The Emerging Roles of γ-Glutamyl Peptides Produced by γ-Glutamyltransferase and the Glutathione Synthesis System.

L-γ-Glutamyl-L-cysteinyl-glycine is commonly referred to as glutathione (GSH); this ubiquitous thiol plays essential roles in animal life. Conjugation and electron donation to enzymes such as glutathione peroxidase (GPX) are prominent functions of GSH. Cellular glutathione balance is robustly maintained via regulated synthesis, which is catalyzed via the coordination of γ-glutamyl-cysteine synthetase (γ-GCS) and glutathione synthetase, as well as by reductive recycling by glutathione reductase. A prevailing short supply of L-cysteine (Cys) tends to limit glutathione synthesis, which leads to the production of various other γ-glutamyl peptides due to the unique enzymatic properties of γ-GCS. Extracellular degradation of glutathione by γ-glutamyltransferase (GGT) is a dominant source of Cys for some cells. GGT catalyzes the hydrolytic removal of the γ-glutamyl group of glutathione or transfers it to amino acids or to dipeptides outside cells. Such processes depend on an abundance of acceptor substrates. However, the physiological roles of extracellularly preserved γ-glutamyl peptides have long been unclear. The identification of γ-glutamyl peptides, such as glutathione, as allosteric modulators of calcium-sensing receptors (CaSRs) could provide insights into the significance of the preservation of γ-glutamyl peptides. It is conceivable that GGT could generate a new class of intercellular messaging molecules in response to extracellular microenvironments.

Generation of Rat Monoclonal Antibody for Human Nucleolin.

Nucleolin (NCL) is a multifunctional phosphoprotein that is mainly localized in the nucleolus, but it is also found in the nucleoplasm, cytoplasm, and cell membrane. The principal functions of NCL involve DNA and RNA metabolism, gene transcription and translation, ribosome biogenesis, and mRNA stability. It was also reported that the localization of human NCL (hNCL) is related to tumor malignancy. Therefore, analyzing the cellular dynamics of NCL could be useful. In this article, we describe rat monoclonal antibody (mAb) 6F9A6 that was generated against a hNCL peptide. This mAb recognizes endogenous human, monkey, dog, and mouse NCL and was shown to be useful in immunofluorescence staining, immunoprecipitation, and immunoblotting experiments in several cancer cell lines. We anticipate that the mAb 6F9A6 will be useful for functional analyses of hNCL in cancer cells.

Defense systems to avoid ferroptosis caused by lipid peroxidation-mediated membrane damage.

The presence of hydrogen peroxide along with ferrous iron produces hydroxyl radicals that preferably oxidize polyunsaturated fatty acids (PUFA) to alkyl radicals (L•). The reaction of L• with an oxygen molecule produces lipid peroxyl radical (LOO•) that collectively trigger chain reactions, which results in the accumulation of lipid peroxidation products (LOOH). Oxygenase enzymes, such as lipoxygenase, also stimulate the peroxidation of PUFA. The production of phospholipid hydroperoxides (P-LOOH) can result in the destruction of the architecture of cell membranes and ultimate cell death. This iron-dependent regulated cell death is generally referred to as ferroptosis. Radical scavengers, which include tocopherol and nitric oxide (•NO), react with lipid radicals and terminate the chain reaction. When tocopherol reductively detoxifies lipid radicals, the resultant tocopherol radicals are recycled via reduction by coenzyme Q or ascorbate. CoQ radicals are reduced back by the anti-ferroptotic enzyme FSP1. •NO reacts with lipid radicals and produces less reactive nitroso compounds. The resulting P-LOOH is reductively detoxified by the action of glutathione peroxidase 4 (GPX4) or peroxiredoxin 6 (PRDX6). The hydrolytic removal of LOOH from P-LOOH by calcium-independent phospholipase A2 leads the preservation of membrane structure. While the expression of such protective genes or the presence of these anti-oxidant compounds serve to maintain a healthy condition, tumor cells employ them to make themselves resistant to anti-tumor treatments. Thus, these defense mechanisms against ferroptosis are protective in ordinary cells but are also potential targets for cancer treatment.

Critical Roles of the Cysteine-Glutathione Axis in the Production of γ-Glutamyl Peptides in the Nervous System.

γ-Glutamyl moiety that is attached to the cysteine (Cys) residue in glutathione (GSH) protects it from peptidase-mediated degradation. The sulfhydryl group of the Cys residue represents most of the functions of GSH, which include electron donation to peroxidases, protection of reactive sulfhydryl in proteins via glutaredoxin, and glutathione conjugation of xenobiotics, whereas Cys-derived sulfur is also a pivotal component of some redox-responsive molecules. The amount of Cys that is available tends to restrict the capacity of GSH synthesis. In in vitro systems, cystine is the major form in the extracellular milieu, and a specific cystine transporter, xCT, is essential for survival in most lines of cells and in many primary cultivated cells as well. A reduction in the supply of Cys causes GPX4 to be inhibited due to insufficient GSH synthesis, which leads to iron-dependent necrotic cell death, ferroptosis. Cells generally cannot take up GSH without the removal of γ-glutamyl moiety by γ-glutamyl transferase (GGT) on the cell surface. Meanwhile, the Cys-GSH axis is essentially common to certain types of cells; primarily, neuronal cells that contain a unique metabolic system for intercellular communication concerning γ-glutamyl peptides. After a general description of metabolic processes concerning the Cys-GSH axis, we provide an overview and discuss the significance of GSH-related compounds in the nervous system.

Biological Action of Singlet Molecular Oxygen from the Standpoint of Cell Signaling, Injury and Death.

Energy transfer to ground state triplet molecular oxygen results in the generation of singlet molecular oxygen (1O2), which has potent oxidizing ability. Irradiation of light, notably ultraviolet A, to a photosensitizing molecule results in the generation of 1O2, which is thought to play a role in causing skin damage and aging. It should also be noted that 1O2 is a dominant tumoricidal component that is generated during the photodynamic therapy (PDT). While type II photodynamic action generates not only 1O2 but also other reactive species, endoperoxides release pure 1O2 upon mild exposure to heat and, hence, are considered to be beneficial compounds for research purposes. Concerning target molecules, 1O2 preferentially reacts with unsaturated fatty acids to produce lipid peroxidation. Enzymes that contain a reactive cysteine group at the catalytic center are vulnerable to 1O2 exposure. Guanine base in nucleic acids is also susceptible to oxidative modification, and cells carrying DNA with oxidized guanine units may experience mutations. Since 1O2 is produced in various physiological reactions in addition to photodynamic reactions, overcoming technical challenges related to its detection and methods used for its generation would allow its potential functions in biological systems to be better understood.

Loss of Peroxiredoxin IV Protects Mice from Azoxymethane/Dextran Sulfate Sodium-Induced Colorectal Cancer Development.

Peroxiredoxin IV (Prx4), a typical two-cysteine-containing member of the peroxidase family, functions as an antioxidant to maintain cellular redox homeostasis through the reduction of reactive oxygen species (ROS) via cycles of oxidation-reduction reactions. Under oxidative stress, all Prxs including Prx4 are inactivated as their catalytic cysteines undergo hyperoxidation, and hyperoxidized two-cysteine Prxs can be exclusively repaired and revitalized through the reduction cycle catalyzed by sulfiredoxin (Srx). Previously, we showed that Prx4 is a preferred substrate of Srx, and knockout of Srx in mice leads to resistance to azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colon carcinogenesis. To further understand the significance of the Srx/Prx4 axis in colorectal cancer development, Prx4-/- mice were established and subjected to standard AOM/DSS protocol. Compared with wildtype littermates, mice with Prx4-/- genotype had significantly fewer and smaller tumors. Histopathological analysis revealed that loss of Prx4 leads to increased cell death through lipid peroxidation and lower infiltration of inflammatory cells in the knockout tumors compared to wildtype. Treatment with DSS alone also showed decreased infiltration of macrophages and lymphocytes in the colon of knockout mice, suggesting a role for Prx4 in inflammatory response. In addition, loss of Prx4 caused alterations in plasma cytokines and chemokines after DSS and AOM/DSS treatments. These findings suggest that loss of Prx4 protects mice from AOM/DSS-induced colon tumorigenesis. Thus, targeting Prx4 may provide novel strategies for colon cancer prevention and treatment.

Critical Role of the Sulfiredoxin-Peroxiredoxin IV Axis in Urethane-Induced Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC), the most common type of lung cancer, etiologically associates with tobacco smoking which mechanistically contributes to oxidative stress to facilitate the occurrence of mutations, oncogenic transformation and aberrantly activated signaling pathways. Our previous reports suggested an essential role of Sulfiredoxin (Srx) in promoting the development of lung cancer in humans, and was causally related to Peroxiredoxin IV (Prx4), the major downstream substrate and mediator of Srx-enhanced signaling. To further explore the role of the Srx-Prx4 axis in de novo lung tumorigenesis, we established Prx4-/- and Srx-/-/Prx4-/- mice in pure FVB/N background. Together with wild-type litter mates, these mice were exposed to carcinogenic urethane and the development of lung tumorigenesis was evaluated. We found that disruption of the Srx-Prx4 axis, either through knockout of Srx/Prx4 alone or together, led to a reduced number and size of lung tumors in mice. Immunohistological studies found that loss of Srx/Prx4 led to reduced rate of cell proliferation and less intratumoral macrophage infiltration. Mechanistically, we found that exposure to urethane increased the levels of reactive oxygen species, activated the expression of and Prx4 in normal lung epithelial cells, while knockout of Prx4 inhibited urethane-induced cell transformation. Moreover, bioinformatics analysis found that the Srx-Prx4 axis is activated in many human cancers, and their increased expression is tightly correlated with poor prognosis in NSCLC patients.

[A Case of Multiple Postoperative Recurrence of Hepatocellular Carcinoma Successfully Treated with Atezolizumab plus Bevacizumab].

We report a case of postoperative multiple recurrence of hepatocellular carcinoma(HCC)treated with atezolizumab plus bevacizumab. A 73-year-old man with a chief complaint of abdominal distention was indicated a 90-mm-sized tumor extending from the lateral hepatic segment to the extrahepatic region by a contrast-enhanced CT scan of the abdomen. He underwent a laparoscopic liver resection of the lateral segment for suspected HCC, and was diagnosed as pStage Ⅱ HCC. Six months after surgery, multiple recurrent at intrahepatic lesions and suspected lymph node recurrence or peritoneal dissemination were observed, and tumor markers were markedly elevated. The patient was diagnosed with multiple intrahepatic and extrahepatic recurrences of postoperative HCC and started combination chemotherapy with atezolizumab (1,200 mg/body)plus bevacizumab(15 mg/kg). After the initiation of the therapy, tumor size reduction and normalization of tumor markers were observed, and at 17 months postoperatively, tumor size reduction has been maintained and tumor markers were in the normal range. We report a case of postoperative intrahepatic and extrahepatic multiple recurrences of he patocellular carcinoma treated with atezolizumab plus bevacizumab.

Involvement of Nitric Oxide in Protecting against Radical Species and Autoregulation of M1-Polarized Macrophages through Metabolic Remodeling.

When the expression of NOS2 in M1-polarized macrophages is induced, huge amounts of nitric oxide (•NO) are produced from arginine and molecular oxygen as the substrates. While anti-microbial action is the primary function of M1 macrophages, excessive activation may result in inflammation being aggravated. The reaction of •NO with superoxide produces peroxynitrite, which is highly toxic to cells. Alternatively, however, this reaction eliminates radial electrons and may occasionally alleviate subsequent radical-mediated damage. Reactions of •NO with lipid radicals terminates the radical chain reaction in lipid peroxidation, which leads to the suppression of ferroptosis. •NO is involved in the metabolic remodeling of M1 macrophages. Enzymes in the tricarboxylic acid (TCA) cycle, notably aconitase 2, as well as respiratory chain enzymes, are preferential targets of •NO derivatives. Ornithine, an alternate compound produced from arginine instead of citrulline and •NO, is recruited to synthesize polyamines. Itaconate, which is produced from the remodeled TCA cycle, and polyamines function as defense systems against overresponses of M1 macrophages in a feedback manner. Herein, we overview the protective aspects of •NO against radical species and the autoregulatory systems that are enabled by metabolic remodeling in M9-polarized macrophages.

Ascorbate Is a Primary Antioxidant in Mammals.

Ascorbate (vitamin C in primates) functions as a cofactor for a number of enzymatic reactions represented by prolyl hydroxylases and as an antioxidant due to its ability to donate electrons, which is mostly accomplished through non-enzymatic reaction in mammals. Ascorbate directly reacts with radical species and is converted to ascorbyl radical followed by dehydroascorbate. Ambiguities in physiological relevance of ascorbate observed during in vivo situations could be attributed in part to presence of other redox systems and the pro-oxidant properties of ascorbate. Most mammals are able to synthesize ascorbate from glucose, which is also considered to be an obstacle to verify its action. In addition to animals with natural deficiency in the ascorbate synthesis, such as guinea pigs and ODS rats, three strains of mice with genetic removal of the responsive genes (GULO, RGN, or AKR1A) for the ascorbate synthesis have been established and are being used to investigate the physiological roles of ascorbate. Studies using these mice, along with ascorbate transporter (SVCT)-deficient mice, largely support its ability in protection against oxidative insults. While combined actions of ascorbate in regulating epigenetics and antioxidation appear to effectively prevent cancer development, pharmacological doses of ascorbate and dehydroascorbate may exert tumoricidal activity through redox-dependent mechanisms.

Flow cytometric determination of ferroptosis using a rat monoclonal antibody raised against ferroptotic cells.

Ferroptosis, a type of iron-dependent necrotic cell death, is specifically associated with increased lipid peroxidation. The dysfunction of the glutathione (GSH) production via the starvation of cysteine or the inhibition of phospholipid hydroperoxide glutathione peroxidase (GPX4) typically results in the accumulation of lipid peroxidation products and, consequently, the development of ferroptosis. We recently reported on the production of a rat monoclonal antibody, referred to as FerAb, against mouse-derived Hepa 1-6 cells that had been cultivated in cystine-deprived medium. Immunocytological analyses by means of fluorescence microscopy revealed that FerAb binds to fixed ferroptotic cells regardless of the species from which they were obtained, but not to apoptotic cells. We report herein on an in-depth characterization of the reactivity of FerAb with respect to unfixed cells by means of flow cytometry. The binding of FerAb to the cells was stimulated by incubating the cells in cystine deprived culture medium or treatment with RSL3, a GPX4 inhibitor, while treatment with staurosporine, an apoptosis inducer, had no effect on its binding to the cells. Supplementation with ferrostatin-1, a ferroptosis inhibitor, effectively suppressed the binding of FerAb to cells that had been cultivated in cystine-deprived medium or treated with RSL3, further confirming the specific binding of FerAb to ferroptotic cells. Thus, FerAb combined with a flow cytometry can be used to distinguish ferroptotic cells from living cells or apoptotic cells without the need for fixation. Applications of this combined technique will enable the quantitative evaluation of ferroptotic cells under a variety of patho-physiological conditions and will contribute to our understanding of the roles of ferroptosis in the body as well as cultured cells.

d-Cysteine supplementation partially protects against ferroptosis induced by xCT dysfunction via increasing the availability of glutathione.

Glutathione (GSH) is synthesized from three amino acids and the overall process is highly dependent on the availability of l-cysteine (l-Cys). GSH serves as an essential cofactor for glutathione peroxidase 4 (Gpx4), which reduces phospholipid hydroperoxides. The inactivation of Gpx4 or an insufficient supply of l-Cys results in the accumulation of lipid hydroperoxides, eventually leading to iron-dependent cell death, ferroptosis. In this study, we investigated the anti-ferroptotic properties of d-cysteine (d-Cys) under conditions of dysfunction in cystine transporter, xCT. l-Cys supplementation completely rescued ferroptosis that had been induced by the erastin-mediated inhibition of xCT in Hepa 1-6 cells. Upon d-Cys supplementation, the erastin-treated cells remained completely viable for periods of up to 24 h but eventually died after 48 h. d-Cys supplementation suppressed the production of lipid peroxides, thereby ferroptosis. The addition of d-Cys sustained intracellular Cys and GSH levels to a certain extent. When Hepa 1-6 cells were treated with a combination of buthionine sulfoximine and erastin, the anti-ferroptotic effect of d-Cys was diminished. These collective results indicate that, although d-Cys is not the direct source of GSH, d-Cys supplementation protects cells from ferroptosis in a manner that is dependent on GSH synthesis via stimulating the uptake of l-Cys.

The concerted elevation of conjugation reactions is associated with the aggravation of acetaminophen toxicity in Akr1a-knockout mice with an ascorbate insufficiency.

AIMS: Acetaminophen (APAP) is a relatively safe analgesic drug, but overdosing can cause acute liver failure. Ingested APAP is detoxified by metabolic conversion through conjugation reactions with glucuronate, sulfate, or glutathione (GSH). The consumption of GSH through conjugation as well as mitochondrial dysfunction is considered to be responsible for the increased susceptibility to APAP-induced hepatotoxicity. Compared to wild-type (WT) mice, Akr1a-knockout (KO) mice are vulnerable to developing hepatotoxicity due to the fact that ascorbate synthesis is attenuated. We used such KO mice to investigate how these conjugation reactions are involved in the hepatotoxicity caused by an overdose of APAP under ascorbate-deficient conditions. MAIN METHODS: APAP (400 mg/kg) was intraperitoneally administered to WT mice and KO mice. In addition to histological and blood biochemical analyses, metabolites in the liver, blood plasma, and urine were measured at several time points by liquid chromatography-mass spectrometry. KEY FINDINGS: Liver damage occurred earlier in the KO mice than in the WT mice. The levels of APAP-Cys, a final metabolite of GSH-conjugated APAP, as well as glucuronidated APAP and sulfated APAP were all higher in the KO mice compared to the WT mice. Treatment of the APAP-administered KO mice with N-acetylcysteine or supplementation of ascorbate suppressed the conjugation reactions at 6 h after APAP had been administrated, which mitigated the degree of liver damage. SIGNIFICANCE: An ascorbate deficiency coordinately stimulates conjugation reactions of APAP, which, combined with the mitochondrial damage caused by APAP metabolites, collectively results in the aggravation of the acute liver failure.

Methionine Deprivation Reveals the Pivotal Roles of Cell Cycle Progression in Ferroptosis That Is Induced by Cysteine Starvation.

Ferroptosis, a type of iron-dependent necrotic cell death, is triggered by the accumulation of excessive lipid peroxides in cells. Glutathione (GSH), a tripeptide redox molecule that contains a cysteine (Cys) unit in the center, plays a pivotal role in protection against ferroptosis. When the transsulfuration pathway is activated, the sulfur atom of methionine (Met) is utilized to generate Cys, which can then suppress Cys-starvation-induced ferroptosis. In the current study, we cultured HeLa cells in Met- and/or cystine (an oxidized Cys dimer)- deprived medium and investigated the roles of Met in ferroptosis execution. The results indicate that, in the absence of cystine or Met, ferroptosis or cell cycle arrest, respectively, occurred. Contrary to our expectations, however, the simultaneous deprivation of both Met and cystine failed to induce ferroptosis, although the intracellular levels of Cys and GSH were maintained at low levels. Supplementation with S-adenosylmethionine (SAM), a methyl group donor that is produced during the metabolism of Met, caused the cell cycle progression to resume and lipid peroxidation and the subsequent induction of ferroptosis was also restored under conditions of Met/cystine double deprivation. DNA methylation appeared to be involved in the resumption in the SAM-mediated cell cycle because its downstream metabolite S-adenosylhomocysteine failed to cause either cell cycle progression or ferroptosis to be induced. Taken together, our results suggest that elevated lipid peroxidation products that are produced during cell cycle progression are involved in the execution of ferroptosis under conditions of Cys starvation.

Peroxisome proliferator-activated receptor δ rescues xCT-deficient cells from ferroptosis by targeting peroxisomes.

Ferroptosis is a recently recognized process of cell death characterized by accumulation of iron-dependent lipid peroxides. Herein, we demonstrate that peroxisome proliferator-activated receptor δ (PPARδ) inhibits ferroptosis of mouse embryonic fibroblasts (MEFs) derived from cysteine/glutamate transporter (xCT)-knockout mice. Activation of PPARδ by the specific ligand GW501516 led to a dose-dependent decrease in ferroptotic cell death triggered by xCT deficiency, along with decreased levels of intracellular iron accumulation and lipid peroxidation. These effects of GW501516 were abolished by PPARδ-targeting small interfering RNA (siRNA) and the PPARδ inhibitor GSK0660, indicating that PPARδ inhibits xCT deficiency-induced ferroptosis. In addition, GW501516-activated PPARδ time- and dose-dependently upregulated catalase expression at both the mRNA and protein levels. This PPARδ-mediated upregulation of catalase was markedly attenuated in cells treated with PPARδ-targeting siRNA and GSK0660, indicating that expression of catalase is dependent on PPARδ. Consistently, the effects of GW501516 on ferroptosis of xCT-deficient MEFs were counteracted in the presence of 3-amino-1,2,4-triazole, a specific inhibitor of catalase, suggesting that catalase is essential for the effect of PPARδ on ferroptosis triggered by xCT deficiency. GW501516-activated PPARδ stabilized peroxisomes through catalase upregulation by targeting peroxisomal hydrogen peroxide-mediated lysosomal rupture, which led to ferroptosis of xCT-deficient MEFs. Collectively, these results demonstrate that PPARδ modulates ferroptotic signals in xCT-deficient MEFs by regulating catalase expression.

Ascorbate is a multifunctional micronutrient whose synthesis is lacking in primates.

Ascorbate (vitamin C) is an essential micronutrient in primates, and exhibits multiple physiological functions. In addition to antioxidative action, ascorbate provides reducing power to α-ketoglutarate-dependent non-heme iron dioxygenases, such as prolyl hydroxylases. Demethylation of histones and DNA with the aid of ascorbate results in the reactivation of epigenetically silenced genes. Ascorbate and its oxidized form, dehydroascorbate, have attracted interest in terms of their roles in cancer therapy. The last step in the biosynthesis of ascorbate is catalyzed by l-gulono-γ-lactone oxidase whose gene Gulo is commonly mutated in all animals that do not synthesize ascorbate. One common explanation for this deficiency is based on the increased availability of ascorbate from foods. In fact, pathways for ascorbate synthesis and the detoxification of xenobiotics by glucuronate conjugation share the metabolic processes up to UDP-glucuronate, which prompts another hypothesis, namely, that ascorbate-incompetent animals might have developed stronger detoxification systems in return for their lack of ability to produce ascorbate, which would allow them to cope with their situation. Here, we overview recent advances in ascorbate research and propose that an enhanced glucuronate conjugation reaction may have applied positive selection pressure on ascorbate-incompetent animals, thus allowing them to dominate the animal kingdom.

LAT1 inhibitor JPH203 sensitizes cancer cells to radiation by enhancing radiation-induced cellular senescence.

L-type amino acid transporter 1 (LAT1) is important for transporting neutral amino acids into cells. LAT1 expression is correlated with cancer malignancy, suggesting that LAT1 is a promising target for cancer therapy. JPH203, a potential novel drug targeting LAT1, has been shown to suppress tumor growth in various cancer cell lines. However, a combination study of JPH203 and radiation therapy has not been reported. Here, we examined the effects of JPH203 on radiosensitivity after irradiation in A549 and MIA Paca-2 cells. We showed that X-irradiation increased cellular neutral amino acid uptake via LAT1 in both cell lines. JPH203 inhibited the radiation-induced increase in neutral amino acid uptake. We demonstrated that JPH203, at minimally toxic concentrations, significantly sensitized cancer cells to radiation. JPH203 significantly downregulated mTOR activity and enhanced cellular senescence post-irradiation without reducing ATP and GSH levels. These results indicate that LAT1 inhibition by JPH203 sensitizes cancer cells to radiation by enhancing cellular senescence via mTOR downregulation. Thus, JPH203 may be a potent anti-cancer drug in combination with radiation therapy.

Nitric oxide protects against ferroptosis by aborting the lipid peroxidation chain reaction.

Ferroptosis is a type of iron-dependent necrotic cell death, which is typically triggered by the depletion of intracellular glutathione (GSH), which is associated with increased lipid peroxidation. Nitric oxide (NO) is a highly reactive gaseous radical mediator with anti-oxidation properties that terminates lipid peroxidation reactions. In the current study, we report the anti-ferroptotic action of NOC18, an NO donor that spontaneously releases NO, in cells under various ferroptotic conditions in vitro. Our results indicate that, when mouse hepatoma Hepa 1-6 cells are incubated with NOC18, cell death induced by various ferroptotic stimuli such as cysteine (Cys) starvation, the inhibition of glutathione peroxidase 4 (GPX4) and treatment with tertiary-butyl hydroperoxide (TBHP) is significantly reduced. Treatment with NOC18 failed to improve the decrease in the levels of Cys or GSH and the accumulation of ferrous iron upon ferroptotic stimuli. The fluorescent intensity of C11-BODIPY581/591, a probe that is used to detect lipid peroxidation products, was increased somewhat by treatment with NOC18 under conditions of Cys starvation, and the accumulation of lipid peroxidation end-products, as evidenced by the levels of 4-hydroxynonenal, were effectively suppressed. The pre-incubation of TBHP with NOC7, a short-lived NO donor completely eliminated its ability to trigger ferroptosis. These collective results indicate that NO exerts a cytoprotective action against various ferroptotic stimuli by aborting the lipid peroxidation chain reaction.

Carnosine dipeptidase II (CNDP2) protects cells under cysteine insufficiency by hydrolyzing glutathione-related peptides.

The knockout (KO) of the cystine transporter xCT causes ferroptosis, a type of iron-dependent necrotic cell death, in mouse embryonic fibroblasts, but this does not occur in macrophages. In this study, we explored the gene that supports cell survival under a xCT deficiency using a proteomics approach. Analysis of macrophage-derived peptides that were tagged with iTRAQ by liquid chromatography-mass spectrometry revealed a robust elevation in the levels of carnosine dipeptidase II (CNDP2) in xCT KO macrophages. The elevation in the CNDP2 protein levels was confirmed by immunoblot analyses and this elevation was accompanied by an increase in hydrolytic activity towards cysteinylglycine, the intermediate degradation product of glutathione after the removal of the γ-glutamyl group, in xCT KO macrophages. Supplementation of the cystine-free media of Hepa1-6 cells with glutathione or cysteinylglycine extended their survival, whereas the inclusion of bestatin, an inhibitor of CNDP2, counteracted the effects of these compounds. We established CNDP2 KO mice by means of the CRISPR/Cas9 system and found a decrease in dipeptidase activity in the liver, kidney, and brain. An acetaminophen overdose (350 mg/kg) showed not only aggravated hepatic damage but also renal injury in the CNDP2 KO mice, which was not evident in the wild-type mice that were receiving the same dose. The aggravated renal damage in the CNDP2 KO mice was consistent with the presence of abundant levels of CNDP2 in the kidney, the organ prone to developing ferroptosis. These collective data imply that cytosolic CNDP2, in conjugation with the removal of the γ-glutamyl group, recruits Cys from extracellular GSH and supports redox homeostasis of cells, particularly in epithelial cells of proximal tubules that are continuously exposed to oxidative insult from metabolic wastes that are produced in the body.

Pleiotropic Actions of Aldehyde Reductase (AKR1A).

We provide an overview of the physiological roles of aldehyde reductase (AKR1A) and also discuss the functions of aldose reductase (AKR1B) and other family members when necessary. Many types of aldehyde compounds are cytotoxic and some are even carcinogenic. Such toxic aldehydes are detoxified via the action of AKR in an NADPH-dependent manner and the resulting products may exert anti-diabetic and anti-tumorigenic activity. AKR1A is capable of reducing 3-deoxyglucosone and methylglyoxal, which are reactive intermediates that are involved in glycation, a non-enzymatic glycosylation reaction. Accordingly, AKR1A is thought to suppress the formation of advanced glycation end products (AGEs) and prevent diabetic complications. AKR1A and, in part, AKR1B are responsible for the conversion of d-glucuronate to l-gulonate which constitutes a process for ascorbate (vitamin C) synthesis in competent animals. AKR1A is also involved in the reduction of S-nitrosylated glutathione and coenzyme A and thereby suppresses the protein S-nitrosylation that occurs under conditions in which the production of nitric oxide is stimulated. As the physiological functions of AKR1A are currently not completely understood, the genetic modification of Akr1a could reveal the latent functions of AKR1A and differentiate it from other family members.