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1.
Immunity ; 57(5): 941-956, 2024 May 14.
Article in English | MEDLINE | ID: mdl-38749397

ABSTRACT

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.


Subject(s)
Ferroptosis , Iron , Ferroptosis/immunology , Humans , Animals , Iron/metabolism , Neoplasms/immunology , Neoplasms/metabolism , Lipid Peroxidation/immunology , Autoimmune Diseases/immunology , Immunity , Oxidative Stress/immunology , Mitochondria/metabolism , Mitochondria/immunology
2.
Cell ; 172(3): 409-422.e21, 2018 01 25.
Article in English | MEDLINE | ID: mdl-29290465

ABSTRACT

Selenoproteins are rare proteins among all kingdoms of life containing the 21st amino acid, selenocysteine. Selenocysteine resembles cysteine, differing only by the substitution of selenium for sulfur. Yet the actual advantage of selenolate- versus thiolate-based catalysis has remained enigmatic, as most of the known selenoproteins also exist as cysteine-containing homologs. Here, we demonstrate that selenolate-based catalysis of the essential mammalian selenoprotein GPX4 is unexpectedly dispensable for normal embryogenesis. Yet the survival of a specific type of interneurons emerges to exclusively depend on selenocysteine-containing GPX4, thereby preventing fatal epileptic seizures. Mechanistically, selenocysteine utilization by GPX4 confers exquisite resistance to irreversible overoxidation as cells expressing a cysteine variant are highly sensitive toward peroxide-induced ferroptosis. Remarkably, concomitant deletion of all selenoproteins in Gpx4cys/cys cells revealed that selenoproteins are dispensable for cell viability provided partial GPX4 activity is retained. Conclusively, 200 years after its discovery, a specific and indispensable role for selenium is provided.


Subject(s)
Apoptosis , Glutathione Peroxidase/metabolism , Seizures/metabolism , Selenium/metabolism , Animals , Cell Survival , Cells, Cultured , Female , Glutathione Peroxidase/genetics , HEK293 Cells , Humans , Hydrogen Peroxide/toxicity , Interneurons/metabolism , Lipid Peroxidation , Male , Mice , Mice, Inbred C57BL , Phospholipid Hydroperoxide Glutathione Peroxidase , Seizures/etiology
3.
Cell ; 175(1): 101-116.e25, 2018 09 20.
Article in English | MEDLINE | ID: mdl-30220459

ABSTRACT

IDH1 mutations are common in low-grade gliomas and secondary glioblastomas and cause overproduction of (R)-2HG. (R)-2HG modulates the activity of many enzymes, including some that are linked to transformation and some that are probably bystanders. Although prior work on (R)-2HG targets focused on 2OG-dependent dioxygenases, we found that (R)-2HG potently inhibits the 2OG-dependent transaminases BCAT1 and BCAT2, likely as a bystander effect, thereby decreasing glutamate levels and increasing dependence on glutaminase for the biosynthesis of glutamate and one of its products, glutathione. Inhibiting glutaminase specifically sensitized IDH mutant glioma cells to oxidative stress in vitro and to radiation in vitro and in vivo. These findings highlight the complementary roles for BCATs and glutaminase in glutamate biosynthesis, explain the sensitivity of IDH mutant cells to glutaminase inhibitors, and suggest a strategy for maximizing the effectiveness of such inhibitors against IDH mutant gliomas.


Subject(s)
Glioma/metabolism , Glutamic Acid/biosynthesis , Transaminases/physiology , Cell Line, Tumor , Glioma/physiopathology , Glutamic Acid/drug effects , Glutarates/metabolism , Glutarates/pharmacology , Homeostasis/drug effects , Humans , Isocitrate Dehydrogenase/genetics , Isocitrate Dehydrogenase/physiology , Minor Histocompatibility Antigens/genetics , Minor Histocompatibility Antigens/physiology , Mutation , Oxidation-Reduction/drug effects , Pregnancy Proteins/genetics , Pregnancy Proteins/physiology , Transaminases/antagonists & inhibitors , Transaminases/genetics
4.
Annu Rev Biochem ; 86: 777-797, 2017 06 20.
Article in English | MEDLINE | ID: mdl-28654321

ABSTRACT

Severe changes in the environmental redox potential, and resulting alterations in the oxidation states of intracellular metabolites and enzymes, have historically been considered negative stressors, requiring responses that are strictly defensive. However, recent work in diverse organisms has revealed that more subtle changes in the intracellular redox state can act as signals, eliciting responses with benefits beyond defense and detoxification. Changes in redox state have been shown to influence or trigger chromosome segregation, sporulation, aerotaxis, and social behaviors, including luminescence as well as biofilm establishment and dispersal. Connections between redox state and complex behavior allow bacteria to link developmental choices with metabolic state and coordinate appropriate responses. Promising future directions for this area of study include metabolomic analysis of species- and condition-dependent changes in metabolite oxidation states and elucidation of the mechanisms whereby the redox state influences circadian regulation.


Subject(s)
Biofilms/growth & development , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial , Membrane Proteins/metabolism , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Spores, Bacterial/metabolism , Aliivibrio fischeri/genetics , Aliivibrio fischeri/growth & development , Aliivibrio fischeri/metabolism , Bacillus subtilis/genetics , Bacillus subtilis/growth & development , Bacillus subtilis/metabolism , Caulobacter crescentus/genetics , Caulobacter crescentus/growth & development , Caulobacter crescentus/metabolism , Escherichia coli/genetics , Escherichia coli/growth & development , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Glutathione/metabolism , Membrane Proteins/genetics , Oxidation-Reduction , Protein Kinases/genetics , Protein Serine-Threonine Kinases/genetics , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/growth & development , Pseudomonas aeruginosa/metabolism , Signal Transduction , Spores, Bacterial/genetics , Spores, Bacterial/growth & development , Streptomyces/genetics , Streptomyces/growth & development , Streptomyces/metabolism
5.
Cell ; 171(2): 273-285, 2017 Oct 05.
Article in English | MEDLINE | ID: mdl-28985560

ABSTRACT

Ferroptosis is a form of regulated cell death characterized by the iron-dependent accumulation of lipid hydroperoxides to lethal levels. Emerging evidence suggests that ferroptosis represents an ancient vulnerability caused by the incorporation of polyunsaturated fatty acids into cellular membranes, and cells have developed complex systems that exploit and defend against this vulnerability in different contexts. The sensitivity to ferroptosis is tightly linked to numerous biological processes, including amino acid, iron, and polyunsaturated fatty acid metabolism, and the biosynthesis of glutathione, phospholipids, NADPH, and coenzyme Q10. Ferroptosis has been implicated in the pathological cell death associated with degenerative diseases (i.e., Alzheimer's, Huntington's, and Parkinson's diseases), carcinogenesis, stroke, intracerebral hemorrhage, traumatic brain injury, ischemia-reperfusion injury, and kidney degeneration in mammals and is also implicated in heat stress in plants. Ferroptosis may also have a tumor-suppressor function that could be harnessed for cancer therapy. This Primer reviews the mechanisms underlying ferroptosis, highlights connections to other areas of biology and medicine, and recommends tools and guidelines for studying this emerging form of regulated cell death.


Subject(s)
Cell Death , Animals , Apoptosis , Humans , Iron/metabolism , Oxidation-Reduction , Reactive Oxygen Species/metabolism
6.
Mol Cell ; 84(1): 23-33, 2024 Jan 04.
Article in English | MEDLINE | ID: mdl-38029751

ABSTRACT

Scientists in this field often joke, "If you don't have a mechanism, say it's ROS." Seemingly connected to every biological process ever described, reactive oxygen species (ROS) have numerous pleiotropic roles in physiology and disease. In some contexts, ROS act as secondary messengers, controlling a variety of signaling cascades. In other scenarios, they initiate damage to macromolecules. Finally, in their worst form, ROS are deadly to cells and surrounding tissues. A set of molecules with detoxifying abilities, termed antioxidants, is the direct counterpart to ROS. Notably, antioxidants exist in the public domain, touted as a "cure-all" for diseases. Research has disproved many of these claims and, in some cases, shown the opposite. Of all the diseases, cancer stands out in its paradoxical relationship with antioxidants. Although the field has made numerous strides in understanding the roles of antioxidants in cancer, many questions remain.


Subject(s)
Antioxidants , Neoplasms , Humans , Reactive Oxygen Species , Oxidative Stress , Neoplasms/genetics , Signal Transduction
7.
Mol Cell ; 84(4): 802-810.e6, 2024 Feb 15.
Article in English | MEDLINE | ID: mdl-38157846

ABSTRACT

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.


Subject(s)
Iron , Mitochondria , Animals , Humans , ATPases Associated with Diverse Cellular Activities/metabolism , ATP-Dependent Proteases/metabolism , Iron/metabolism , Mitochondria/genetics , Mitochondria/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Homeostasis , Glutathione/metabolism , Mammals/metabolism , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Membrane Transport Proteins/metabolism
8.
Immunity ; 53(6): 1168-1181.e7, 2020 12 15.
Article in English | MEDLINE | ID: mdl-33326766

ABSTRACT

Viruses have evolved multiple strategies to evade elimination by the immune system. Here we examined the contribution of host long noncoding RNAs (lncRNAs) in viral immune evasion. By functional screening of lncRNAs whose expression decreased upon viral infection of macrophages, we identified a lncRNA (lncRNA-GM, Gene Symbol: AK189470.1) that promoted type I interferon (IFN-I) production and inhibited viral replication. Deficiency of lncRNA-GM in mice increased susceptibility to viral infection and impaired IFN-I production. Mechanistically, lncRNA-GM bound to glutathione S-transferase M1 (GSTM1) and blocked GSTM1 interaction with the kinase TBK1, reducing GSTM1-mediated S-glutathionylation of TBK1. Decreased S-glutathionylation enhanced TBK1 activity and downstream production of antiviral mediators. Viral infection reprogrammed intracellular glutathione metabolism and furthermore, an oxidized glutathione mimetic could inhibit TBK1 activity and promote viral replication. Our findings reveal regulation of TBK1 by S-glutathionylation and provide insight into the viral mediated metabolic changes that impact innate immunity and viral evasion.


Subject(s)
Glutathione/metabolism , Immune Evasion , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/metabolism , RNA, Long Noncoding/metabolism , Animals , Glutathione Transferase/metabolism , Humans , Immunity, Innate , Interferon Regulatory Factor-3/metabolism , Interferon Type I/metabolism , Macrophages/immunology , Macrophages/metabolism , Mice , RNA, Long Noncoding/genetics , Signal Transduction , Virus Diseases/genetics , Virus Diseases/immunology , Virus Diseases/metabolism , Virus Replication
9.
Mol Cell ; 81(15): 3216-3226.e8, 2021 08 05.
Article in English | MEDLINE | ID: mdl-34161757

ABSTRACT

Glutamate receptor-like channels (GLRs) play vital roles in various physiological processes in plants, such as wound response, stomatal aperture control, seed germination, root development, innate immune response, pollen tube growth, and morphogenesis. Despite the importance of GLRs, knowledge about their molecular organization is limited. Here we use X-ray crystallography and single-particle cryo-EM to solve structures of the Arabidopsis thaliana GLR3.4. Our structures reveal the tetrameric assembly of GLR3.4 subunits into a three-layer domain architecture, reminiscent of animal ionotropic glutamate receptors (iGluRs). However, the non-swapped arrangement between layers of GLR3.4 domains, binding of glutathione through S-glutathionylation of cysteine C205 inside the amino-terminal domain clamshell, unique symmetry, inter-domain interfaces, and ligand specificity distinguish GLR3.4 from representatives of the iGluR family and suggest distinct features of the GLR gating mechanism. Our work elaborates on the principles of GLR architecture and symmetry and provides a molecular template for deciphering GLR-dependent signaling mechanisms in plants.


Subject(s)
Arabidopsis Proteins/chemistry , Arabidopsis Proteins/metabolism , Receptors, Glutamate/chemistry , Receptors, Glutamate/metabolism , Animals , Arabidopsis Proteins/genetics , Binding Sites , COS Cells , Calcium/metabolism , Chlorocebus aethiops , Cryoelectron Microscopy , Crystallography, X-Ray , Cysteine/metabolism , Glutathione/metabolism , HEK293 Cells , Humans , Models, Molecular , Plants, Genetically Modified , Protein Domains , Receptors, Glutamate/genetics
10.
Genes Dev ; 34(7-8): 544-559, 2020 04 01.
Article in English | MEDLINE | ID: mdl-32079653

ABSTRACT

Excessive reactive oxygen species (ROS) can cause oxidative stress and consequently cell injury contributing to a wide range of diseases. Addressing the critical gaps in our understanding of the adaptive molecular events downstream ROS provocation holds promise for the identification of druggable metabolic vulnerabilities. Here, we unveil a direct molecular link between the activity of two estrogen-related receptor (ERR) isoforms and the control of glutamine utilization and glutathione antioxidant production. ERRα down-regulation restricts glutamine entry into the TCA cycle, while ERRγ up-regulation promotes glutamine-driven glutathione production. Notably, we identify increased ERRγ expression/activation as a hallmark of oxidative stress triggered by mitochondrial disruption or chemotherapy. Enhanced tumor antioxidant capacity is an underlying feature of human breast cancer (BCa) patients that respond poorly to treatment. We demonstrate that pharmacological inhibition of ERRγ with the selective inverse agonist GSK5182 increases antitumor efficacy of the chemotherapeutic paclitaxel on poor outcome BCa tumor organoids. Our findings thus underscore the ERRs as novel redox sensors and effectors of a ROS defense program and highlight the potential therapeutic advantage of exploiting ERRγ inhibitors for the treatment of BCa and other diseases where oxidative stress plays a central role.


Subject(s)
Breast Neoplasms/physiopathology , Drug Resistance, Neoplasm/drug effects , Oxidative Stress , Reactive Oxygen Species/metabolism , Receptors, Estrogen/metabolism , Signal Transduction/physiology , Animals , Antineoplastic Agents/pharmacology , Biosensing Techniques , Breast Neoplasms/drug therapy , Female , Gene Expression Regulation, Neoplastic/drug effects , Glutamine/metabolism , Glutathione/metabolism , Humans , Mice , Oxidative Stress/drug effects , Oxidative Stress/physiology , Paclitaxel/pharmacology , Receptors, Estrogen/genetics , Rotenone/pharmacology , Tamoxifen/analogs & derivatives , Tamoxifen/pharmacology , ERRalpha Estrogen-Related Receptor
11.
Proc Natl Acad Sci U S A ; 121(42): e2403450121, 2024 Oct 15.
Article in English | MEDLINE | ID: mdl-39388265

ABSTRACT

Aging is the biggest risk factor for Parkinson's disease (PD), suggesting that age-related changes in the brain promote dopamine neuron vulnerability. It is unclear, however, whether aging alone is sufficient to cause significant dopamine neuron loss, and if so, how this intersects with PD-related neurodegeneration. Here, through examining a large collection of naturally varying Drosophila strains, we find a strong relationship between life span and age-related dopamine neuron loss. Strains with naturally short-lived animals exhibit a loss of dopamine neurons without generalized neurodegeneration, while animals from long-lived strains retain dopamine neurons across age. Metabolomic profiling reveals lower glutathione levels in short-lived strains which is associated with elevated levels of reactive oxygen species (ROS), sensitivity to oxidative stress, and vulnerability to silencing the familial PD gene parkin. Strikingly, boosting neuronal glutathione levels via glutamate-cysteine ligase (Gcl) overexpression is sufficient to normalize ROS levels, extend life span, and block dopamine neurons loss in short-lived backgrounds, demonstrating that glutathione deficiencies are central to neurodegenerative phenotypes associated with short longevity. These findings may be relevant to human PD pathogenesis, where glutathione depletion is reported to occur in the idiopathic PD patient brain through unknown mechanisms. Building on this, we find reduced expression of the Gcl catalytic subunit in both Drosophila strains vulnerable to age-related dopamine neuron loss and in the human brain from familial PD patients harboring the common LRRK2 G2019S mutation. Our study across Drosophila and human PD systems suggests that glutathione synthesis and levels play a conserved role in regulating age-related dopamine neuron health.


Subject(s)
Aging , Dopaminergic Neurons , Drosophila Proteins , Glutathione , Longevity , Parkinson Disease , Reactive Oxygen Species , Animals , Glutathione/metabolism , Dopaminergic Neurons/metabolism , Dopaminergic Neurons/pathology , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Parkinson Disease/metabolism , Parkinson Disease/pathology , Parkinson Disease/genetics , Aging/metabolism , Aging/pathology , Reactive Oxygen Species/metabolism , Drosophila melanogaster/metabolism , Oxidative Stress , Humans , Glutamate-Cysteine Ligase/metabolism , Glutamate-Cysteine Ligase/genetics , Nerve Degeneration/pathology , Nerve Degeneration/metabolism , Nerve Degeneration/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Drosophila/metabolism , Male
12.
Proc Natl Acad Sci U S A ; 121(21): e2400740121, 2024 May 21.
Article in English | MEDLINE | ID: mdl-38743629

ABSTRACT

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.


Subject(s)
Cytosol , Glutaredoxins , Glutathione , Iron-Sulfur Proteins , Mitochondria , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Cytosol/metabolism , Iron-Sulfur Proteins/metabolism , Humans , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Glutathione/metabolism , Mitochondria/metabolism , Glutaredoxins/metabolism , Glutaredoxins/genetics , ATP-Binding Cassette Transporters/metabolism , Mitochondrial Proteins/metabolism
13.
Trends Biochem Sci ; 47(7): 558-560, 2022 07.
Article in English | MEDLINE | ID: mdl-35292185

ABSTRACT

Tricarboxylic acid (TCA) cycle is a major hub for catabolic and anabolic reactions, yet cellular metabolic adaptations following its inhibition are largely unknown. Using multi-tiered omics approaches, Ryan et al. have shown convergent activation of the integrated stress response (ISR) through ATF4-mediated rewiring of cellular amino acid and redox metabolic pathways.


Subject(s)
Amino Acids , Citric Acid Cycle , Homeostasis , Metabolic Networks and Pathways , Oxidation-Reduction
14.
Immunity ; 46(4): 675-689, 2017 04 18.
Article in English | MEDLINE | ID: mdl-28423341

ABSTRACT

Activated T cells produce reactive oxygen species (ROS), which trigger the antioxidative glutathione (GSH) response necessary to buffer rising ROS and prevent cellular damage. We report that GSH is essential for T cell effector functions through its regulation of metabolic activity. Conditional gene targeting of the catalytic subunit of glutamate cysteine ligase (Gclc) blocked GSH production specifically in murine T cells. Gclc-deficient T cells initially underwent normal activation but could not meet their increased energy and biosynthetic requirements. GSH deficiency compromised the activation of mammalian target of rapamycin-1 (mTOR) and expression of NFAT and Myc transcription factors, abrogating the energy utilization and Myc-dependent metabolic reprogramming that allows activated T cells to switch to glycolysis and glutaminolysis. In vivo, T-cell-specific ablation of murine Gclc prevented autoimmune disease but blocked antiviral defense. The antioxidative GSH pathway thus plays an unexpected role in metabolic integration and reprogramming during inflammatory T cell responses.


Subject(s)
Glutamate-Cysteine Ligase/deficiency , Glutathione/metabolism , Inflammation/metabolism , T-Lymphocytes/metabolism , Animals , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/metabolism , Energy Metabolism/genetics , Glutamate-Cysteine Ligase/genetics , Glutamine/metabolism , Glycolysis , Immunoblotting , Inflammation/genetics , Mice, Inbred C57BL , Mice, Knockout , NFATC Transcription Factors/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction/genetics , TOR Serine-Threonine Kinases/metabolism
15.
Proc Natl Acad Sci U S A ; 120(39): e2306288120, 2023 09 26.
Article in English | MEDLINE | ID: mdl-37729198

ABSTRACT

Nonsmall cell lung cancer (NSCLC) is highly malignant with limited treatment options, platinum-based chemotherapy is a standard treatment for NSCLC with resistance commonly seen. NSCLC cells exploit enhanced antioxidant defense system to counteract excessive reactive oxygen species (ROS), which contributes largely to tumor progression and resistance to chemotherapy, yet the mechanisms are not fully understood. Recent studies have suggested the involvement of histones in tumor progression and cellular antioxidant response; however, whether a major histone variant H1.2 (H1C) plays roles in the development of NSCLC remains unclear. Herein, we demonstrated that H1.2 was increasingly expressed in NSCLC tumors, and its expression was correlated with worse survival. When crossing the H1c knockout allele with a mouse NSCLC model (KrasLSL-G12D/+), H1.2 deletion suppressed NSCLC progression and enhanced oxidative stress and significantly decreased the levels of key antioxidant glutathione (GSH) and GCLC, the catalytic subunit of rate-limiting enzyme for GSH synthesis. Moreover, high H1.2 was correlated with the IC50 of multiple chemotherapeutic drugs and with worse prognosis in NSCLC patients receiving chemotherapy; H1.2-deficient NSCLC cells presented reduced survival and increased ROS levels upon cisplatin treatment, while ROS scavenger eliminated the survival inhibition. Mechanistically, H1.2 interacted with NRF2, a master regulator of antioxidative response; H1.2 enhanced the nuclear level and stability of NRF2 and, thus, promoted NRF2 binding to GCLC promoter and the consequent transcription; while NRF2 also transcriptionally up-regulated H1.2. Collectively, these results uncovered a tumor-driving role of H1.2 in NSCLC and indicate an "H1.2-NRF2" antioxidant feedforward cycle that promotes tumor progression and chemoresistance.


Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Animals , Mice , Humans , Histones/genetics , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Antioxidants , NF-E2-Related Factor 2/genetics , Reactive Oxygen Species , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Glutathione , Disease Models, Animal
16.
J Biol Chem ; 300(5): 107289, 2024 May.
Article in English | MEDLINE | ID: mdl-38636663

ABSTRACT

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.


Subject(s)
Glutathione , Rats , Transcobalamins , Vitamin B 12 , Animals , Crystallography, X-Ray , Glutathione/metabolism , Glutathione/analogs & derivatives , Glutathione/chemistry , Protein Binding , Transcobalamins/metabolism , Transcobalamins/chemistry , Vitamin B 12/metabolism , Vitamin B 12/analogs & derivatives , Vitamin B 12/chemistry
17.
J Biol Chem ; 300(10): 107741, 2024 Aug 31.
Article in English | MEDLINE | ID: mdl-39222686

ABSTRACT

Transition metal (TM) distribution through the phloem is an essential part of plant metabolism and is required for systemic signaling and balancing source-to-sink relationships. Due to their reactivity, TMs are expected to occur in complexes within the phloem sap; however, metal speciation in the phloem sap remains largely unexplored. Here, we isolated phloem sap from Brassica napus and analyzed it via size exclusion chromatography coupled online to sector-field ICP-MS. Our data identified known TM-binding proteins and molecules including metallothioneins (MT), glutathione, and nicotianamine. While the main peak of all metals was low MW (∼1.5 kD), additional peaks ∼10 to 15 kD containing Cu, Fe, S, and Zn were also found. Further physicochemical analyses of MTs with and without affinity tags corroborated that MTs can form complexes of diverse molecular weights. We also identified and characterized potential artifacts in the TM-biding ability of B. napus MTs between tagged and non-tagged MTs. That is, the native BnMT2 binds Zn, Cu, and Fe, while MT3a and MT3b only bind Cu and Zn. In contrast, his-tagged MTs bind less Cu and were found to bind Co and Mn and aggregated to oligomeric forms to a greater extent compared to the phloem sap. Our data indicates that TM chemistry in the phloem sap is more complex than previously anticipated and that more systematic analyses are needed to establish the precise speciation of TM and TM-ligand complexes within the phloem sap.

18.
J Biol Chem ; 300(3): 105746, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38354787

ABSTRACT

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.


Subject(s)
Protein Disulfide-Isomerases , Protein Folding , Saccharomycetales , Disulfides/chemistry , Glutathione/metabolism , Oxidation-Reduction , Protein Disulfide-Isomerases/chemistry , Protein Disulfide-Isomerases/metabolism , Protein Structure, Tertiary , Saccharomycetales/enzymology , Thioredoxins/metabolism
19.
J Biol Chem ; 300(4): 107123, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38417796

ABSTRACT

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.


Subject(s)
Carica , Glutathione Transferase , Thiram , Carica/enzymology , Carica/genetics , Fungicides, Industrial/metabolism , Glutathione Transferase/metabolism , Glutathione Transferase/genetics , Glutathione Transferase/chemistry , Mutagenesis, Site-Directed , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/metabolism , Thiram/metabolism , Escherichia coli/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
20.
J Biol Chem ; 300(2): 105645, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38218225

ABSTRACT

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.


Subject(s)
Glutamate-Cysteine Ligase , Glutathione , Animals , Mice , Buthionine Sulfoximine/pharmacology , Disease Models, Animal , Glutamate-Cysteine Ligase/genetics , Glutamate-Cysteine Ligase/metabolism , Glutathione/metabolism , Cell Line, Tumor , Humans
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