Ironing out the role of ferroptosis in immunity.

Ferroptosis is a type of regulated cell death that drives the pathophysiology of many diseases. Oxidative stress is detectable in many types of regulated cell death, but only ferroptosis involves lipid peroxidation and iron dependency. Ferroptosis originates and propagates from several organelles, including the mitochondria, endoplasmic reticulum, Golgi, and lysosomes. Recent data have revealed that immune cells can both induce and undergo ferroptosis. A mechanistic understanding of how ferroptosis regulates immunity is critical to understanding how ferroptosis controls immune responses and how this is dysregulated in disease. Translationally, more work is needed to produce ferroptosis-modulating immunotherapeutics. This review focuses on the role of ferroptosis in immune-related diseases, including infection, autoimmune diseases, and cancer. We discuss how ferroptosis is regulated in immunity, how this regulation contributes to disease pathogenesis, and how targeting ferroptosis may lead to novel therapies.

Dual regulation of SLC25A39 by AFG3L2 and iron controls mitochondrial glutathione homeostasis.

Organelle transporters define metabolic compartmentalization, and how this metabolite transport process can be modulated is poorly explored. Here, we discovered that human SLC25A39, a mitochondrial transporter critical for mitochondrial glutathione uptake, is a short-lived protein under dual regulation at the protein level. Co-immunoprecipitation mass spectrometry and CRISPR knockout (KO) in mammalian cells identified that mitochondrial m-AAA protease AFG3L2 is responsible for degrading SLC25A39 through the matrix loop 1. SLC25A39 senses mitochondrial iron-sulfur cluster using four matrix cysteine residues and inhibits its degradation. SLC25A39 protein regulation is robust in developing and mature neurons. This dual transporter regulation, by protein quality control and metabolic sensing, allows modulating mitochondrial glutathione level in response to iron homeostasis, opening avenues for exploring regulation of metabolic compartmentalization. Neuronal SLC25A39 regulation connects mitochondrial protein quality control, glutathione, and iron homeostasis, which were previously unrelated biochemical features in neurodegeneration.

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.

The structure of the rat vitamin B12 transporter TC and its complex with glutathionylcobalamin.

Vitamin B12 (cobalamin or Cbl) functions as a cofactor in two important enzymatic processes in human cells, and life is not sustainable without it. B12 is obtained from food and travels from the stomach, through the intestine, and into the bloodstream by three B12-transporting proteins: salivary haptocorrin (HC), gastric intrinsic factor, and transcobalamin (TC), which all bind B12 with high affinity and require proteolytic degradation to liberate Cbl. After intracellular delivery of dietary B12, Cbl in the aquo/hydroxocobalamin form can coordinate various nucleophiles, for example, GSH, giving rise to glutathionylcobalamin (GSCbl), a naturally occurring form of vitamin B12. Currently, there is no data showing whether GSCbl is recognized and transported in the human body. Our crystallographic data shows for the first time the complex between a vitamin B12 transporter and GSCbl, which compared to aquo/hydroxocobalamin, binds TC equally well. Furthermore, sequence analysis and structural comparisons show that TC recognizes and transports GSCbl and that the residues involved are conserved among TCs from different organisms. Interestingly, haptocorrin and intrinsic factor are not structurally tailored to bind GSCbl. This study provides new insights into the interactions between TC and Cbl.

Komagataella phaffii Erp41 is a protein disulfide isomerase with unprecedented disulfide bond catalyzing activity when coupled to glutathione.

In the methylotrophic yeast Komagataella phaffii, we identified an endoplasmic reticulum-resident protein disulfide isomerase (PDI) family member, Erp41, with a peculiar combination of active site motifs. Like fungal ERp38, it has two thioredoxin-like domains which contain active site motifs (a and a'), followed by an alpha-helical ERp29c C-terminal domain (c domain). However, while the a domain has a typical PDI-like active site motif (CGHC), the a' domain instead has CGYC, a glutaredoxin-like motif which confers to the protein an exceptional affinity for GSH/GSSG. This combination of active site motifs has so far been unreported in PDI-family members. Homology searches revealed ERp41 is present in the genome of some plants, fungal parasites, and a few nonconventional yeasts, among which are Komagataella spp. and Yarrowia lipolytica. These yeasts are both used for the production of secreted recombinant proteins. Here, we analyzed the activity of K. phaffii Erp41. We report that it is nonessential in K. phaffii, and that it can catalyze disulfide bond formation in partnership with the sulfhydryl oxidase Ero1 in vitro with higher turnover rates than the canonical PDI from K. phaffii, Pdi1, but slower activation times. We show how Erp41 has unusually fast glutathione-coupled oxidation activity and relate it to its unusual combination of active sites in its thioredoxin-like domains. We further describe how this determines its unusually efficient catalysis of dithiol oxidation in peptide and protein substrates.

G-site residue S67 is involved in the fungicide-degrading activity of a tau class glutathione S-transferase from Carica papaya.

Thiram is a toxic fungicide extensively used for the management of pathogens in fruits. Although it is known that thiram degrades in plant tissues, the key enzymes involved in this process remain unexplored. In this study, we report that a tau class glutathione S-transferase (GST) from Carica papaya can degrade thiram. This enzyme was easily obtained by heterologous expression in Escherichia coli, showed low promiscuity toward other thiuram disulfides, and catalyzed thiram degradation under physiological reaction conditions. Site-directed mutagenesis indicated that G-site residue S67 shows a key influence for the enzymatic activity toward thiram, while mutation of residue S13, which reduced the GSH oxidase activity, did not significantly affect the thiram-degrading activity. The formation of dimethyl dithiocarbamate, which was subsequently converted into carbon disulfide, and dimethyl dithiocarbamoylsulfenic acid as the thiram degradation products suggested that thiram undergoes an alkaline hydrolysis that involves the rupture of the disulfide bond. Application of the GST selective inhibitor 4-chloro-7-nitro-2,1,3-benzoxadiazole reduced papaya peel thiram-degrading activity by 95%, indicating that this is the main degradation route of thiram in papaya. GST from Carica papaya also catalyzed the degradation of the fungicides chlorothalonil and thiabendazole, with residue S67 showing again a key influence for the enzymatic activity. These results fill an important knowledge gap in understanding the catalytic promiscuity of plant GSTs and reveal new insights into the fate and degradation products of thiram in fruits.

Development of a mouse model expressing a bifunctional glutathione-synthesizing enzyme to study glutathione limitation in vivo.

Glutathione (GSH) is a highly abundant tripeptide thiol that performs diverse protective and biosynthetic functions in cells. While changes in GSH availability are associated with inborn errors of metabolism, cancer, and neurodegenerative disorders, studying the limiting role of GSH in physiology and disease has been challenging due to its tight regulation. To address this, we generated cell and mouse models that express a bifunctional glutathione-synthesizing enzyme from Streptococcus thermophilus (GshF), which possesses both glutamate-cysteine ligase and glutathione synthase activities. GshF expression allows efficient production of GSH in the cytosol and mitochondria and prevents cell death in response to GSH depletion, but not ferroptosis induction, indicating that GSH is not a limiting factor under lipid peroxidation. CRISPR screens using engineered enzymes further revealed genes required for cell proliferation under cellular and mitochondrial GSH depletion. Among these, we identified the glutamate-cysteine ligase modifier subunit, GCLM, as a requirement for cellular sensitivity to buthionine sulfoximine, a glutathione synthesis inhibitor. Finally, GshF expression in mice is embryonically lethal but sustains postnatal viability when restricted to adulthood. Overall, our work identifies a conditional mouse model to investigate the limiting role of GSH in physiology and disease.

The integrated stress response in metabolic adaptation.

The integrated stress response (ISR) refers to signaling pathways initiated by stress-activated eIF2α kinases. Distinct eIF2α kinases respond to different stress signals, including amino acid deprivation and mitochondrial stress. Such stress-induced eIF2α phosphorylation attenuates general mRNA translation and, at the same time, stimulates the preferential translation of specific downstream factors to orchestrate an adaptive gene expression program. In recent years, there have been significant new advances in our understanding of ISR during metabolic stress adaptation. Here, I discuss those advances, reviewing among others the ISR activation mechanisms in response to amino acid deprivation and mitochondrial stress. In addition, I review how ISR regulates the amino acid metabolic pathways and how changes in the ISR impact the physiology and pathology of various disease models.

N2-Acetylornithine deacetylase functions as a Cys-Gly dipeptidase in the cytosolic glutathione degradation pathway in Arabidopsis thaliana.

Glutathione (GSH) is required for various physiological processes in plants, including redox regulation and detoxification of harmful compounds. GSH also functions as a repository for assimilated sulfur and is actively catabolized in plants. In Arabidopsis, GSH is mainly degraded initially by cytosolic enzymes, γ-glutamyl cyclotransferase, and γ-glutamyl peptidase, which release cysteinylglycine (Cys-Gly). However, the subsequent enzyme responsible for catabolizing this dipeptide has not been identified to date. In the present study, we identified At4g17830 as a Cys-Gly dipeptidase, namely cysteinylglycine peptidase 1 (CGP1). CGP1 complemented the phenotype of the yeast mutant that cannot degrade Cys-Gly. The Arabidopsis cgp1 mutant had lower Cys-Gly degradation activity than the wild type and showed perturbed concentrations of thiol compounds. Recombinant CGP1 showed reasonable Cys-Gly degradation activity in vitro. Metabolomic analysis revealed that cgp1 exhibited signs of severe sulfur deficiency, such as elevated accumulation of O-acetylserine (OAS) and the decrease in sulfur-containing metabolites. Morphological changes observed in cgp1, including longer primary roots of germinating seeds, were also likely associated with sulfur starvation. Notably, At4g17830 has previously been reported to encode an N2-acetylornithine deacetylase (NAOD) that functions in the ornithine biosynthesis. The cgp1 mutant did not show a decrease in ornithine content, whereas the analysis of CGP1 structure did not rule out the possibility that CGP1 has Cys-Gly dipeptidase and NAOD activities. Therefore, we propose that CGP1 is a Cys-Gly dipeptidase that functions in the cytosolic GSH degradation pathway and may play dual roles in GSH and ornithine metabolism.

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.

Glutathione safeguards TET-dependent DNA demethylation and is critical for the acquisition of totipotency and pluripotency during preimplantation development.

During early development, both genome-wide epigenetic reprogramming and metabolic remodeling are hallmark changes of normal embryogenesis. However, little is known about their relationship and developmental functions during the preimplantation window, which is essential for the acquisition of totipotency and pluripotency. Herein, we reported that glutathione (GSH), a ubiquitous intracellular protective antioxidant that maintains mitochondrial function and redox homeostasis, plays a critical role in safeguarding postfertilization DNA demethylation and is essential for establishing developmental potential in preimplantation embryos. By profiling mitochondria-related transcriptome that coupled with different pluripotency, we found GSH is a potential marker that is tightly correlated with full pluripotency, and its beneficial effect on prompting developmental potential was functionally conformed using in vitro fertilized mouse and bovine embryos as the model. Mechanistic study based on preimplantation embryos and embryonic stem cells further revealed that GSH prompts the acquisition of totipotency and pluripotency by facilitating ten-eleven-translocation (TET)-dependent DNA demethylation, and ascorbic acid (AsA)-GSH cycle is implicated in the process. In addition, we also reported that GSH serves as an oviductal paracrine factor that supports development potential of preimplantation embryos. Thus, our results not only advance the current knowledge of functional links between epigenetic reprogramming and metabolic remodeling during preimplantation development but also provided a promising approach for improving current in vitro culture system for assisted reproductive technology.

Brusatol hinders the progression of bladder cancer by Chac1/Nrf2/SLC7A11 pathway.

Bladder cancer is a common tumor that impacts the urinary system and marked by a significant fatality rate and an unfavorable prognosis. Promising antineoplastic properties are exhibited by brusatol, which is obtained from the dried ripe fruit of Brucea javanica. The present study aimed to evaluate the influence of brusatol on the progression of bladder cancer and uncover the molecular mechanism involved. We used Cell Counting Kit-8, colony formation and EdU assays to detect cell numbers, viability and proliferation. We used transwell migration assay to detect cell migration ability. The mechanism of brusatol inhibition of bladder cancer proliferation was studied by flow cytometry and western blotting. It was revealed that brusatol could reduce the viability and proliferation of T24 and 5637 cells. The transwell migration assay revealed that brusatol was able to attenuate the migration of T24 and 5637 cells. We found that treatment with brusatol increased the levels of reactive oxygen species, malondialdehyde and Fe2+, thereby further promoting ferroptosis in T24 and 5637 cells. In addition, treatment with RSL3 (an agonistor of ferroptosis) ferrostatin-1 (a selective inhibitor of ferroptosis) enhanced or reversed the brusatol-induced inhibition. In vivo, treatment with brusatol significantly suppressed the tumor growth in nude mice. Mechanistically, brusatol induced ferroptosis by upregulating the expression of ChaC glutathione-specific gamma-glutamylcyclotransferase (Chac1) and decreasing the expression of SLC7A11 and Nrf2 in T24 and 5637 cells. To summarize, the findings of this research demonstrated that brusatol hindered the growth of bladder cancer and triggered ferroptosis via the Chac1/Nrf2/SLC7A11 pathway.

Myocardial reperfusion injury exacerbation due to ALDH2 deficiency is mediated by neutrophil extracellular traps and prevented by leukotriene C4 inhibition.

BACKGROUND AND AIMS: The Glu504Lys polymorphism in the aldehyde dehydrogenase 2 (ALDH2) gene is closely associated with myocardial ischaemia/reperfusion injury (I/RI). The effects of ALDH2 on neutrophil extracellular trap (NET) formation (i.e. NETosis) during I/RI remain unknown. This study aimed to investigate the role of ALDH2 in NETosis in the pathogenesis of myocardial I/RI. METHODS: The mouse model of myocardial I/RI was constructed on wild-type, ALDH2 knockout, peptidylarginine deiminase 4 (Pad4) knockout, and ALDH2/PAD4 double knockout mice. Overall, 308 ST-elevation myocardial infarction patients after primary percutaneous coronary intervention were enrolled in the study. RESULTS: Enhanced NETosis was observed in human neutrophils carrying the ALDH2 genetic mutation and ischaemic myocardium of ALDH2 knockout mice compared with controls. PAD4 knockout or treatment with NETosis-targeting drugs (GSK484, DNase1) substantially attenuated the extent of myocardial damage, particularly in ALDH2 knockout. Mechanistically, ALDH2 deficiency increased damage-associated molecular pattern release and susceptibility to NET-induced damage during myocardial I/RI. ALDH2 deficiency induced NOX2-dependent NETosis via upregulating the endoplasmic reticulum stress/microsomal glutathione S-transferase 2/leukotriene C4 (LTC4) pathway. The Food and Drug Administration-approved LTC4 receptor antagonist pranlukast ameliorated I/RI by inhibiting NETosis in both wild-type and ALDH2 knockout mice. Serum myeloperoxidase-DNA complex and LTC4 levels exhibited the predictive effect on adverse left ventricular remodelling at 6 months after primary percutaneous coronary intervention in ST-elevation myocardial infarction patients. CONCLUSIONS: ALDH2 deficiency exacerbates myocardial I/RI by promoting NETosis via the endoplasmic reticulum stress/microsomal glutathione S-transferase 2/LTC4/NOX2 pathway. This study hints at the role of NETosis in the pathogenesis of myocardial I/RI, and pranlukast might be a potential therapeutic option for attenuating I/RI, particularly in individuals with the ALDH2 mutation.

Hypoxia exposure blunts angiogenic signaling and upregulates the antioxidant system in endothelial cells derived from elephant seals.

BACKGROUND: Elephant seals exhibit extreme hypoxemic tolerance derived from repetitive hypoxia/reoxygenation episodes they experience during diving bouts. Real-time assessment of the molecular changes underlying protection against hypoxic injury in seals remains restricted by their at-sea inaccessibility. Hence, we developed a proliferative arterial endothelial cell culture model from elephant seals and used RNA-seq, functional assays, and confocal microscopy to assess the molecular response to prolonged hypoxia. RESULTS: Seal and human endothelial cells exposed to 1% O2 for up to 6 h respond differently to acute and prolonged hypoxia. Seal cells decouple stabilization of the hypoxia-sensitive transcriptional regulator HIF-1α from angiogenic signaling. Rapid upregulation of genes involved in glutathione (GSH) metabolism supports the maintenance of GSH pools, and intracellular succinate increases in seal but not human cells. High maximal and spare respiratory capacity in seal cells after hypoxia exposure occurs in concert with increasing mitochondrial branch length and independent from major changes in extracellular acidification rate, suggesting that seal cells recover oxidative metabolism without significant glycolytic dependency after hypoxia exposure. CONCLUSIONS: We found that the glutathione antioxidant system is upregulated in seal endothelial cells during hypoxia, while this system remains static in comparable human cells. Furthermore, we found that in contrast to human cells, hypoxia exposure rapidly activates HIF-1 in seal cells, but this response is decoupled from the canonical angiogenesis pathway. These results highlight the unique mechanisms that confer extraordinary tolerance to limited oxygen availability in a champion diving mammal.

Nanoparticle-Catalyzed Transamination under Tumor Microenvironment Conditions: A Novel Tool to Disrupt the Pool of Amino Acids and GSSG in Cancer Cells.

Catalytic cancer therapy targets cancer cells by exploiting the specific characteristics of the tumor microenvironment (TME). TME-based catalytic strategies rely on the use of molecules already present in the TME. Amino groups seem to be a suitable target, given the abundance of proteins and peptides in biological environments. Here we show that catalytic CuFe2O4 nanoparticles are able to foster transaminations with different amino acids and pyruvate, another key molecule present in the TME. We observed a significant in cellulo decrease in glutamine and alanine levels up to 48 h after treatment. In addition, we found that di- and tripeptides also undergo catalytic transamination, thereby extending the range of the effects to other molecules such as glutathione disulfide (GSSG). Mechanistic calculations for GSSG transamination revealed the formation of an imine between the oxo group of pyruvate and the free -NH2 group of GSSG. Our results highlight transamination as alternative to the existing toolbox of catalytic therapies.

Three-Step Depletion Strategy of Glutathione: Tunable Metal-Organic-Framework-Engineered Nanozymes for Driving Oxidative/Nitrative Stress to Maximize Ferroptosis Therapy.

Ferroptosis is a novel type of nonapoptotic programmed cell death involving the accumulation of lipid peroxidation (LPO) to a lethal threshold. Herein, we propose tunable zeolitic imidazolate framework (ZIFs)-engineered biodegradable nanozymes for ferroptosis mediated by both reactive oxygen species (ROS) and nitrogen species (RNS). l-Arginine is utilized as an exogenous nitric oxide donor and loaded into hollow ZIFs@MnO2 artificial nanozymes, which are formed by etching ZIFs with potassium permanganate and simultaneously generating a MnO2 shell in situ. The constructed nanozymes with multienzyme-like activities including peroxidase, oxidase, and catalase can release satisfactory ROS and RNS through a cascade reaction, consequently promoting the accumulation of LPO. Furthermore, it can improve the efficiency of ferroptosis through a three-step strategy of glutathione (GSH) depletion; that is, the outer MnO2 layer consumes GSH under slightly acidic conditions and RNS downregulates SLC7A11 and glutathione reductase, thus directly inhibiting GSH biosynthesis and indirectly preventing GSH regeneration.

Ultrabright Green-Emissive Nanodots for Precise Biological Visualization.

Developing general methods to fabricate water-dispersible and biocompatible fluorescent probes will promote different biological visualization applications. Herein, we report a metal-facilitated method to fabricate ultrabright green-emissive nanodots via the one-step solvothermal treatment of rose bengal, ethanol, and various metal ions. These metal-doped nanodots show good water dispersity, ultrahigh photoluminescence quantum yields (PLQYs) (e.g., the PLQY of Fe-doped nanodots (FeNDs) was ∼97%), and low phototoxicity. Owing to the coordination effect of metal ions, the FeNDs realize glutathione detection with outstanding properties. Benefiting from the high endoplasmic reticulum (ER) affinity of the chloride group, the FeNDs can act as an ER tracker with long ER imaging capacity (FeNDs: >24 h; commercial ER tracker: ∼1 h) and superb photostability and can achieve tissue visualization in living Caenorhabditis elegans. The metal-doped nanodots represent a general nanodot preparation method and may shed new light on diverse biological visualization uses.

Redox biomarkers in asymptomatic latent human tuberculosis: a comparison with active disease.

BACKGROUND: The latent TB infection (LTBI) is an asymptomatic infection caused by Mycobacterium tuberculosis (M.bt). Previous studies have shown a host-protective role for Heme oxygenase-1 (HO-1) during Mtb infection and an important involvement of Glutathione peroxidase-4 (Gpx4) in the necrotic pathology of the disease. Furthermore, increasing evidence suggested a crucial role for Glutathione in the granulomatous response to M. tb infection, with altered GSH levels associated to decreased host resistance. The aim of this study was to provide additional tools for discriminating the pathologic TB state and the asymptomatic infection. METHODS: We analyzed the gene expression of HO-1 and Gpx4 enzymes in blood of subjects with LTBI, active TB and healthy controls, and we also measured blood levels of the reduced (GSH) and oxidized (GSSG) forms of glutathione, together with the evaluation of GCL expression, the gene responsible for the GSH de novo synthesis. RESULTS: Our findings highlight a shift of glutathione homeostasis towards a more reducing conditions in LTBI, and a different modulation of GSH-dependent genes and HO-1 expression respect to active TB. CONCLUSION: This study can provide useful tools to understand the redox background that address the infection toward the asymptomatic or active disease.

The role of metals in hypothiocyanite resistance in Escherichia coli.

The innate immune system employs a variety of antimicrobial oxidants to control and kill host-associated bacteria. Hypothiocyanite/hypothiocyanous acid (-OSCN/HOSCN) is one such antimicrobial oxidant that is synthesized by lactoperoxidase, myeloperoxidase, and eosinophil peroxidase at sites throughout the human body. HOSCN has potent antibacterial activity while being largely non-toxic toward human cells. The molecular mechanisms by which bacteria sense and defend themselves against HOSCN have only recently begun to be elaborated, notably by the discovery of bacterial HOSCN reductase (RclA), an HOSCN-degrading enzyme widely conserved among bacteria that live on epithelial surfaces. In this paper, I show that Ni2+ sensitizes Escherichia coli to HOSCN by inhibiting glutathione reductase and that inorganic polyphosphate protects E. coli against this effect, probably by chelating Ni2+ ions. I also found that RclA is very sensitive to inhibition by Cu2+ and Zn2+, metals that are accumulated to high levels by innate immune cells, and that, surprisingly, thioredoxin and thioredoxin reductase are not involved in HOSCN stress resistance in E. coli. These results advance our understanding of the contribution of different oxidative stress responses and redox buffering pathways to HOSCN resistance in E. coli and illustrate important interactions between metal ions and the enzymes bacteria use to defend themselves against oxidative stress. IMPORTANCE: Hypothiocyanite (HOSCN) is an antimicrobial oxidant produced by the innate immune system. The molecular mechanisms by which host-associated bacteria defend themselves against HOSCN have only recently begun to be understood. The results in this paper are significant because they show that the low molecular weight thiol glutathione and enzyme glutathione reductase are critical components of the Escherichia coli HOSCN response, working by a mechanism distinct from that of the HOSCN-specific defenses provided by the RclA, RclB, and RclC proteins and that metal ions (including nickel, copper, and zinc) may impact the ability of bacteria to resist HOSCN by inhibiting specific defensive enzymes (e.g., glutathione reductase or RclA).

Glutathione-triggered prodrugs: Design strategies, potential applications, and perspectives.

The burgeoning prodrug strategy offers a promising avenue toward improving the efficacy and specificity of cytotoxic drugs. Elevated intracellular levels of glutathione (GSH) have been regarded as a hallmark of tumor cells and characteristic feature of the tumor microenvironment. Considering the pivotal involvement of elevated GSH in the tumorigenic process, a diverse repertoire of GSH-triggered prodrugs has been developed for cancer therapy, facilitating the attenuation of deleterious side effects associated with conventional chemotherapeutic agents and/or the attainment of more efficacious therapeutic outcomes. These prodrug formulations encompass a spectrum of architectures, spanning from small molecules to polymer-based and organic-inorganic nanomaterial constructs. Although the GSH-triggered prodrugs have been gaining increasing interests, a comprehensive review of the advancements made in the field is still lacking. To fill the existing lacuna, this review undertakes a retrospective analysis of noteworthy research endeavors, based on a categorization of these molecules by their diverse recognition units (i.e., disulfides, diselenides, Michael acceptors, and sulfonamides/sulfonates). This review also focuses on explaining the distinct benefits of employing various chemical architecture strategies in the design of these prodrug agents. Furthermore, we highlight the potential for synergistic functionality by incorporating multiple-targeting conjugates, theranostic entities, and combinational treatment modalities, all of which rely on the GSH-triggering. Overall, an extensive overview of the emerging field is presented in this review, highlighting the obstacles and opportunities that lie ahead. Our overarching goal is to furnish methodological guidance for the development of more efficacious GSH-triggered prodrugs in the future. By assessing the pros and cons of current GSH-triggered prodrugs, we expect that this review will be a handful reference for prodrug design, and would provide a guidance for improving the properties of prodrugs and discovering novel trigger scaffolds for constructing GSH-triggered prodrugs.