-
Autophagy Jul 2023Ferroptosis is a newly characterized form of programmed cell death, which is driven by the lethal accumulation of lipid peroxides catalyzed by the intracellular...
Ferroptosis is a newly characterized form of programmed cell death, which is driven by the lethal accumulation of lipid peroxides catalyzed by the intracellular bioactive iron. Targeted induction of ferroptotic cell death holds great promise for therapeutic design against other therapy-resistant cancers. To date, multiple post-translational modifications have been elucidated to impinge on the ferroptotic sensitivity. Here we report that the Ser/Thr protein kinase ATM, the major sensor of DNA double-strand break damage, is indispensable for ferroptosis execution. Pharmacological inhibition or genetic ablation of ATM significantly antagonizes ferroptosis. Besides, ATM ablation-induced ferroptotic resistance is largely independent of its downstream target TRP53, as cells defective in both and are still more insensitive to ferroptotic inducers than the single knockout cells. Mechanistically, ATM dominates the intracellular labile free iron by phosphorylating NCOA4, facilitating NCOA4-ferritin interaction and therefore sustaining ferritinophagy, a selective type of macroautophagy/autophagy specifically degrading ferritin for iron recycling. Our results thus uncover a novel regulatory circuit of ferroptosis comprising ATM-NCOA4 in orchestrating ferritinophagy and iron bioavailability. AMPK: AMP-activated protein kinase; ATM: ataxia telangiectasia mutated; BSO: buthionine sulphoximine; CDKN1A: cyclin-dependent kinase inhibitor 1A (P21); CQ: chloroquine; DFO: deferoxamine; DFP: deferiprone; Fer: ferrostatin-1; FTH1: ferritin heavy polypeptide 1; GPX4: glutathione peroxidase 4; GSH: glutathione; MEF: mouse embryonic fibroblast; NCOA4: nuclear receptor coactivator 4; PFTα: pifithrin-α; PTGS2: prostaglandin-endoperoxide synthase 2; Slc7a11: solute carrier family 7 member 11; Sul: sulfasalazine; TFRC: transferrin receptor; TRP53: transformation related protein 53.
Topics: Animals; Mice; Ferroptosis; Autophagy; Fibroblasts; Transcription Factors; Ferritins; Iron; Buthionine Sulfoximine
PubMed: 36752571
DOI: 10.1080/15548627.2023.2170960 -
Pharmacological Research Jul 2023Oxidative disruption of dopaminergic neurons is regarded as a crucial pathogenesis in Parkinson's disease (PD), eventually causing neurodegenerative progression....
Oxidative disruption of dopaminergic neurons is regarded as a crucial pathogenesis in Parkinson's disease (PD), eventually causing neurodegenerative progression. (-)-Clausenamide (Clau) is an alkaloid isolated from plant Clausena lansium (Lour.), which is well-known as a scavenger of lipid peroxide products and exhibiting neuroprotective activities both in vivo and in vitro, yet with the in-depth molecular mechanism unrevealed. In this study, we evaluated the protective effects and mechanisms of Clau on dopaminergic neuron. Our results showed that Clau directly interacted with the Ser663 of ALOX5, the PKCα-phosphorylation site, and thus prevented the nuclear translocation of ALOX5, which was essential for catalyzing the production of toxic lipids 5-HETE. LC-MS/MS-based phospholipidomics analysis demonstrated that the oxidized membrane lipids were involved in triggering ferroptotic death in dopaminergic neurons. Furthermore, the inhibition of ALOX5 was found to significantly improving behavioral defects in PD mouse model, which was confirmed associated with the effects of attenuating the accumulation of lipid peroxides and neuronal damages. Collectively, our findings provide an attractive strategy for PD therapy by targeting ALOX5 and preventing ferroptosis in dopaminergic neurons.
Topics: Animals; Mice; Dopaminergic Neurons; Ferroptosis; Chromatography, Liquid; Tandem Mass Spectrometry; Parkinson Disease
PubMed: 37121496
DOI: 10.1016/j.phrs.2023.106779 -
Biomedicine & Pharmacotherapy =... Aug 2023Vitamin A (retinol) is a lipid-soluble vitamin that acts as a precursor for several bioactive compounds, such as retinaldehyde (retinal) and isomers of retinoic acid....
Vitamin A (retinol) is a lipid-soluble vitamin that acts as a precursor for several bioactive compounds, such as retinaldehyde (retinal) and isomers of retinoic acid. Retinol and all-trans-retinoic acid (atRA) penetrate the blood-brain barrier and are reported to be neuroprotective in several animal models. We characterised the impact of retinol and its metabolites, all-trans-retinal (atRAL) and atRA, on ferroptosis-a programmed cell death caused by iron-dependent phospholipid peroxidation. Ferroptosis was induced by erastin, buthionine sulfoximine or RSL3 in neuronal and non-neuronal cell lines. We found that retinol, atRAL and atRA inhibited ferroptosis with a potency superior to α-tocopherol, the canonical anti-ferroptotic vitamin. In contrast, we found that antagonism of endogenous retinol with anhydroretinol sensitises ferroptosis induced in neuronal and non-neuronal cell lines. Retinol and its metabolites atRAL and atRA directly interdict lipid radicals in ferroptosis since these compounds displayed radical trapping properties in a cell-free assay. Vitamin A, therefore, complements other anti-ferroptotic vitamins, E and K; metabolites of vitamin A, or agents that alter their levels, may be potential therapeutics for diseases where ferroptosis is implicated.
Topics: Animals; Vitamin A; Ferroptosis; Lipid Peroxidation; Tretinoin; Vitamins; Retinaldehyde; Lipids
PubMed: 37236031
DOI: 10.1016/j.biopha.2023.114930 -
Redox Biology Nov 2023Glutathione (GSH) depletion, and impaired redox homeostasis have been observed in experimental animal models and patients with epilepsy. Pleiotropic strategies that...
Glutathione (GSH) depletion, and impaired redox homeostasis have been observed in experimental animal models and patients with epilepsy. Pleiotropic strategies that elevate GSH levels via transcriptional regulation have been shown to significantly decrease oxidative stress and seizure frequency, increase seizure threshold, and rescue certain cognitive deficits. Whether elevation of GSH per se alters neuronal hyperexcitability remains unanswered. We previously showed that thiols such as dimercaprol (DMP) elevate GSH via post-translational activation of glutamate cysteine ligase (GCL), the rate limiting GSH biosynthetic enzyme. Here, we asked if elevation of cellular GSH by DMP altered neuronal hyperexcitability in-vitro and in-vivo. Treatment of primary neuronal-glial cerebrocortical cultures with DMP elevated GSH and inhibited a voltage-gated potassium channel blocker (4-aminopyridine, 4AP) induced neuronal hyperexcitability. DMP increased GSH in wildtype (WT) zebrafish larvae and significantly attenuated convulsant pentylenetetrazol (PTZ)-induced acute 'seizure-like' swim behavior. DMP treatment increased GSH and inhibited convulsive, spontaneous 'seizure-like' swim behavior in the Dravet Syndrome (DS) zebrafish larvae (scn1Lab). Furthermore, DMP treatment significantly decreased spontaneous electrographic seizures and associated seizure parameters in scn1Lab zebrafish larvae. We investigated the role of the redox-sensitive mammalian target of rapamycin (mTOR) pathway due to the presence of several cysteine-rich proteins and their involvement in regulating neuronal excitability. Treatment of primary neuronal-glial cerebrocortical cultures with 4AP or l-buthionine-(S,R)-sulfoximine (BSO), an irreversible inhibitor of GSH biosynthesis, significantly increased mTOR complex I (mTORC1) activity which was rescued by pre-treatment with DMP. Furthermore, BSO-mediated GSH depletion oxidatively modified the tuberous sclerosis protein complex (TSC) consisting of hamartin (TSC1), tuberin (TSC2), and TBC1 domain family member 7 (TBC1D7) which are critical negative regulators of mTORC1. In summary, our results suggest that DMP-mediated GSH elevation by a novel post-translational mechanism can inhibit neuronal hyperexcitability both in-vitro and in-vivo and a plausible link is the redox sensitive mTORC1 pathway.
Topics: Animals; Humans; Zebrafish; Glutathione; Glutamate-Cysteine Ligase; TOR Serine-Threonine Kinases; Mechanistic Target of Rapamycin Complex 1; Seizures; Buthionine Sulfoximine; Mammals
PubMed: 37769522
DOI: 10.1016/j.redox.2023.102895 -
Blood Advances Jan 2024Cysteine is a nonessential amino acid required for protein synthesis, the generation of the antioxidant glutathione, and for synthesizing the nonproteinogenic amino acid...
Cysteine is a nonessential amino acid required for protein synthesis, the generation of the antioxidant glutathione, and for synthesizing the nonproteinogenic amino acid taurine. Here, we highlight the broad sensitivity of leukemic stem and progenitor cells to cysteine depletion. By CRISPR/CRISPR-associated protein 9-mediated knockout of cystathionine-γ-lyase, the cystathionine-to-cysteine converting enzyme, and by metabolite supplementation studies upstream of cysteine, we functionally prove that cysteine is not synthesized from methionine in acute myeloid leukemia (AML) cells. Therefore, although perhaps nutritionally nonessential, cysteine must be imported for survival of these specific cell types. Depletion of cyst(e)ine increased reactive oxygen species (ROS) levels, and cell death was induced predominantly as a consequence of glutathione deprivation. nicotinamide adenine dinucleotide phosphate hydrogen oxidase inhibition strongly rescued viability after cysteine depletion, highlighting this as an important source of ROS in AML. ROS-induced cell death was mediated via ferroptosis, and inhibition of glutathione peroxidase 4 (GPX4), which functions in reducing lipid peroxides, was also highly toxic. We therefore propose that GPX4 is likely key in mediating the antioxidant activity of glutathione. In line, inhibition of the ROS scavenger thioredoxin reductase with auranofin also impaired cell viability, whereby we find that oxidative phosphorylation-driven AML subtypes, in particular, are highly dependent on thioredoxin-mediated protection against ferroptosis. Although inhibition of the cystine-glutamine antiporter by sulfasalazine was ineffective as a monotherapy, its combination with L-buthionine-sulfoximine (BSO) further improved AML ferroptosis induction. We propose the combination of either sulfasalazine or antioxidant machinery inhibitors along with ROS inducers such as BSO or chemotherapy for further preclinical testing.
Topics: Humans; Cysteine; Reactive Oxygen Species; Antioxidants; Ferroptosis; Cystathionine; Sulfasalazine; Amino Acids; Glutathione; Buthionine Sulfoximine; Leukemia, Myeloid, Acute
PubMed: 37906522
DOI: 10.1182/bloodadvances.2023010786 -
Materials Today. Bio Jun 2023Easy recurrence and bacteria infected-wound healing after surgery excision pose severe challenges to clinical melanoma therapy. Herein, an injectable CuO...
Easy recurrence and bacteria infected-wound healing after surgery excision pose severe challenges to clinical melanoma therapy. Herein, an injectable CuO nanodots-engineered thermosensitive chitosan hydrogel (CuO-BSO@Gel) for enhanced melanoma chemo-sonodynamic therapy and improved infected wound healing was rationally constructed by facilely integrating the CuO nanodots and L-Buthionine-(S, R)-sulfoximine (BSO) with thermoresponsive hydrogel. Favored by the Fenton catalytic activity of Cu, the CuO nanodots can achieve enhanced chemodynamic therapy (CDT) by self-supplying HO under acidic tumor microenvironment. Simultaneously, the CuO nanodots with a narrow bandgap (2.29 eV) were proven to be the efficient sonosensitizers, and the corresponding quantum yield of singlet oxygen (O) could be boosted by the O generation during Fenton-like reactions. Additionally, combining with the glutathione (GSH) depletion of loaded BSO, intracellular oxidative stress induced by SDT and CDT was further amplified, leading to the specific ferroptosis. Importantly, this multifunctional hydrogel significantly promoted the proliferation of normal skin cells and accelerated the bacteria-infected wound healing by the effective chemo-sonodynamic antibacterial activity and the enhanced angiogenesis. Thus, the engineered thermogel features the distinct chemo-sonodynamic performance, desirable biocompatibility and bioactivity, providing a competitive strategy for eradicating melanoma and infected wound healing.
PubMed: 37056919
DOI: 10.1016/j.mtbio.2023.100621 -
The Journal of Biological Chemistry Feb 2024Glutathione (GSH) is a highly abundant tripeptide thiol that performs diverse protective and biosynthetic functions in cells. While changes in GSH availability are...
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.
Topics: Animals; Mice; Buthionine Sulfoximine; Disease Models, Animal; Glutamate-Cysteine Ligase; Glutathione; Cell Line, Tumor; Humans
PubMed: 38218225
DOI: 10.1016/j.jbc.2024.105645 -
Frontiers in Microbiology 2023Although cellular metabolic states have been shown to modulate bacterial susceptibility to antibiotics, the interaction between glutamate (Glu) and chloramphenicol (CAP)...
Although cellular metabolic states have been shown to modulate bacterial susceptibility to antibiotics, the interaction between glutamate (Glu) and chloramphenicol (CAP) resistance remains unclear because of the specificity of antibiotics and bacteria. We found that the level of Glu was upregulated in the CAP-resistant strain of according to a comparative metabolomics approach based on LC-MS/MS. Furthermore, we verified that exogenous metabolites related to Glu, the tricarboxylic acid (TCA) cycle, and glutathione (GSH) metabolism could promote CAP resistance in survival assays. If GSH metabolism or the TCA cycle is inhibited by L-buthionine sulfoximine or propanedioic acid, the promotion of CAP resistance by Glu in the corresponding pathway disappears. According to metabolomic analysis, exogenous Glu could change pantothenate metabolism, affecting GSH biosynthesis and the TCA cycle. These results showed that the glutamate-pantothenate pathway could promote CAP resistance by being involved in the synthesis of GSH, entering the TCA cycle by direct deamination, or indirectly affecting the metabolism of the two pathways by pantothenate. These results extend our knowledge of the effect of Glu on antibiotic resistance and suggest that the potential effect, which may aggravate antibiotic resistance, should be considered before Glu and GSH administration in the clinic.
PubMed: 37779691
DOI: 10.3389/fmicb.2023.1264602 -
BioRxiv : the Preprint Server For... Feb 2024Ninjurin-1 (NINJ1), initially identified as a stress-induced protein in neurons, recently emerged as a key mediator of plasma membrane rupture during apoptosis,...
Ninjurin-1 (NINJ1), initially identified as a stress-induced protein in neurons, recently emerged as a key mediator of plasma membrane rupture during apoptosis, necrosis, and pyroptosis. However, its involvement in ferroptosis remains unknown. Here, we demonstrate that NINJ1 also plays a crucial role in ferroptosis, but through a distinct mechanism. NINJ1 knockdown significantly protected cancer cells against ferroptosis induced by xCT inhibitors but no other classes of ferroptosis-inducing compounds (FINs). Glycine, known to inhibit canonical NINJ1-mediated membrane rupture in other cell deaths, had no impact on ferroptosis. A compound screen revealed that NINJ1-mediated ferroptosis protection can be abolished by pantothenate kinase inhibitor (PANKi), buthionine sulfoximine (BSO), and diethylmaleate (DEM). These results suggest that this ferroptosis protection is mediated via Coenzyme A (CoA) and glutathione (GSH), both of which were found to be elevated upon NINJ1 knockdown. Furthermore, we discovered that NINJ1 interacts with the xCT antiporter, which is responsible for cystine uptake for the biosynthesis of CoA and GSH. The removal of NINJ1 increased xCT levels and stability, enhanced cystine uptake, and contributed to elevated CoA and GSH levels, collectively contributing to ferroptosis protection. These findings reveal that NINJ1 regulates ferroptosis via a non-canonical mechanism, distinct from other regulated cell deaths.
PubMed: 38464226
DOI: 10.1101/2024.02.22.581432 -
BioRxiv : the Preprint Server For... Jul 2023Reduction oxidation (redox) reactions are central in life and altered redox state is associated with a spectrum of human diseases. Glutathione (GSH) is the most abundant...
Reduction oxidation (redox) reactions are central in life and altered redox state is associated with a spectrum of human diseases. Glutathione (GSH) is the most abundant antioxidant in eukaryotic cells and plays critical roles in maintaining redox homeostasis. Thus, measuring intracellular GSH level is an important method to assess the redox state of organism. The currently available GSH probes are based on irreversible chemical reactions with glutathione and can't monitor the real-time glutathione dynamics. Our group developed the first reversible reaction based fluorescent probe for glutathione, which can measure glutathione levels at high resolution using a confocal microscope and in the bulk scale with a flow cytometry. Most importantly it can quantitatively monitor the real-time GSH dynamics in living cells. Using the 2 generation of GSH probe, RealThiol (RT), this study measured the GSH level in living Hela cells after treatment with varying concentrations of DL-Buthionine sulfoximine (BSO) which inhibits GSH synthesis, using a high throughput imaging system, Cytation™ 5 cell imaging reader. The results revealed that GSH probe RT at the concentration of 2.0 µM accurately monitored the BSO treatment effect on GSH level in the Hela cells. The present results demonstrated that the GSH probe RT is sensitive and precise in GSH measurement in living cells at a high throughput imaging platform and has the potential to be applied to any cell lines.
PubMed: 37503234
DOI: 10.1101/2023.07.11.548586