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Peroxiredoxin 4 (Prdx4) is responsible for the oxidative folding of new proteins that are synthesized in the endoplasmic reticulum (ER). It has recently been suggested that increased ER stress is associated with neurodegenerative diseases, including Alzheimer's disease. Prdx4 is widely distributed throughout the brain, and is also expressed in hippocampal neurons and oligodendrocytes, suggesting that it is associated with learning and memory. We previously established Prdx4-knockout (KO) mice but did not examine the behavioral phenotypes. In the present study, we report on the learning and memory abilities of Prdx4-KO mice based on Morris water maze and the Y-maze tests. The findings indicate that Prdx4-KO mice showed a lower spatial memory ability in both tests. In contrast, the results of the open field test indicated that locomotor activity is significantly increased in Prdx4-KO mice. We then performed mRNA analyses of the brains of Prdx4-KO mice and found an increased expression of genes related to the ER-associated degradation (ERAD) mechanism, which is an important protein quality control system for the maintenance of ER homeostasis. Finally, proteomic analyses of the brains of Prdx4-KO mice showed an aberrant expression in the proteins, which have been suggested to be related to calcium homeostasis and synaptogenesis in neurons. Our collective results suggest that the Prdx4 ablation perturbs oxidative protein folding in the ER, thus leading to aberrant ER homeostasis in neuronal cells, ultimately leading to impaired spatial memory formation.

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.

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.

The defining characteristic of prion diseases is conversion of a cellular prion protein (PrPC) to an abnormal prion protein (PrPSc). The exogenous attachment of PrPSc to the surface of a target cell is critical for infection. However, the initial interaction of PrPSc with the cell surface is poorly characterized. In the current study, we specifically focused on the association of PrPSc with cells during the early phase of infection, using an acute infection model. First, we treated mouse neuroblastoma N2a-58 cells with prion strain 22 L-infected brain homogenates and revealed that PrPSc was associated with membrane fractions within three hours, a short exposure time. These results were also observed in PrPC-deficient hippocampus cell lines. We also demonstrate here that PrPSc from 22 L-infected brain homogenates was associated with lipid rafts during the early phase of infection. Furthermore, we revealed that DS500, a glycosaminoglycan mimetic, inhibited both the attachment of PrPSc to membrane fractions and subsequent prion transmission, suggesting that the early association of prions with cell surface is important for prion infection.

Superoxide dismutase 1 (Sod1) plays pivotal roles in antioxidation via accelerating the conversion of superoxide anion radicals into hydrogen peroxide, thus inhibiting the subsequent radical chain reactions. While Sod1 deficient cells inevitably undergo death in culture conditions, Sod1-knockout (KO) mice show relatively mild phenotypes and live approximately two years. We hypothesized that the presence of abundant levels of ascorbic acid (AsA), which is naturally produced in mice, contributes to the elimination of reactive oxygen species (ROS) in Sod1-KO mice. To verify this hypothesis, we employed mice with a genetic ablation of aldehyde reductase (Akr1a), an enzyme that is involved in the biosynthesis of AsA, and established double knockout (DKO) mice that lack both Sod1 and Akr1a. Supplementation of AsA (1.5 mg/ml in drinking water) was required for the DKO mice to breed, and, upon terminating the AsA supplementation, they died within approximately two weeks regardless of age or gender. We explored the etiology of the death from pathophysiological standpoints in principal organs of the mice. Marked changes were observed in the lungs in the form of macroscopic damage after the AsA withdrawal. Histological and immunological analyses of the lungs indicated oxidative damage of tissue and activated immune responses. Thus, preferential oxidative injury that occurred in pulmonary tissues appeared to be primary cause of the death in the mice. These collective results suggest that the pivotal function of AsA in coping with ROS in vivo, is largely in pulmonary tissues that are exposed to a hyperoxygenic microenvironment.

The ubiquitin-proteasome system (UPS) and autophagy are two major intracellular proteolytic systems that are closely associated with each other. Because UPS and autophagy are involved in the clearance of oxidised and/or aggregated proteins, it would be logical to assume that alterations in proteolysis would accompany pathological conditions. Indeed, both systems are themselves susceptible to oxidative modification and therefore could be a prominent target of reactive oxygen species (ROS). Oxidative stress appears to be a common underlying factor in the development of and the pathogenesis of various metabolic diseases, including non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes (T2D). Recent studies, using obesity and hyperglycaemia model mice, reported that both UPS and autophagy systems are inhibited in these mice and that this inhibition is accompanied by lipid accumulation, insulin resistance, and tissue damage. However, the detailed molecular mechanisms that are responsible for regulating intracellular proteolysis in metabolic diseases are not well understood. In the current review, we discuss the correlation between oxidative stress, defective proteolysis, and metabolic diseases. An understanding of how ROS affects intracellular proteolysis may provide new perspectives on the development of and control of diseases.

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.

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.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.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.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.

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.

New Zealand white (NZW) mouse is a mutant strain that has a larval defect in the immune system, and a F1 hybrid between NZW and New Zealand Black mouse spontaneously develops systemic lupus erythematosus (SLE). In meantime, the ablation of superoxide dismutase 1 (Sod1) causes autoimmune haemolytic anaemia, a clinical condition of SLE, in mice with a C57BL/6 background. On the basis of our previous studies, we hypothesised that oxidative stress may trigger this aberrant autoimmunity in NZW mice without crossing with another strain. To validate this, we attempted to establish Sod1−/−/NZW mice but this attempt failed to obtain any objective mouse. The congenic Sod1+/−/NZW male mice were completely infertile because of severe oligozoospermia attributed to a defect in spermatogenesis. The levels of the SOD1 protein were about a half in the testes of the Sod1+/−/NZW mice. Sperm from the Sod1+/−/NZW mice were largely defective and showed quite low fertilising ability in in vitro fertilisation assays. Concomitant with an increase in the oxidatively modified proteins, spermatogenic cells underwent more cell death in the testes of the Sod1+/−/NZW mice compared to those of WT/NZW mice. An examination of immunocompetent cells from Sod1+/−/NZW mice indicated an abnormality in T-cell responses. These collective results suggest that the oxidative stress caused by an SOD1 haploinsufficiency exerts deleterious effects on the testis, either directly on spermatogenic cells or via the destabilisation of the autoimmune response in Sod1+/−/NZW mice.

Free iron has long been assumed to be a deteriorating factor in an oxidative insult and was recently found to be directly associated with ferroptosis, a specific type of cell death. The free iron-involved production of lipid peroxides activates the fatal pathway, resulting in nonapoptotic, programed cell death. Lipid peroxides appear to destroy membrane integrity, leading to cell rupture. Glutathione (GSH) is a major redox molecule that functions to protect against ferroptosis by its ability to donate an electron to glutathione peroxidase 4 (GPX4), the sole enzyme that reduces phospholipid hydroperoxides. The availability of free cysteine (Cys) determines the levels of GSH synthesis, and, hence, its deprivation causes ferroptosis. Free iron is provided via ferritinophagy, the chaperone-mediated autophagic degradation of ferritin, but GPX4 also undergoes degradation via chaperone-mediated autophagy. Activated Nrf2 and ATF4 induce the expression of the cystine transporter xCT to cope with ferroptosis. To the contrary, the excessive activation of p53 induces ferroptosis by suppressing the expression of xCT in genetic and nongenetic manners. It therefore appears that xCT functions as the gatekeeper for determining cellular survival by regulating the availability of Cys in the cell. The issue of the extent of involvement of ferroptosis in an in vivo situation largely remains ambiguous. Establishing tools for specifying ferroptotic cells in situ would facilitate our understanding of its roles in pathogenesis.

Background and Aims Peroxiredoxin 4 (PRDX4), a secretory protein that is preferentially retained in the endoplasmic reticulum (ER), is encoded by a gene located on the X chromosome and highly expressed in colonic tissue. In this study, we investigated the role of PRDX4 by means of male PRDX4-knockout (PRDX4-/y) mice in the development of intestinal inflammation using a dextran sulfate sodium (DSS)-induced colitis model. Materials and methods Acute colitis was induced with DSS (2.5% in drinking water) in wild-type (WT) and PRDX4-/y male C57BL/6 mice. Histological and biochemical analyses were performed on the colonic tissues. Results PRDX4 was mainly localized in the colonic epithelial cells in WT mice. The disease activity index (DAI) scores of PRDX4-/y mice were significantly higher compared to those of WT mice. Specifically, PRDX4-/y mice showed marked body weight loss and shortening of colon length compared to WT mice, whereas the myeloperoxidase levels were increased in PRDX4-/y compared to WT mice. In addition, the mRNA expression levels of TNF-α and IFN-γ were significantly higher in the colonic mucosa of PRDX4-/y compared to WT mice. Moreover, the levels of CHOP and activated caspase 3 were higher in the colonic tissues of PRDX4-/y compared to WT mice following treatment with DSS. The ER also showed greater expansion in PRDX4-/y than WT mice, which was consistent with severe ER stress under PRDX4 deficiency. Conclusion Our results demonstrated that the lack of PRDX4 aggravated the colonic mucosal damage induced by DSS. Because PRDX4 functions as an ER thiol oxidase as well as an antioxidant, DSS induced oxidative damage and ER stress to a greater degree in PRDX4-/y than WT mice. These findings suggest that PRDX4 may represent a novel therapeutic molecule in intestinal inflammation.

Nitric oxide (NO) reacts with superoxide to produce peroxynitrite, a potent oxidant and reportedly exerts cytotoxic action. Herein we validated the hypothesis that interaction of NO with superoxide exerts protection against superoxide toxicity using macrophages from mice with a knockout (KO) of inducible NO synthase (NOS2) and superoxide dismutase 1 (SOD1), either individually or both. While no difference was observed in viability between wild-type (WT) and NOS2KO macrophages, SOD1KO and SOD1-and NOS2-double knockout (DKO) macrophages were clearly vulnerable and cell death was observed within four days. A lipopolysaccharide (LPS) treatment induced the formation of NOS2, which resulted in NO production in WT and these levels were even higher in SOD1KO macrophages. The viability of the DKO macrophages but not SOD1KO macrophages were decreased by the LPS treatment. Supplementation of NOC18, a NO donor, improved the viability of SOD1KO and DKO macrophages both with and without the LPS treatment. The NOS2 inhibitor nitro-l-arginine methyl ester consistently decreased the viability of LPS-treated SOD1KO macrophages but not WT macrophages. Thus, in spite of the consequent production of peroxynitrite in LPS-stimulated macrophages, the coordinated elevation of NO appears to exert anti-oxidative affects by coping with superoxide cytotoxicity upon conditions of inflammatory stimuli., Highlights • SOD1 deficiency decreases the life span of primary cultured macrophages. • A NO donor prolongs the survival of SOD1-deficient macrophages. • NOS2 inhibition shortens the life span of SOD1-deficient macrophages. • NO reduces superoxide cytotoxicity under conditions of an SOD1 deficiency.

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.

Ferroptosis is a type of iron-dependent, non-apoptotic cell death, which is typically induced by cysteine starvation or by the inhibition of glutathione peroxidase 4 (GPX4) activity with the accompanying elevation in lipid peroxidation product levels. Despite the central role of mitochondria in oxidative metabolism and hence, as main sources of superoxide, the issue of whether mitochondrial superoxide participates in the execution of ferroptosis remains unclear. To gain additional insights into this issue, we employed suppressors of the site IQ electron leak (S1QEL) and suppressors of the site IIIQo electron leak (S3QEL), small molecules that suppress mitochondrial superoxide production from complex I and III, respectively. The findings indicate that S3QEL, but not S1QEL, significantly protected mouse hepatoma Hepa 1–6 cells from lipid peroxidation and the subsequent ferroptosis induced by cysteine (Cys) starvation (cystine deprivation from culture media or xCT inhibition by erastin). The intracellular levels of Cys and GSH remained low irrespective of life or death. Moreover, S3QEL also suppressed ferroptosis in xCT-knockout mouse-derived embryonic fibroblasts, which usually die under conventional cultivating conditions due to the absence of intracellular Cys and GSH. Although it has been reported that erastin induces the hyperpolarization of the mitochondrial membrane potential, no correlation was observed between hyperpolarization and cell death in xCT-knockout cells. Collectively, these results indicate that superoxide production from complex III plays a pivotal role in the ferroptosis that is induced by Cys starvation, suggesting that protecting mitochondria is a promising therapeutic strategy for the treatment of multiple diseases featuring ferroptosis.

PRDX4, a member of peroxiredoxin family, is largely concentrated in the endoplasmic reticulum (ER) and plays a pivotal role in the redox relay during oxidative protein folding as well as in peroxidase reactions. A testis-specific PRDX4 variant transcript (PRDX4t) lacks the conventional exon 1, which encodes the signal peptide that is required for entry into the ER lumen, but instead carries alternative exon 1, which is transcribed from the upstream promoter in a testis-specific manner and results in the PRDX4t protein being localized in the cytosol. However, the potential roles of PRDX4t in male genital action remain unknown. Using a CRISPR/Cas9 system, we first disrupted the testis-specific promoter/exon 1 and generated mice that were specifically deficient in PRDX4t. The resulting PRDX4t knockout (KO) mice underwent normal spermatogenesis and showed no overt abnormalities in the testis. Mating PRDX4t KO male mice with wild-type (WT) female mice produced normal numbers of offspring, indicating that a PRDX4t deficiency alone had no effect on fertility in the male mice. We then generated mice lacking both PRDX4 and PRDX4t by disrupting exon 2, which is communal to these variants. The resulting double knockout (DKO) mice were again fertile, and mature sperm isolated from the epididymis of DKO mice exhibited a normal fertilizing ability in vitro. In the meantime, the protein levels of glutathione peroxidase 4 (GPX4), which plays an essential role in the disulfide bond formation during spermatogenesis, were significantly increased in the testis and caput epididymis of the DKO mice compared with the WT mice. Based on these results, we conclude that the disruption of the function of PRDX4t in the spermatogenic process appears to be compensated by other factors including GPX4.

Ferroptosis is a type of iron-dependent, non-apoptotic cell death, which is typically induced by the suppression of phospholipid hydroperoxide-glutathione peroxidase (GPX4) activity and a corresponding elevation in lipid peroxidation products. While the inhibition of the xCT-mediated uptake of cystine commonly causes ferroptosis, the sensitivity of cells to the inhibition of the glutathione (GSH) synthesis by buthionine sulfoximine (BSO) varies considerably. The objective of this study was to clarify the underlying mechanism responsible for these differential responses to the targeted inhibition of either the cysteine (Cys) supply to or GSH synthesis in the cells. While intracellular GSH levels were depleted when mouse hepatoma Hepa 1-6 cells were treated with BSO, intracellular Cys levels rather increased and viability of the cells remained unchanged, suggesting that Cys has a role in this resistance to ferroptosis under conditions of GSH depletion. When the cells were treated with pioglitazone (PGZ), a potent inhibitor of CDGSH iron sulphur domain-containing proteins (CISDs), PGZ alone had no effect on either cell viability or GSH levels but induced ferroptosis under conditions of GSH depletion by the BSO treatment. In the case of the co-treatment with PGZ and BSO, ferrous iron and the levels of lipid peroxides were robustly increased in the cells, but neither endoplasmic reticulum stress nor apoptosis was evident. Collectively, CISDs appeared to exert an anti-ferroptotic function by suppressing free iron toxicity and the subsequent lipid peroxidation with assistance provided by Cys.

Using cell culture and animal models of prion diseases, Ishibashi et al. show that type I interferon signalling interferes with prion infection in mammals. A selective type I interferon receptor agonist inhibits prion invasion and prolongs survival of prion-infected mice, suggesting potential clinical applications., Infectious prions comprising abnormal prion protein, which is produced by structural conversion of normal prion protein, are responsible for transmissible spongiform encephalopathies including Creutzfeldt-Jakob disease in humans. Prions are infectious agents that do not possess a genome and the pathogenic protein was not thought to evoke any immune response. Although we previously reported that interferon regulatory factor 3 (IRF3) was likely to be involved in the pathogenesis of prion diseases, suggesting the protective role of host innate immune responses mediated by IRF3 signalling, this remained to be clarified. Here, we investigated the reciprocal interactions of type I interferon evoked by IRF3 activation and prion infection and found that infecting prions cause the suppression of endogenous interferon expression. Conversely, treatment with recombinant interferons in an ex vivo model was able to inhibit prion infection. In addition, cells and mice deficient in type I interferon receptor (subunit interferon alpha/beta receptor 1), exhibited higher susceptibility to 22L-prion infection. Moreover, in in vivo and ex vivo prion-infected models, treatment with RO8191, a selective type I interferon receptor agonist, inhibited prion invasion and prolonged the survival period of infected mice. Taken together, these data indicated that the interferon signalling interferes with prion propagation and some interferon-stimulated genes might play protective roles in the brain. These findings may allow for the development of new strategies to combat fatal diseases.

AKR1A, an aldo-keto reductase, is involved in the synthesis of ascorbic acid as well as the reduction of a variety of aldehyde compounds. AKR1A−/− mice produce considerably less ascorbic acid (about 10%) compared to AKR1A+/+ mice and require ascorbic acid supplementation in order to breed. To elucidate the roles played by AKR1A in spatial memory, AKR1A−/− male mice were weaned at 4 weeks of age and groups that received ascorbic acid supplementation and no supplementation were subjected to a Morris water maze test. Juvenile AKR1A−/− mice that received no supplementation showed impaired spatial memory formation, even though about 70% of the ascorbic acid remained in the brains of the AKR1A−/− mice at day 7 after weaning. To the contrary, the young adult AKR1A−/− mice at 13–15 weeks of age maintained only 15% of ascorbic acid but showed no significant difference in the spatial memory compared with the AKR1A+/+ mice or ascorbic acid-supplemented AKR1A−/− mice. It is conceivable that juvenile mice require more ascorbic acid for the appropriate level of formation of spatial memory and that maturation of the neural system renders the memory forming process less sensitive to an ascorbic acid insufficiency.

Aldehyde reductase (Akr1a) has been reported to be involved in detoxification of reactive aldehydes as well as in the synthesis of bioactive compounds such as ascorbic acid (AsA). Because Akr1a is expressed at high levels in the liver and is involved in xenobiotic metabolism, our objective was to investigate the hepato-protective role of Akr1a in a thioacetamide (TAA)-induced hepatotoxicity model using Akr1a-deficient (Akr1a−/−) mice. Wild-type (WT) and Akr1a−/− mice were injected intraperitoneally with TAA and the extent of liver injury in the acute phase was assessed. Intriguingly, the extent of TAA-induced liver damage was less in the Akr1a−/− mice than in the WT mice. Biomarkers for the ER stress-induced apoptosis pathway were markedly decreased in the livers of Akr1a−/− mice, whereas AsA levels in plasma did not change significantly in any of the mice. In the liver, TAA is converted to reactive metabolites such as TAA S-oxide and then to TAA S, S-dioxide via the action of CYP2E1. In Akr1a−/− mice, CYP2E1 activity was relatively lower than WT mice at the basal level, leading to reactive TAA metabolites being produced at lower levels after the TAA treatment. The levels of liver proteins that were modified with these metabolites were also lower in the Akr1a−/− mice than the WT mice after the TAA treatment. Furthermore, after a lethal dose of a TAA challenge, the WT mice all died within 36 h, whereas almost all of the Akr1a−/− mice survived. These collective results suggest that Akr1a−/− mice are resistant to TAA-induced liver injury, and it follows that the absence of Akr1a might modulate TAA bioactivation.

The amino acid transport system xc− is important for maintaining intracellular glutathione levels and extracellular redox balance. The main component of system xc−, xCT, is strongly induced by various stimuli, including oxidative stress and bacterial lipopolysaccharides (LPS) in macrophages. In the present study, we investigated the production of nitric oxide by LPS-stimulated mouse peritoneal macrophages isolated from both xCT-deficient and wild-type mice. After culturing macrophages in the presence of LPS for 24–48 h, nitrite levels in the medium of xCT-deficient macrophages were significantly decreased compared to that of wild-type cells. However, the transport activity of arginine, a precursor of nitric oxide, and the expression of nitric oxide synthase 2 in xCT-deficient macrophages were similar to those of wild-type cells. When wild-type macrophages were cultured in the medium that contained no cystine, nitric oxide production was decreased to the level similar to that of the xCT-deficient macrophages. When xCT-deficient macrophages were cultured with 2-mercaptoethanol, intracellular cysteine levels were increased and nitrite accumulation in the medium was significantly increased. On the other hand, when these cells were cultured with buthionine sulfoximine, an inhibitor of glutathione synthesis, nitrite accumulation in the medium was essentially unchanged, although intracellular glutathione levels were very low. Reactive oxygen species levels in xCT-deficient macrophages were higher than those of wild-type cells, and treatment with LPS caused an increase in oxidative stress in both cells. These results suggest that intracellular cysteine supplied by xCT contributes to nitric oxide production and the reduction of oxidative stress in macrophages.

Extracellular cystine, the oxidized form of cysteine (Cys), is taken up by cells via the cystine transporter xCT. xCT is not expressed in the liver but is induced in primary hepatocytes under conventional cultured conditions. However, compared to wild-type hepatocytes those from the xCT-knockout mouse showed no evidence of an abnormality and the levels of both Cys and glutathione (GSH) remained unchanged. The levels of ophthalmic acid (OPT), which is produced as an alternative compound by the GSH-synthesizing pathway, became increased during the culturing of hepatocytes. It therefore appears that, in primary hepatocytes, Cys is provided by systems other than xCT, most likely via the transsulfuration pathway, but the levels that are produced are not sufficient. We also employed mouse hepatoma-derived Hepa1-6 cells, which constitutively express xCT. When Hepa 1-6 cells were cultivated in Cys-free media, the levels of intracellular Cys and GSH were decreased, compared to cells cultured in conventional media, leading to cell death accompanied by an increase in the levels of reactive oxygen species and lipid peroxidation products with characteristics similar to ferroptosis. While OPT levels were increased by only to a limited extent in Hepa 1-6 cells, primary hepatocytes cultured in Cys- and Met-free media showed a marked elevation in OPT, reaching levels nearly equivalent to the GSH levels when the cells were cultured in conventional media. Thus, OPT may become a marker for Cys insufficiency and might be used to predict pathological conditions of cells with elevated oxidative stress.

Ferroptosis is regulated, non-apoptotic cell death in which ferrous iron and lipid peroxidation products play essential roles. While the ferroptotic pathway is now becoming unveiled, it is difficult to determine its involvement in situ because no unique marker for ferroptotic cells is known. In this study, we report on raising a rat monoclonal antibody against mouse-derived Hepa 1-6 cells that had been cultivated in cystine-deprived media. Binding of the resulting antibody, designated as FerAb, increased during advancing ferroptosis which was caused, not only by cystine deprivation but also treatment with erastin or RSL3, while apoptotic cell death induced by a staurosporine treatment had no effect on the binding. The FerAb was found to bind to 4-hydroxy-2-nonenal (HNE)-modified bovine serum albumin, but no specific protein was detected in ferroptotic cells in an immunoblot analysis. These results indicate that non-proteinaceous, HNE-like structural moiety was part of the antigen for FerAb, although the binding profiles of FerAb to ferroptotic cells were different from those of the currently available anti-HNE antibody. Immunocytological detection revealed inhomogenous staining within cells and partial co-localization with peripheral mitochondria and other cellular components. FerAb was found to be applicable for ferroptotic cells in other mouse cells and cultured human cells that were examined. Thus, the properties of the rat monoclonal antibody FerAb established in this study promise to be useful for the characterization of ferroptotic cell death.

Cellular prion protein (PrP) is a membrane protein that is highly conserved among mammals and mainly expressed on the cell surface of neurons. Despite its reported interactions with various membrane proteins, no functional studies have so far been carried out on it, and its physiological functions remain unclear. Neuronal cell death has been observed in a PrP-knockout mouse model expressing Doppel protein, suggesting that PrP might be involved in Ca

Aldehyde reductase encoded by the Akr1a gene catalyzes the NADPH-dependent reduction of a variety of aldehyde compounds, and it plays a role in the biosynthesis of ascorbic acid (AsA) by converting D-glucuronate to L-gulonate. Although supplementing drinking water with AsA (1.5 mg/mL) ameliorates the fertility of Akr1a −/− (KO) female mice, litter sizes in the KO mice are typically smaller than those for Akr1a +/+ (WT) mice, and about one-third of the neonates have a reduced stature. Half of the neonates in the smallest, developmentally retarded group died before weaning, and the remaining half (less than 6 g in weight) also barely grew to adulthood. While no difference was found in the number of fetuses between the KO and WT mice at 14.5-embryonic days, the sizes of the KO fetuses had already diverged. Among the organs of these retarded KO neonates at 30 d, the spleen and thymus were characteristically small. While an examination of spleen cells showed the normal proportion of immune cells, apoptotic cell death was increased in the thymus, which would lead to thymic atrophy in the retarded KO neonates. Plasma AsA levels were lower in the small neonates despite the fact that their mothers had received sufficient AsA supplementation, and the corticosterone levels were inversely higher compared to wild-type mice. Thus, insufficient AsA contents together with a defect in corticosterone metabolism might be the cause of the retarded growth of the AKR1A-deficient mice embryos and neonates.

Aims Aldehyde reductase (AKR1A) is involved in the synthesis of ascorbic acid (AsA) as well as the detoxification of aldehydes. AKR1A−/− (KO) mice produce about 10% of the normal amounts of AsA compared to AKR1A+/+ (WT) mice. We investigated physiologic roles of AKR1A in running using the KO mice. Main methods The KO mice were subjected to a treadmill test under either restricted AsA production or a sufficiency by supplementation and compared the results with those of WT mice. Contents of glucose, aspartate aminotransferase, AsA and free fatty acids in blood were measured. Glycogen contents were measured in the liver and skeletal muscle, and hepatic proteins were examined by immunoblot analyses. Key findings Running performance was higher in the KO mice than the WT mice irrespective of the AsA status. After the exercise period, blood glucose levels were decreased in the WT mice but were preserved in the KO mice. Liver glycogen levels were also consistently preserved in the KO mice after exercise. Free fatty acid levels tended to be originally high in blood plasma compared to those of the WT mice and were increased to similar extent in them. A key regulator of energy metabolism, PGC-1α, and the products of downstream target genes that encode for glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphatase, were constitutively at high levels in the KO mice. Significance The genetic ablation of AKR1A activates the PGC-1α pathway and spare glucose, which would consequently confer exercise endurance.

Superoxide dismutase 1 suppresses oxidative stress within cells by decreasing the levels of superoxide anions. A dysfunction of the ovary and/or an aberrant production of sex hormones are suspected causes for infertility in superoxide dismutase 1-knockout mice. We report on attempts to rescue the infertility in female knockout mice by providing two antioxidants, ascorbic acid and/or coenzyme Q10, as supplements in the drinking water of the knockout mice after weaning and on an investigation of their reproductive ability. On the first parturition, 80% of the untreated knockout mice produced smaller litter sizes compared with wild-type mice (average 2.8 vs 7.3 pups/mouse), and supplementing with these antioxidants failed to improve these litter sizes. However, in the second parturition of the knockout mice, the parturition rate was increased from 18% to 44–75% as the result of the administration of antioxidants. While plasma levels of progesterone at 7.5 days of pregnancy were essentially the same between the wild-type and knockout mice and were not changed by the supplementation of these antioxidants, sizes of corpus luteum cells, which were smaller in the knockout mouse ovaries after the first parturition, were significantly ameliorated in the knockout mouse with the administration of the antioxidants. Moreover, the impaired vasculogenesis in uterus/placenta was also improved by ascorbic acid supplementation. We thus conclude that ascorbic acid and/or coenzyme Q10 are involved in maintaining ovarian and uterus/placenta homeostasis against insults that are augmented during pregnancy and that their use might have positive effects in terms of improving female fertility.

Patients with nonalcoholic fatty liver disease may subsequently develop nonalcoholic steatohepatitis after suffering from a second insult, such as oxidative stress. Aim of this study was to investigate the pathogenesis of the liver injury caused when lipids accumulate under conditions of intrinsic oxidative stress using mice that are deficient in superoxide dismutase 1 (SOD1) and the leptin receptor (Lepr). We established Sod1-/-::Leprdb/db mice and carried out analyses of four groups of genetically modified mice, namely, wild type, Sod1-/-, Leprdb/db and Sod1-/-::Leprdb/db mice. Mice with defects in the SOD1 or Lepr gene are vulnerable to developing fatty livers, even when fed a normal diet. Feeding a high-fat diet (HFD) caused an increase in the number of lipid droplets in the liver to different extents in each genotypic mouse. an HFD caused the accelerated death of db/db mice, but contradictory to our expectations, the death rates for the Sod1-deficient mice were decreased by feeding HFD. Consistent with the improved probability of survival, liver damage was significantly ameliorated by feeding an HFD compared to a normal diet in the mice with an Sod1-deficient background. Oxidative stress markers, hyperoxidized peroxiredoxin and lipid peroxidation products, were decreased somewhat in Sod1-/- mice by feeding HFD. We conclude that lipids reacted with reactive oxygen species and eliminated them in the livers of the young mice, which resulted in the alleviation of oxidative stress, but in advanced age oxidized products accumulated, leading to the aggravation of the liver injury and an increase in fatality rate.

Aldehyde reductase (AKR1A) plays a role in the biosynthesis of ascorbic acid (AsA), and AKR1A-deficient mice produce about 10-15% of the AsA that is produced by wild-type mice. We found that acetaminophen (AAP) hepatotoxicity was aggravated in AKR1A-deficient mice. The pre-administration of AsA in the drinking water markedly ameliorated the AAP hepatotoxicity in the AKR1A-deficient mice. Treatment of the mice with AAP decreased both glutathione and AsA levels in the liver in the early phase after AAP administration, and an AsA deficiency delayed the recovery of the glutathione content in the healing phase. While in cysteine supply systems; a neutral amino acid transporter ASCT1, a cystine transporter xCT, enzymes for the transsulfuration pathway, and autophagy markers, were all elevated in the liver as the result of the AAP treatment, the AsA deficiency suppressed their induction. Thus, AsA appeared to exert a protective effect against AAP hepatotoxicity by ameliorating the supply of cysteine that is available for glutathione synthesis as a whole. Because some drugs produce reactive oxygen species, resulting in the consumption of glutathione during the metabolic process, the intake of sufficient amounts of AsA would be beneficial for protecting against the hepatic damage caused by such drugs.

Proteasomes play a key role in maintaining cellular homeostasis by the proteolytic removal of proteins, including ubiquitinated proteins and/or oxidatively-damaged proteins. The proteasome inhibitor bortezomib (BTZ) has been reported to exert testicular toxicity in mice. In the current study, we treated SOD1-knockout (KO) mice with BTZ and investigated the issue of whether oxidative stress is involved in the development of testicular toxicity. The BTZ treatment significantly increased superoxide production and cell death in the testes of SOD1-KO mice compared to wild-type (WT) mice. We also found that high levels of both ubiquitinated proteins and p62 accumulated and underwent aggregation in the seminiferous tubules of BTZ-injected SOD1-KO mice. Furthermore, the proteolytic activities of proteasomes were significantly decreased in the testes of BTZ-injected SOD1-KO mice compared to their WT counterparts. These results suggest that a combination of oxidative stress caused by an SOD1 deficiency and proteasome inhibition by BTZ accelerates the impairment of proteasomes, which results in severe testicular damage in SOD1-KO mice.

The two major proteolysis systems that are responsible for the clearance of aggregated proteins and damaged organelles in eukaryotic cells are the ubiquitin–proteasome system (UPS) and autophagy. Both UPS and autophagy themselves are susceptible to oxidative modification and therefore could be a prominent target of reactive oxygen species (ROS). Elevated ROS levels in the liver appear to be a common underlying factor in the development and pathogenesis of nonalcoholic fatty liver disease (NAFLD). Recent studies, using NAFLD model mice, have reported that these two systems are inhibited in the liver and that this inhibition is accompanied by lipid accumulation, insulin resistance, and liver damage. However, the detailed molecular mechanisms that are responsible for regulating intracellular proteolysis in NAFLD are not well understood. In the current review, we discuss the correlation between oxidative stress with intracellular proteolysis and NAFLD. An understanding of how ROS affects intracellular proteolysis in NAFLD may provide new perspectives on disease development and control.

Iron-mediated lipid peroxidation has a primary role in ferroptosis. Among a variety of antioxidative systems, glutathione peroxidase 4 (GPX4) is a potent protective enzyme against ferroptosis. Suppling reducing equivalents to GPX4 has been found to be important in terms of the function of glutathione from the standpoint of cell survival against ferroptosis. A cysteine deficiency results in elevated levels of free iron via ferritinophagy, resulting in a decrease in GPX4 activity caused by a decrease in glutathione production. Ferritinophagy releases redox-inactive ferric iron, which is then converted to redox-active ferrous iron by accepting an electron from radical compounds and results in lipid peroxidation. In many cases the availability of cysteine can hold the key to determining cell fate in vivo. The electron used in the lipid peroxidation reaction appears to be from the mitochondrial electron transport system under conditions of a cysteine deficiency but can also be obtained from other sources, depending on the circumstances.

Ppm1b, a metal‑dependent serine/threonine protein phosphatase, catalyzes the dephosphorylation of a variety of phosphorylated proteins. Ppm1b‑/‑ mouse embryos die at the fertilized oocyte stage, whereas Ppm1b+/‑ mice with a C57BL/6 background exhibit no phenotypic abnormalities. Because the C57BL/6 strain produces a limited number of pups, in an attempt to produce Ppm1b‑/‑ mice, congenic Ppm1b+/‑ mice with an ICR background were established, which are more fertile and gave birth to more pups. As a result, however, no Ppm1b‑/‑ offspring were obtained when pairs of Ppm1b+/‑ ICR mice were bred again. Ppm1b+/‑ male and female ICR mice were analyzed from the viewpoint of fecundity. The Ppm1b haploinsufficiency had no effect on testicular weight or the number of sperm in male mice. Despite the fact that the levels of Ppm1b protein in the ovaries of sexually mature Ppm1b+/‑ mice were decreased compared with those of Ppm1b+/+ mice, there appeared to be no significant difference in the histological appearance of the ovaries, litter sizes or plasma progesterone levels at the estrous stage. When superovulation was induced by stimulation using a hormone treatment, the number of ovulated oocytes were the same for Ppm1b+/‑ and Ppm1b+/+ mice at 4 weeks of age when the estrous cycle did not proceed, however, the number of ovulated oocytes was lower in sexually mature Ppm1b+/‑ mice at 11 weeks of age compared with Ppm1b+/+ mice in the first and the second superovulation cycles. These collective results suggest that follicle development is excessive in Ppm1b+/‑ mice, and that this leads to a partial depletion of matured follicles and a corresponding decrease in the number of ovulated oocytes.

Oxidative stress triggers the formation of lipid droplets in the liver by stimulating lipogenesis and simultaneously suppresses lipoprotein secretion under hypernutritional conditions. Herein we report on the observation of systemic organ failure that is associated with lipid droplet accumulation in fasting, SOD1-knockout (KO) mice. Upon a three-day fasting period, the KO mice were observed to be vulnerable, could not be rescued by refeeding and had largely died, while wild-type mice were totally recovered. Visceral fat was rapidly consumed during fasting, which resulted in energy shortage and increased fatality in the KO mice. Lipid droplets had accumulated and continued to remain in KO mouse organs that routinely catalyze fatty acids via β-oxidation, even though the levels of free fatty acids and β-hydroxybutyrate, a ketone body, in blood plasma were less in KO mice compared to WT mice during the fasting period. The fasting-triggered organ failure in the KO mice was effectively mitigated by feeding a high calorie-diet for 2 weeks prior to fasting, even though the mice had an excessive accumulation of lipid droplets in the liver. These collective data suggest that the lipid-catabolizing system is the sensitive target of oxidative stress triggered by fasting conditions in the KO mice.

Peroxiredoxin 4 (PRDX4) is a member of the peroxiredoxin family of antioxidant enzymes. Previously, we reported that PRDX4 can restrain the initiation and progression of nonalcoholic steatohepatitis by reducing local and systemic reactive oxygen species (ROS) levels. Oxidative stress is recognized as a key factor in hepatocarcinogenesis, and a high ROS level has also been found in hepatocellular carcinoma (HCC). Here, our aim is to investigate roles of PRDX4 in the initiation and progression of HCC.In this study, for hepatocarcinogenesis, wild-type (WT), PRDX4 knockout (PRDX4

Glutathione (GSH), an abundant tripeptidyl molecule, plays pivotal roles in protecting cells against oxidative stress-induced cellular damage and in detoxifying xenobiotics and drug metabolism. GSH is now entering a new era of therapeutic applications. Decreased GSH levels are associated with the common features of aging as well as of a wide range of pathological conditions, including neurodegenerative disorders. Notably, GSH depletion and/or alterations in its metabolism appear to be crucial in the onset of Parkinson's disease. Despite the fact that GSH is required for cell survival, the molecular mechanism that links GSH depletion to cell death remains poorly understood. Recently, considerable attention has been focused on a newly defined type of cell death: irondependent cell death, also referred to as "ferroptosis". The iron chelator deferoxamine nearly abolishes ferroptosis induced by inhibiting GSH synthesis or cystine uptake by the xCT transporter. Deferoxamine preferentially abrogates the intralysosomal accumulation of iron and inhibits oxidative stress-induced lysosomal membrane permeabilization and cell death. The use of GSH and a prodrug derived from it can be useful, since the dysfunction of the GSH redox system appears to cause a variety of diseases including neurodegenerative disorders. However, the effectiveness of GSH as a therapeutic agent is limited because of its low bioavailability. We also review trials that have been designed to cope with this difficulty; e.g. the use of precursors such as N-acetyl cysteine and chemical modification such as methylation.

We previously demonstrated that elevated levels of ROS in red blood cells (RBCs) are responsible for anemia in SOD1-deficient mice, suggesting that the oxidative stress-induced massive destruction of RBCs is an underlying mechanism for autoimmune hemolytic anemia. In the current study, we examined the issue of how elevated ROS are involved in the destruction of RBCs and the onset of anemia from the view point of the proteolytic removal of oxidatively-damaged proteins. We found that poly-ubiquitinated proteins had accumulated and had undergone aggregation in RBCs from SOD1-deficient mice and from phenylhydrazine-induced anemic mice. Although the protein levels of the three catalytic components of the proteasome, β1, β2, and β5, were not significantly altered, their proteolytic activities were decreased in the SOD1-deficient RBCs. These data suggest that oxidative-stress triggers the dysfunction of the proteasomal system, which results in the accumulation of the aggregation of poly-ubiquitinated proteins. We conclude that an oxidative stress-induced malfunction in the scavenging activity of proteasomes accelerates the accumulation of damaged proteins, leading to a shortened lifespan of RBCs and, hence, anemia.

A deficiency of superoxide dismutase 1 (SOD1) or peroxiredoxin (Prx) 2 causes anemia in mice due to elevated oxidative stress. In the current study, we investigated whether intrinsic oxidative stress caused by a SOD1 deficiency affected the redox status of Prx2 and other isoforms in red blood cells (RBCs) and several organs of mice. We observed a marked elevation in hyperoxidized Prx2 levels in RBCs from SOD1-deficient mice. Hyperoxidized Prx2 reportedly undergoes a rhythmic change in isolated RBCs under culture conditions. We confirmed such changes in RBCs from wild-type mice but observed no evident changes in SOD1-deficient RBCs. In addition, an elevation in hyperoxidized Prxs, notably Prx2 and Prx3, was observed in several organs from SOD1-deficient mice. However, a SOD1 deficiency had no impact on the wheel-running activity of the mice. Thus, although the redox status of some Prxs is systemically shifted to a more oxidized state as the result of a SOD1 deficiency, which is associated with anemia and some diseases, a redox imbalance appears to have no detectable effect on the circadian activity of mice.

The cystine/glutamate transporter, designated as system xc−, is important for maintaining intracellular glutathione levels and extracellular redox balance. The substrate-specific component of system xc−, xCT, is strongly induced by various stimuli, including oxidative stress, whereas it is constitutively expressed only in specific brain regions and immune tissues, such as the thymus and spleen. Although cystine and glutamate are the well established substrates of system xc− and the knockout of xCT leads to alterations of extracellular redox balance, nothing is known about other potential substrates. We thus performed a comparative metabolite analysis of tissues from xCT-deficient and wild-type mice using capillary electrophoresis time-of-flight mass spectrometry. Although most of the analyzed metabolites did not show significant alterations between xCT-deficient and wild-type mice, cystathionine emerged as being absent specifically in the thymus and spleen of xCT-deficient mice. No expression of either cystathionine β-synthase or cystathionine γ-lyase was observed in the thymus and spleen of mice. In embryonic fibroblasts derived from wild-type embryos, cystine uptake was significantly inhibited by cystathionine in a concentration-dependent manner. Wild-type cells showed an intracellular accumulation of cystathionine when incubated in cystathionine-containing buffer, which concomitantly stimulated an increased release of glutamate into the extracellular space. By contrast, none of these effects could be observed in xCT-deficient cells. Remarkably, unlike knock-out cells, wild-type cells could be rescued from cystine deprivation-induced cell death by cystathionine supplementation. We thus conclude that cystathionine is a novel physiological substrate of system xc− and that the accumulation of cystathionine in immune tissues is exclusively mediated by system xc−.

The amino acid transport system x

The ubiquitin-proteasome system (UPS) is an important intracellular proteolytic pathway responsible for the degradation of proteins and oxidative damage; hence it plays a central role in maintaining homeostasis of red blood cells (RBCs). The present study investigated the levels of polyubiquitination, the function of proteasomes and effect of hydroxycarbamide (HC) therapy in RBCs from sickle cell disease (SCD) patients. Polyubiquitinated proteins were found to be elevated in untreated SCD (UT-SCD) patients compared to those in HC-treated SCD patients (HC-SCD) and controls. Activities of β1 and β2 subunits were a little higher in UT-SCD patients, and much higher proteolytic activities were observed in all three subunits (β1, β2 and β5) of RBCs in HC-SCD patients compared to those of UT-SCD patients and controls, although the protein levels of these subunits remained approximately the same. It is notable that, despite HC therapy, some patients showed persistent complications and accumulation of polyubiquitinated proteins. The enhanced proteasomal activity among HC-treated patients might remove the polyubiquitinated protein and could be one of the important mechanisms of therapeutic action. These findings could be useful to understand the pathophysiology of SCD and its clinical heterogeneity and identify a suitable therapeutic target for the better management of these patients.

Mice that are deficient in superoxide dismutase 1 (Sod1), an antioxidative enzyme, are susceptible to developing liver steatosis. Peroxiredoxin 4 (Prdx4) catalyzes disulfide bond formation in proteins via the action of hydrogen peroxide and hence decreases oxidative stress and supports oxidative protein folding for the secretion of lipoproteins. Because elevated reactive oxygen species induce endoplasmic reticulum stress, this negative chain reaction is likely involved in the development of nonalcoholic fatty liver diseases and more advanced steatohepatitis (NASH). In the current study, we generated Prdx4 and Sod1 double knockout (DKO; Prdx4−/ySod1−/−) mice and examined whether the combined deletion of Prdx4 and Sod1 aggravated liver pathology compared to single knockout and wild-type mice. The secretion of triglyceride-rich lipoprotein was strikingly impaired in the DKO mice, leading to aggravated liver steatosis. Simultaneously, the activation of caspase-3 in the liver was observed. The hyperoxidation of Prdxs, a hallmark of oxidative stress, occurred in different isoforms that are uniquely associated with Sod1−/− and Prdx4−/y mice, and the effect was additive in DKO mouse livers. Because DKO mice spontaneously develop severe liver failure at a relatively young stage, they have the potential for use as a model for hepatic disorders and for testing other potential treatments.