Antiporter Subunit Research Articles

Water extract of moschus alleviates erastin-induced ferroptosis by regulating the Keap1/Nrf2 pathway in HT22 cells

Ethnopharmacological relevanceMoschus, first described in the Shennong's Classic of the Materia medicine, is a scarce and precious animal medicine. Modern pharmacological researches have suggested that Moschus has neuroprotective actions, and its mechanism is related to anti-inflammatory, antioxidant, and anti-apoptosis effects. Ferroptosis is one of the major pathologies of Alzheimer's disease (AD) and is widely implicated in the pathogenesis and progression of AD. Although previous studies have suggested that Moschus possesses neuroprotective effect, whether Moschus could mitigate neuronal damages by inhibiting the onset of ferroptosis is unknown in model cells of AD. Aim of the studyThe aim of study was to explore the water extract of Moschus (WEM) on ferroptosis caused by erastin and the potential mechanism. Materials and methodsErastin was used to stimulate HT22 cells to form ferroptosis model to evaluate the anti-ferroptosis effect of WEM by cell counting kit-8 and lactic dehydrogenase (LDH) tests. The malondialdehyde (MDA) and glutathione (GSH) kits are used for detection of MDA and GSH levels, and 2′,7′-dichlorofluorescein diacetate and C11 BODIPY 581/591 fluorescence probe are used for evaluation of reactive oxygen species (ROS) and lipid peroxide (LOOH) levels. And Western blot was used to test nuclear factor erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), heme oxygenase-1 (HO-1), and ferroptosis associated proteins including glutathione peroxidase 4 (GPX4), cystine/glutamate antiporter subunit (SLC7A11), ferritin heavy chain 1 (FTH1), ferroportin1 (FPN1), transferrin receptor (TFRC). In addition, the Nrf2 inhibitor ML385 was applied to verify whether WEM prevents erastin-induced ferroptosis by activating the Keap1/Nrf2 pathway. ResultsAfter WEM treatment, erastin-induced HT22 cell survival was significantly elevated, the accumulation of intracellular MDA, ROS, and LOOH were significantly reduced, the level of GSH and expressions of ferroptosis inhibitors GPX4 and SLC7A11 were significantly increased, and iron metabolism-related proteins TFRC, FPN1, and FTH1 were regulated. These effects of WEM are implemented by activating the Keap1/Nrf2 pathway. ConclusionsThis study demonstrated that WEM could perform neuroprotective effects by alleviating ferroptosis, verified that WEM treatment of AD can be mediated by the Keap1/Nrf2 pathway, and provided theoretical support for the application of WEM in the treatment of AD.

Metagenomic analysis of the soil microbial composition and salt tolerance mechanism in Yuncheng Salt Lake, Shanxi Province.

The soil in Yuncheng Salt Lake has serious salinization and the biogeographic environment affects the composition and distribution of special halophilic and salt-tolerant microbial communities in this area. Therefore, this study collected soils at distances of 15, 30, and 45 m from the Salt Lake and used non-saline soil (60 m) as a control to explore the microbial composition and salt tolerance mechanisms using metagenomics technology. The results showed that the dominant species and abundance of salt-tolerant microorganisms changed gradually with distance from Salt Lake. The salt-tolerant microorganisms can increase the expression of the Na+/H+ antiporter by upregulating the Na+/H+ antiporter subunit mnhA-G to respond to salt stress, simultaneously upregulating the genes in the betaine/proline transport system to promote the conversion of choline into betaine, while also upregulating the trehalose/maltose transport system encode genes to promote the synthesis of trehalose to resist a high salt environment.

Abstract 2019: Drug repositioning to induce the synthetic lethality in cancer cells with GSH-depleting therapy

Abstract The major cellular antioxidant glutathione (GSH) protects cancer cells from oxidative damage that can lead to the induction of ferroptosis. Inhibitor of the GSH synthesis including buthionine sulfoximine (BSO) and cystine-glutamate antiporter subunit xCT, which mediates the uptake of extracellular cystine and thereby promotes GSH synthesis, thus potential anticancer agents. However, the efficacy of xCT or GSH synthesis-targeted therapy has been found to be diminished by metabolic reprogramming that affects redox status in cancer cells. In this study, we performed a synthetic lethal screen of a drug library to identify agents that sensitize the GSH deficiency-resistant cancer cells to the xCT inhibitor sulfasalazine (SSZ). This screen identified the oral anesthetic dyclonine (DYC) which act as a covalent inhibitor for aldehyde dehydrogenases (ALDHs). Treatment with DYC induced intracellular accumulation of the toxic aldehyde 4-hydroxynonenal (4-HNE) in a cooperative manner with SSZ. The combination of DYC and SSZ cooperatively suppressed the growth of SSZ-resistant tumors. Furthermore, we recently found that a vasodilator oxyfedrine (OXY) also possess the similar structure with dyclonine and induce cell death in BSO-resistant cancer cells both in vitro and in vivo. Microarray analysis of tumor xenograft tissue showed cyclooxygenase-2 expression as a potential biomarker for the efficacy of such combination therapy. Moreover, OXY-mediated ALDH inhibition was found to sensitize cancer cells to GSH depletion induced by radiation therapy in vitro. Our findings thus establish a rationale for repurposing of OXY as a sensitizing drug for cancer treatment with agents that induce GSH depletion. Citation Format: Yuji Otsuki, Osamu Nagano, Hideyuki Saya. Drug repositioning to induce the synthetic lethality in cancer cells with GSH-depleting therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2019.

Comparative phylo-pangenomics reveals generalist lifestyles in representative Acinetobacter species and proposes candidate gene markers for species identification.

Acinetobacter species have the potential to invade and colonize immunocompromised patients, therefore being well-known as opportunistic pathogens. Among these bacteria, the species of the Acinetobacter calcoaceticus-Acinetobacter baumannii "complex" (Acb members) emerge as the main often isolated bacteria in clinical specimens. The unequivocal taxonomy is crucial to correctly identify these species and associated with comparative genomic analyses aids to understand their life-styles as well. In this study, all publicly available Acinetobacter species at the date of this study preparation were analyzed. The results revealed that the Acb members are in fact a complex when phenotypic methods are confronted, while for comparative and phylogenomics analyses this term is misleading, since they composed a monophyletic group instead. Nine best gene markers (response regulator, recJ, recG, phosphomannomutase, pepSY, monovalent cation/H+antiporter subunit D, mnmE, glnE, and bamA) were selected for identification of Acinetobacter species. Moreover, representative strains of each species were split according their isolation sources in the categories: environmental, human, insect and non-human vertebrate. Neither niche-specific genome signature nor niche-associated functional and pathogenic potential were associated with their isolation source, meaning it is not the main force acting on Acinetobacter adaptation in a given niche and corroborating that their ubiquitous distribution is a reflex of their generalist life-styles.

Vasodilator oxyfedrine inhibits aldehyde metabolism and thereby sensitizes cancer cells to xCT-targeted therapy.

The major cellular antioxidant glutathione (GSH) protects cancer cells from oxidative damage that can lead to the induction of ferroptosis, an iron‐dependent form of cell death triggered by the aberrant accumulation of lipid peroxides. Inhibitors of the cystine‐glutamate antiporter subunit xCT, which mediates the uptake of extracellular cystine and thereby promotes GSH synthesis, are thus potential anticancer agents. However, the efficacy of xCT‐targeted therapy has been found to be diminished by metabolic reprogramming that affects redox status in cancer cells. Identification of drugs for combination with xCT inhibitors that are able to overcome resistance to xCT‐targeted therapy might thus provide the basis for effective cancer treatment. We have now identified the vasodilator oxyfedrine (OXY) as a sensitizer of cancer cells to GSH‐depleting agents including the xCT inhibitor sulfasalazine (SSZ). Oxyfedrine contains a structural motif required for covalent inhibition of aldehyde dehydrogenase (ALDH) enzymes, and combined treatment with OXY and SSZ was found to induce accumulation of the cytotoxic aldehyde 4‐hydroxynonenal and cell death in SSZ‐resistant cancer cells both in vitro and in vivo. Microarray analysis of tumor xenograft tissue showed cyclooxygenase‐2 expression as a potential biomarker for the efficacy of such combination therapy. Furthermore, OXY‐mediated ALDH inhibition was found to sensitize cancer cells to GSH depletion induced by radiation therapy in vitro. Our findings thus establish a rationale for repurposing of OXY as a sensitizing drug for cancer treatment with agents that induce GSH depletion.

Influx rate of 18F-fluoroaminosuberic acid reflects cystine/glutamate antiporter expression in tumour xenografts.

18F-fluoroaminosuberic acid (18F-FASu) is a recently developed amino acid tracer for positron emission tomography (PET) of oxidative stress that may offer improved tumour assessment over the conventional tracer 18F-fluorodeoxyglucose (18F-FDG). Our aim was to evaluate and relate dynamic 18F-FASu and 18F-FDG uptake with pharmacokinetic modelling to transporter protein expression levels in a panel of diverse tumour xenograft lines. Four different tumour xenograft lines were implanted in female athymic nude mice: MAS98.12 and HBCx3 (breast), TPMX (osteosarcoma) and A549 (lung). Dynamic PET over 60min was performed on a small animal unit. The time-activity curves (TACs) for 18F-FASu and 18F-FDG in individual tumours were used to extract early (SUVE; 2min p.i.) and late (SUVL; 55min p.i.) standardised uptake values. Pharmacokinetic two-tissue compartment models were applied to the TACs to estimate rate constants K1-k4 and blood volume fraction vB. Relative levels of cystine/glutamate antiporter subunit xCT were assessed by western blotting, and expression of GLUT1 and CD31 by immunohistochemistry. 18F-FASu showed higher SUVE, whilst 18F-FDG exhibited higher SUVL. Influx rate K1 for 18F-FASu was significantly correlated with xCT levels (p = 0.001) and was significantly higher than K1 for 18F-FDG (p < 0.001). K1 for 18F-FDG was significantly correlated with GLUT1 levels (p = 0.002). vB estimated from 18F-FASu and 18F-FDG TACs was highly consistent and significantly correlated (r = 0.85, p < 0.001). Two qualitatively different 18F-FASu uptake profiles were identified: type α with low xCT expression and low K1 (A549 and HBCx3), and type β with high xCT expression and high K1 (MAS98.12 and TPMX). The influx rate of 18F-FASu reflects xCT activity in tumour xenografts. Dynamic PET with pharmacokinetic modelling is needed to fully appraise 18F-FASu distribution routes.

Inhibiting STAT3 in a murine model of human breast cancer-induced bone pain delays the onset of nociception.

Aggressive breast cancer subtypes utilize system xc−, a membrane antiporter, to import cystine for glutathione synthesis and maintenance of redox homeostasis, in turn releasing glutamate as a metabolic pro-nociceptive by-product. Metastatic breast cancers establish themselves at distal sites including bone, where changes in extracellular glutamate levels contribute to cancer-induced bone pain. We previously established that stearically blocking system xc− activity with sulfasalazine delays the onset of nociceptive behaviours and that xCT, the functional antiporter subunit, is positively regulated by signal transducer and activator of transcription 3 (STAT3). In the current investigation, a murine xenograft cancer-induced bone pain model was applied to examine whether pharmacological inhibition of phosphorylated STAT3 (pSTAT3) induces changes in nociception. A high glutamate-releasing, xCT/pSTAT3 over-expressing human breast cancer cell line was selected for injection into the distal epiphysis of the right femur of female nude mice. A 14-day regimen of intraperitoneal injections with either vehicle or the novel STAT3 inhibitor DR-1–55 commenced three weeks after initial intrafemoral bone injection. Nociceptive behaviours were temporally monitored by automated von Frey, dynamic weight bearing and open-field testing for the duration of the study, beginning at the baseline. Prior to sacrifice and at ethical end point, tumour-induced osteolytic lesions were radiographically assessed. Treatment with DR-1–55 significantly delayed the onset and severity of spontaneous and induced nociceptive behaviours, also decreasing human SLC7A11 (xCT) mRNA levels in tumour-bearing limbs without altering osteolysis. In addition, two pro-inflammatory cytokines released by this cell line, interleukin 6 and interleukin 1β, were also down-regulated at the mRNA level in response to DR-1–55 treatment in vivo, with lower human interleukin 6 levels detected in the host circulation. This study demonstrates that targeting pSTAT3 may be a viable therapeutic means to manage cancer-induced bone pain, alone or in combination with stearic system xc− blockers.

Synthetic lethality of the ALDH3A1 inhibitor dyclonine and xCT inhibitors in glutathione deficiency-resistant cancer cells.

The cystine-glutamate antiporter subunit xCT suppresses iron-dependent oxidative cell death (ferroptosis) and is therefore a promising target for cancer treatment. Given that cancer cells often show resistance to xCT inhibition resulting in glutathione (GSH) deficiency, however, we here performed a synthetic lethal screen of a drug library to identify agents that sensitize the GSH deficiency-resistant cancer cells to the xCT inhibitor sulfasalazine. This screen identified the oral anesthetic dyclonine which has been recently reported to act as a covalent inhibitor for aldehyde dehydrogenases (ALDHs). Treatment with dyclonine induced intracellular accumulation of the toxic aldehyde 4-hydroxynonenal in a cooperative manner with sulfasalazine. Sulfasalazine-resistant head and neck squamous cell carcinoma (HNSCC) cells were found to highly express ALDH3A1 and knockdown of ALDH3A1 rendered these cells sensitive to sulfasalazine. The combination of dyclonine and sulfasalazine cooperatively suppressed the growth of highly ALDH3A1-expressing HNSCC or gastric tumors that were resistant to sulfasalazine monotherapy. Our findings establish a rationale for application of dyclonine as a sensitizer to xCT-targeted cancer therapy.

The study of patterns of development of resistance of Staphylococcus aureus to triclosan

The article presents the results of studying molecular genetic mechanisms of development of resistance to antiseptic Triclosan in strain Staphylococcus aureus АТСС25923. The modifcations of strain S. aureus АТСС25923 (Tr1, Tr2, Tr1С и Tr2С) are obtained resistant to 64 mg/l of Triclosan and stably preserving the given characteristic under cultivation in absence of selective pressure. The strain S. aureus Tr1was characterized by slightly delayed growth and the strain S. aureus Tr2 was characterized by growth velocity comparable with initial strain. In the Triclosan-resistant strains a mutation C284T in gene fabI was detected resulting in amino-acid replacement A95V in enzyme enoyl-acyl protein reductase FabI, triclosan target. Besides, in these strains a stably inheriting mutation was detected in genes associated with transport of substances in cell: hypothetical transport protein HlyC/CorC family transporter, protein-transporter of ions of Na+, K+, Li+ and alkali of Na+/H+ antiporter subunit F, membrane hypothetical protein and ATP-binding protein. It is demonstrated that resistance to triclosan in staphylococci is associated with acquirement of point mutations in genes of enoyl-acyl protein reductase and also in other genes related to transport of substances in bacterial cell.

Novel Metal Cation Resistance Systems from Mutant Fitness Analysis of Denitrifying Pseudomonas stutzeri.

Metal ion transport systems have been studied extensively, but the specificity of a given transporter is often unclear from amino acid sequence data alone. In this study, predicted Cu(2+) and Zn(2+) resistance systems in Pseudomonas stutzeri strain RCH2 are compared with those experimentally implicated in Cu(2+) and Zn(2+) resistance, as determined by using a DNA-barcoded transposon mutant library. Mutant fitness data obtained under denitrifying conditions are combined with regulon predictions to yield a much more comprehensive picture of Cu(2+) and Zn(2+) resistance in strain RCH2. The results not only considerably expand what is known about well-established metal ion exporters (CzcCBA, CzcD, and CusCBA) and their accessory proteins (CzcI and CusF), they also reveal that isolates with mutations in some predicted Cu(2+) resistance systems do not show decreased fitness relative to the wild type when exposed to Cu(2+) In addition, new genes are identified that have no known connection to Zn(2+) (corB, corC, Psest_3226, Psest_3322, and Psest_0618) or Cu(2+) resistance (Mrp antiporter subunit gene, Psest_2850, and Psest_0584) but are crucial for resistance to these metal cations. Growth of individual deletion mutants lacking corB, corC, Psest_3226, or Psest_3322 confirmed the observed Zn-dependent phenotypes. Notably, to our knowledge, this is the first time a bacterial homolog of TMEM165, a human gene responsible for a congenital glycosylation disorder, has been deleted and the resulting strain characterized. Finally, the fitness values indicate Cu(2+)- and Zn(2+)-based inhibition of nitrite reductase and interference with molybdenum cofactor biosynthesis for nitrate reductase. These results extend the current understanding of Cu(2+) and Zn(2+) efflux and resistance and their effects on denitrifying metabolism. In this study, genome-wide mutant fitness data in P. stutzeri RCH2 combined with regulon predictions identify several proteins of unknown function that are involved in resisting zinc and copper toxicity. For zinc, these include a member of the UPF0016 protein family that was previously implicated in Ca(2+)/H(+) antiport and a human congenital glycosylation disorder, CorB and CorC, which were previously linked to Mg(2+) transport, and Psest_3322 and Psest_0618, two proteins with no characterized homologs. Experiments using mutants lacking Psest_3226, Psest_3322, corB, corC, or czcI verified their proposed functions, which will enable future studies of these little-characterized zinc resistance determinants. Likewise, Psest_2850, annotated as an ion antiporter subunit, and the conserved hypothetical protein Psest_0584 are implicated in copper resistance. Physiological connections between previous studies and phenotypes presented here are discussed. Functional and mechanistic understanding of transport proteins improves the understanding of systems in which members of the same protein family, including those in humans, can have different functions.

Decreased expression of the active subunit of the cystine/glutamate antiporter xCT is associated with loss of heterozygosity of 1p in oligodendroglial tumours WHO grade II

Oligodendroglial tumours with loss of heterozygosity on 1p (LOH1p) respond better to treatment than oligodendrogliomas without LOH. Previous reports have assigned a crucial role of glutamate metabolism to glioma growth and invasion. The aim was to study the protein expression of different glutamate transporters in relation to LOH1p in low-grade oligodendroglial tumours. Seventeen oligodendrogliomas World Health Organization (WHO) grade II, 16 oligoastrocytomas WHO grade II and seven astrocytomas WHO grade II were examined. Eleven oligodendrogliomas and five oligoastrocytomas exhibited LOH1p. Immunoreactivity scores (IRS) for glutamate transporters excitatory amino acid transporter (EAAT)-1, -2 and -3 as well as the active cystine/glutamate antiporter subunit xCT were semiquantitatively rated by percentage of positive cells and intensity of immunoreactivity. Reactivity for xCT was lower in tumours with LOH1p than in those without (P = 0.03, Mann-Whitney U-test). No association was found between LOH status and IRS for EAAT-1, -2 or -3. High xCT immunoreactivity was associated with high expression of EAAT-1, -2 or -3. Expression of xCT is significantly reduced in low-grade oligodendroglial tumours harbouring LOH1p. Further studies should investigate a potential beneficial effect by inhibiting xCT in low-grade gliomas.

Functional expression of β2 adrenergic receptors responsible for protection against oxidative stress through promotion of glutathione synthesis after Nrf2 upregulation in undifferentiated mesenchymal C3H10T1/2 stem cells

Adrenaline is believed to play a dual role as a neurotransmitter in the central nervous system and an adrenomedullary hormone in the peripheral tissues. In contrast to accumulating evidence for the involvement in endochondral ossification, osteoblastogenesis, and osteoclastogenesis, little attention has been paid to the role of adrenergic signals in the mechanisms underlying proliferation and differentiation of mesenchymal stem cells with self-renewal capacity and multi-potentiality to differentiate into osteoblast, chondrocyte, adipocyte, and myocyte lineages. Expression of mRNA was seen for different adrenergic receptor (AdR) subtypes, including beta(2)AdR, in the mesenchymal stem cell line C3H10T1/2 cells and mouse bone marrow mesenchymal stem cells before differentiation. Exposure to adrenaline not only increased cAMP formation, phosphorylation of cAMP responsive element (CRE) binding protein (CREB) on serine133 and CRE reporter activity in a manner sensitive to propranolol, but also rendered C3H10T1/2 cells resistant to the cytotoxicity of hydrogen peroxide, but not of either 2,4-dinitirophenol or tunicamycin. Adrenaline induced a rapid but transient increase in mRNA expression of the antioxidative gene nuclear factor E2 p45-related factor-2 (Nrf2) along with an increase in the cystine/glutamate antiporter subunit xCT mRNA expression. Hydrogen peroxide was less cytotoxic in cells overexpressing Nrf2, moreover, while adrenaline significantly increased xCT promoter activity with an increase in endogenous glutathione levels. These results suggest that adrenaline may selectively protect mesenchymal C3H10T1/2 cells from oxidative stress through a mechanism related to the promoted biosynthesis of glutathione in association with transient Nrf2 expression after activation of beta(2)AdR.

S4.26 Evidence for the presence of a Na+/H+ antiporter subunit in Rhodothermus marinus complex I

S4.26 Evidence for the presence of a Na+/H+ antiporter subunit in Rhodothermus marinus complex I

Cooperative action of glutamate transporters and cystine/glutamate antiporter system Xc– protects from oxidative glutamate toxicity

Oxidative glutamate toxicity in the neuronal cell line HT22 is a model for cell death by oxidative stress. In this paradigm, an excess of extracellular glutamate blocks the glutamate/cystine-antiporter system Xc-, depleting the cell of cysteine, a building block of the antioxidant glutathione. Loss of glutathione leads to the accumulation of reactive oxygen species and eventually cell death. We selected cells resistant to oxidative stress, which exhibit reduced glutamate-induced glutathione depletion mediated by an increase in the antiporter subunit xCT and system Xc- activity. Cystine uptake was less sensitive to inhibition by glutamate and we hypothesized that glutamate import via excitatory amino acid transporters and immediate re-export via system Xc- underlies this phenomenon. Inhibition of glutamate transporters by l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) and DL-threo-beta-benzyloxyaspartic acid (TBOA) exacerbated glutamate-induced cell death. PDC decreased intracellular glutamate accumulation and exacerbated glutathione depletion in the presence of glutamate. Transient overexpression of xCT and the glutamate transporter EAAT3 cooperatively protected against glutamate. We conclude that EAATs support system Xc- to prevent glutathione depletion caused by high extracellular glutamate. This knowledge could be of use for the development of novel therapeutics aimed at diseases associated with depletion of glutathione like Parkinson's disease.

New functions for the three subunits of the CzcCBA cation-proton antiporter.

The membrane-bound CzcCBA protein complex mediates heavy metal resistance in Alcaligenes eutrophus by an active cation efflux mechanism driven by cation-proton antiport. The CzcA protein alone is able to mediate weak resistance to zinc and cobalt and is thus the central antiporter subunit. The two histidine-rich motifs in the CzcB subunit are not essential for zinc resistance; however, deletion of both motifs led to a small but significant loss of resistance to this cation. Translation of the czcC gene encoding the third subunit of the CzcCBA complex starts earlier than predicted, and CzcC is probably a periplasmic protein, as judged by the appearance of two bands after expression of czcC in Escherichia coli under control of the phage T7 promoter. Fusions of CzcC and CzcB with alkaline phosphatase and beta-galactosidase are in agreement with a periplasmic location of most parts of both proteins. Both CzcC and CzcB are bound to a membrane, probably the outer membrane, by themselves and do not need either CzcA or each other as an anchoring protein. Based on these data, a new working model for the function of the Czc system is discussed.