regulation is an important point of control in protein structure, interactions, cellular location, and function, with thioredoxins (TRXs) and glutaredoxins (GRXs) being key players in the maintenance of cellular redox homeostasis. The crosstalk between second messengers, ROS, thiol redox signalling, and redox homeostasis-related genes controls almost every aspect of plant development and stress response. We review the emerging roles of TRXs and GRXs in redox-regulated processes interacting with other cell signalling systems such as organellar retrograde communication and gene expression, especially in plants during their development and under stressful environments. This approach will cast light on the specific role of these proteins as redox signalling components, and their importance in different developmental processes during abiotic stress.

Glutaredoxin 1 regulates cholesterol metabolism and gallstone formation by influencing protein S-glutathionylation.

OBJECTIVE: Cholesterol gallstone disease (CGD) is closely related to cholesterol metabolic disorder. Glutaredoxin-1 (Glrx1) and Glrx1-related protein S-glutathionylation are increasingly being observed to drive various physiological and pathological processes, especially in metabolic diseases such as diabetes, obesity and fatty liver. However, Glrx1 has been minimally explored in cholesterol metabolism and gallstone disease. METHODS: We first investigated whether Glrx1 plays a role in gallstone formation in lithogenic diet-fed mice using immunoblotting and quantitative real-time PCR. Then a whole-body Glrx1-deficient (Glrx1-/-) mice and hepatic-specific Glrx1-overexpressing (AAV8-TBG-Glrx1) mice were generated, in which we analyzed the effects of Glrx1 on lipid metabolism upon LGD feeding. Quantitative proteomic analysis and immunoprecipitation (IP) of glutathionylated proteins were performed. RESULTS: We found that protein S-glutathionylation was markedly decreased and the deglutathionylating enzyme Glrx1 was greatly increased in the liver of lithogenic diet-fed mice. Glrx1-/- mice were protected from gallstone disease induced by a lithogenic diet because their biliary cholesterol and cholesterol saturation index (CSI) were reduced. Conversely, AAV8-TBG-Glrx1 mice showed greater gallstone progression with increased cholesterol secretion and CSI. Further studies showed that Glrx1-overexpressing greatly altered bile acid levels and/or composition to increase intestinal cholesterol absorption by upregulating Cyp8b1. In addition, liquid chromatography-mass spectrometry and IP analysis revealed that Glrx1 also affected the function of asialoglycoprotein receptor 1 (ASGR1) by mediating its deglutathionylation, thereby altering the expression of LXRα and controlling cholesterol secretion. CONCLUSION: Our findings present novel roles of Glrx1 and Glrx1-regulated protein S-glutathionylation in gallstone formation through the targeting of cholesterol metabolism. Our data advises Glrx1 significantly increased gallstone formation by simultaneously increase bile-acid-dependent cholesterol absorption and ASGR1- LXRα-dependent cholesterol efflux. Our work suggests the potential effects of inhibiting Glrx1 activity to treat cholelithiasis.