Activity of the Calcium Channel Pore Cch1 Is Dependent on a Modulatory Region of the Subunit Mid1 in Cryptococcus neoformans

Calcium (Ca²⁺)-mediated signaling events in fungal pathogens such as Cryptococcus neoformans are central to physiological processes, including those that mediate stress responses and promote virulence. The Cch1-Mid1 channel (CMC) represents the only high-affinity Ca²⁺ channel in the plasma membrane of fungal cells; consequently, cryptococci cannot survive in low-Ca²⁺ environments in the absence of CMC. Previous electrophysiological characterization revealed that Cch1, the predicted channel pore, and Mid1, a binding partner of Cch1, function as a store-operated Ca²⁺-selective channel gated by depletion of endoplasmic reticulum (ER) Ca²⁺ stores. Cryptococci lacking CMC did not survive ER stress, indicating its critical role in restoring Ca²⁺ homeostasis. Despite the requirement for Mid1 in promoting Ca²⁺ influx via Cch1, identification of the role of Mid1 remains elusive. Here we show that the C-terminal tail of Mid1 is a modulatory region that impinges on Cch1 channel activity directly and mediates the trafficking of Mid1 to the plasma membrane. This region consists of the last 24 residues of Mid1, and the functional expression of Mid1 in a human embryonic cell line (HEK293) and in C. neoformans is dependent on this domain. Substitutions of arginine (R619A) or cysteine (C621A) in the modulatory region failed to target Mid1 to the plasma membrane and prevented CMC activity. Interestingly, loss of a predicted protein kinase C (PKC)-phosphorylated serine residue (S605A) had no effect on Mid1 trafficking but did alter the kinetics of Cch1 channel activity. Thus, establishment of Ca²⁺ homeostasis in C. neoformans is dependent on a modulatory domain of Mid1.     

Roles of Cch1 and Mid1 in Morphogenesis, Oxidative Stress Response and Virulence in Candida albicans

Ca²⁺ channel Cch1, and its subunit Mid1, has been suggested as the protein complex responsible for mediating Ca²⁺ influx, which is often employed by fungal cells to maintain cell survival. The abilities of morphological switch and response to stress conditions are closely related to pathogenicity in Candida albicans. Cch1 and Mid1 activity are required for virulence of Cryptococcus neoformans and Claviceps purpurea, respectively. To investigate whether Cch1 and Mid1 also play a role in the virulence of C. albicans, we constructed cch1Δ/Δ and mid1Δ/Δ mutant strains for functional analysis of CCH1 and MID1. Although both of the mutants displayed the ability of yeast-to-hypha transition, they were defective in hyphae maintenance and invasive growth. Interestingly, deletion of CCH1 or MID1 in C. albicans led to an obvious defect phenotype in oxidative stress response. Moreover, the virulence of the mutants was reduced in a mouse model. Our results demonstrated that Cch1 and Mid1 activity are related to the virulence of C. albicans and may provide a new antifungal target.     

Peroxynitrite and hydrogen peroxide elicit similar cellular stress responses mediated by the Ccp1 sensor protein

Peroxynitrite [ONOO(H)] is an oxidant associated with deleterious effects in cells. Because it is an inorganic peroxide that reacts rapidly with peroxidases, we speculated that cells may respond to ONOO(H) and H₂O₂ challenge in a similar manner. We exposed yeast cells to SIN-1, a well-characterized ONOO(H) generator, and observed stimulation of catalase and peroxiredoxin (Prx) activities. Previously, we reported that H₂O₂ challenge increases these activities in wild-type cells and in cells producing the hyperactive mutant H₂O₂ sensor Ccp1^W191F but not in Ccp1-knockout cells (ccp1Δ). We find here that the response of ccp1Δ and ccp1^W191F cells to SIN-1 mirrors that to H₂O₂, identifying Ccp1 as a sensor of both peroxides. SIN-1 simultaneously releases ^•NO and O₂^•−, which react to form ONOO(H), but exposure of the three strains separately to an ^•NO donor (spermine-NONOate) or an O₂^•− generator (paraquat) mainly depresses catalase or Prx activity, whereas co-challenge with the NONOate and paraquat stimulates these activities. Because Ccp1 appears to sense ONOO(H) in cells, we examined its reaction with ONOO(H) in vitro and found that peroxynitrous acid (ONOOH) rapidly (k₂>10⁶ M⁻¹ s⁻¹) oxidizes purified Ccp1 to an intermediate with spectral and ferrocytochrome-oxidizing properties indistinguishable from those of its well-characterized compound I formed with H₂O₂. Importantly, the nitrite released from ONOOH is not oxidized to ^•NO₂ by Ccp1^׳s compound I, unlike peroxidases involved in immune defense. Overall, our results reveal that yeast cells mount a common antioxidant response to ONOO(H) and H₂O₂, with Ccp1 playing a pivotal role as an inorganic peroxide sensor.     

Cytochrome c peroxidase is a mitochondrial heme-based H2O2 sensor that modulates antioxidant defense

Hydrogen peroxide (H₂O₂) is a key signaling molecule that also induces apoptosis. Thus, cells must rapidly sense and tightly control H₂O₂ levels. Well-characterized cellular responses to exogenous H₂O₂ involve oxidation of specific cytosolic protein-based thiols but sensing of H₂O₂ generated by mitochondrial respiration is less well described. Here we provide substantial biochemical evidence that the heme enzyme Ccp1 (cytochrome c peroxidase), which is targeted to the intermembrane space, functions primarily as a mitochondrial H₂O₂ sensing and signaling protein in Saccharomyces cerevisiae. Key evidence for a sensing role for Ccp1 is the significantly higher H₂O₂ accumulation in ccp1-null cells(ccp1Δ) vs ccp1^W191F cells producing the catalytically inactive Ccp1^W191F variant. In fact, intracellular H₂O₂ levels (ccp1Δ>wildtype >ccp1^W191F) correlate inversely with the activity of the mitochondrial (and peroxisomal) heme catalase, Cta1 (ccp1Δ<wildtype <ccp1^W191F). Mitochondrial Sod2 activity also varies in the three strains (ccp1Δ>wildtype >ccp1^W191F) and ccp1Δ cells exhibit low superoxide levels. Notably, Ccp1^W191F is a more persistent H₂O₂ signaling protein than wild-type Ccp1, and this enhanced mitochondrial H₂O₂ signaling decreases the mitochondrial fitness of ccp1^W191F cells. However, these cells are fully protected from a bolus (0.4 mM) of exogenous H₂O₂ added after 12 h of growth, whereas the viability of ccp1Δ cells drops below 20%, which additionally associates Ccp1 with Yap1-dependent H₂O₂ signaling. Combined, our results strongly implicate Ccp1, independent of its peroxidase activity, in mitochondrial H₂O₂ sensing and signaling to maintain reactive oxygen species homeostasis.     

Over-expression of Trxo1 increases the viability of tobacco BY-2 cells under H2O2 treatment

Background and Aims Reactive oxygen species (ROS), especially hydrogen peroxide, play a critical role in the regulation of plant development and in the induction of plant defence responses during stress adaptation, as well as in plant cell death. The antioxidant system is responsible for controlling ROS levels in these processes but redox homeostasis is also a key factor in plant cell metabolism under normal and stress situations. Thioredoxins (Trxs) are ubiquitous small proteins found in different cell compartments, including mitochondria and nuclei (Trxo1), and are involved in the regulation of target proteins through reduction of disulphide bonds, although their role under oxidative stress has been less well studied. This study describes over-expression of a Trxo1 for the first time, using a cell-culture model subjected to an oxidative treatment provoked by H₂O₂.Methods Control and over-expressing PsTrxo1 tobacco (Nicotiana tabacum) BY-2 cells were treated with 35 mm H₂O₂ and the effects were analysed by studying the growth dynamics of the cultures together with oxidative stress parameters, as well as several components of the antioxidant systems involved in the metabolism of H₂O₂. Analysis of different hallmarks of programmed cell death was also carried out.Key Results Over-expression of PsTrxo1 caused significant differences in the response of TBY-2 cells to high concentrations of H₂O₂, namely higher and maintained viability in over-expressing cells, whilst the control line presented a severe decrease in viability and marked indications of oxidative stress, with generalized cell death after 3 d of treatment. In over-expressing cells, an increase in catalase activity, decreases in H₂O₂ and nitric oxide contents and maintenance of the glutathione redox state were observed.Conclusions A decreased content of endogenous H₂O₂ may be responsible in part for the delayed cell death found in over-expressing cells, in which changes in oxidative parameters and antioxidants were less extended after the oxidative treatment. It is concluded that PsTrxo1 transformation protects TBY-2 cells from exogenous H₂O₂, thus increasing their viability via a process in which not only antioxidants but also Trxo1 seem to be involved.     

Silencing of miR-23a attenuates hydrogen peroxide (H2O2) induced oxidative damages in ARPE-19 cells by upregulating GLS1: an in vitro study

BackgroundOxidative damages contributes to age-related macular degeneration (AMD) caused vision blindness, but the molecular mechanisms are still largely unknown.ObjectivesThis study managed to investigate this issue by conducting in vitro experiments.MethodsOxidative stress were evaluated by L-012 dye, DHE staining and MDA assay. CCK-8 and colony formation assay were conducted to examine cell proliferation. Cell death was evaluated by trypan blue staining and Annexin V-FITC/PI double staining method through flow cytometry (FCM). The binding sites of miR-23a and GLS1 mRNA were predicted by online miRDB database and validated by dual-luciferase reporter gene system. Real-Time qPCR for miR-23a levels and Western Blot for protein expressions.ResultsThe retinal pigment epithelial (RPE) cells (ARPE-19) were subjected to hydrogen peroxide (H₂O₂) stimulation to simulate AMD progression in vitro, and we identified a novel miR-23a/glutaminase-1 (GLS1) pathway that regulated H₂O₂ induced oxidative damages in ARPE-19 cells. Mechanistically, H₂O₂ induced oxidative stress, inhibited cell proliferation and induced cell death in ARPE-19 cells in a dose- and time-dependent manner. Also, H₂O₂ stimulation hindered cell invasion, migration and glutamine uptake in ARPE-19 cells. Interestingly, we proved that H₂O₂ increased miR-23a levels, while downregulated glutaminase-1 (GLS1) in ARPE-19 cells, and miR-23a targeted 3′ untranslated region (3′UTR) of GLS1 mRNA for GLS1 degradation. Finally, our data suggested that silencing miR-23a upregulated GLS1 to reverse the detrimental effects of H₂O₂ treatment on ARPE-19 cells.ConclusionsIn general, analysis of the data suggested that miR-23a ablation upregulated GLS1 to attenuate H₂O₂ stimulation induced oxidative damages in ARPE-19 cells in vitro, and this study broadened our knowledge in this field, which might help to provide novel theranostic signatures for AMD.     

The Saccharomyces cerevisiae Ca2+ channel Cch1pMid1p is essential for tolerance to cold stress and iron toxicity

Cch1p and Mid1p are components of a high-affinity Ca(2+)-permeable channel in the yeast plasma membrane. Here, we show that growth of mutants in the Cch1pMid1p channel is markedly hypersensitive to low temperature and to high iron concentration in the medium. Both phenotypes were suppressed by high Ca(2+) concentration. Iron stress elicited an increased Ca(2+) influx into both wild type and cch1Deltamid1Delta yeast. Inhibition of calcineurin strongly depressed growth of iron-stressed wild type yeast, indicating that calcineurin is a downstream element of the iron stress response. Iron hypersensitivity of the cch1Deltamid1Delta mutant was not associated with an increased iron uptake. An involvement of oxidative stress in the iron-hypersensitive phenotype was indicated by the findings that the antioxidants tocopheryl acetate and (ethyl)glutathione improved growth and viability of the iron-stressed mutant. Further, the degree of glutathione oxidation was increased in the presence of iron. The results indicate that iron stress leads to an increased oxidative poise and that Cch1pMid1p is essential to tolerate this condition.     

Overexpression of a peroxiredoxin gene from <Emphasis Type="Italic">Tamarix hispida</Emphasis>, <Emphasis Type="Italic">ThPrx1</Emphasis>, confers tolerance to oxidative stress in yeast and Arabidopsis

Peroxiredoxins (Prxs) are ubiquitous thiol-specific antioxidant enzymes that are critically involved in cell defense and protect cells from oxidative damage. In this study, a putative Type II Prx (ThPrx1) was identified and characterized from Tamarix hispida. The expression of ThPrx1 is highly induced in response to hydrogen peroxide (H₂O₂) and methyl viologen (MV) stresses. When expressed ectopically, ThPrx1 showed enhanced tolerance against oxidative stress in yeast and Arabidopsis. In addition, transgenic Arabidopsis plants overexpressing ThPrx1 displayed improved seedling survival rates and increased root growth and fresh weight gain under H₂O₂ and MV treatments. Moreover, transgenic Arabidopsis plants showed decreased accumulation of H₂O₂, superoxide (O₂^??) and malondialdehyde (MDA), increased superoxide dismutase (SOD) activity compared to wild-type (WT) plants under oxidative stress. Moreover, transgenic plants maintained higher photosynthesis efficiency and lower electrolyte leakage rates than that of WT plants under stress conditions. These results clearly indicated that ThPrx1 plays an important role in cellular redox homeostasis under stress conditions, leading to the maintenance of membrane integrity and increased tolerance to oxidative stress.     

Comparative Biochemistry of the Oxidative Burst Produced by Rose and French Bean Cells Reveals Two Distinct Mechanisms

Cultured cells of rose (Rosa damascena) treated with an elicitor derived from Phytophthora spp. and suspension-cultured cells of French bean (Phaseolus vulgaris) treated with an elicitor derived from the cell walls of Colletotrichum lindemuthianum both produced H₂O₂. It has been hypothesized that in rose cells H₂O₂ is produced by a plasma membrane NAD(P)H oxidase (superoxide synthase), whereas in bean cells H₂O₂ is derived directly from cell wall peroxidases following extracellular alkalinization and the appearance of a reductant. In the rose/Phytophthora spp. system treated with N,N-diethyldithiocarbamate, superoxide was detected by a N,N′-dimethyl-9,9′-biacridium dinitrate-dependent chemiluminescence; in contrast, in the bean/C. lindemuthianum system, no superoxide was detected, with or without N,N-diethyldithiocarbamate. When rose cells were washed free of medium (containing cell wall peroxidase) and then treated with Phytophthora spp. elicitor, they accumulated a higher maximum concentration of H₂O₂ than when treated without the washing procedure. In contrast, a washing treatment reduced the H₂O₂ accumulated by French bean cells treated with C. lindemuthianum elicitor. Rose cells produced reductant capable of stimulating horseradish (Armoracia lapathifolia) peroxidase to form H₂O₂ but did not have a peroxidase capable of forming H₂O₂ in the presence of reductant. Rose and French bean cells thus appear to be responding by different mechanisms to generate the oxidative burst.     

Sec-containing TrxR1 is essential for self-sufficiency of cells by control of glucose-derived H2O2

It is commonly recognized that diabetic complications involve increased oxidative stress directly triggered by hyperglycemia. The most important cellular protective systems against such oxidative stress have yet remained unclear. Here we show that the selenoprotein thioredoxin reductase 1 (TrxR1), encoded by the Txnrd1 gene, is an essential enzyme for such protection. Individually grown Txnrd1 knockout (Txnrd1^−/−) mouse embryonic fibroblasts (MEFs) underwent massive cell death directly linked to glucose-induced H₂O₂ production. This death and excessive H₂O₂ levels could be reverted by reconstituted expression of selenocysteine (Sec)-containing TrxR1, but not by expression of Sec-devoid variants of the enzyme. Our results show that Sec-containing TrxR1 is absolutely required for self-sufficient growth of MEFs under high-glucose conditions, owing to an essential importance of this enzyme for elimination of glucose-derived H₂O₂. To our knowledge, this is the first time a strict Sec-dependent function of TrxR1 has been identified as being essential for mammalian cells.     

Ataxia telangiectasia mutated inhibits oxidative stress-induced apoptosis by regulating heme oxygenase-1 expression

Ataxia telangiectasia (AT) is caused by mutational inactivation of the ataxia telangiectasia mutated (Atm) gene, which is involved in DNA repair. Increased oxidative stress has been shown in human AT cells and neuronal tissues of Atm-deficient mice. Heme oxygenase-1 (HO-1) is an inducible antioxidant enzyme and protects cells against oxidative stress. The purpose of this study is to determine whether ATM induces antioxidant enzyme HO-1 and protects cells from oxidative stress-mediated apoptosis by driving the activation of PKC-δ and NF-κB, by increasing cell viability, and by downregulating DNA fragmentation and apoptotic indicators (apoptosis-inducing factor and cleaved caspase-3). AT fibroblasts stably transfected with human full-length ATM cDNA (YZ5 cells) or the empty vector (MOCK cells) were treated with H₂O₂ as a source of reactive oxygen species (ROS). As a result, transfection with ATM inhibited ROS-induced cell death and DNA fragmentation in MOCK cells. Transfection with ATM induced expression of HO-1 which was mediated by PKC-δ and NF-κB in H₂O₂-treated MOCK cells. ZnPP, an HO-1 inhibitor, and transfection with HO-1 siRNA increased ROS levels and apoptosis, whereas hemin, an HO-1 activator, reduced ROS levels and apoptosis in H₂O₂-treated YZ5 cells. Rottlerin, a PKC-δ inhibitor, inhibited NF-κB activation and HO-1 expression in H₂O₂-treated YZ5 cells. MOCK cells showed increased cell death, DNA fragmentation, and apoptotic indicators compared to YZ5 cells exposed to H₂O₂. In addition, transfection with p65 siRNA increased ROS levels and DNA fragmentation, but decreased HO-1 protein levels in H₂O₂-treated YZ5 cells. In conclusion, ATM induces HO-1 expression via activation of PKC-δ and NF-κB and inhibits oxidative stress-induced apoptosis. A loss of HO-1 induction may explain why AT patients are vulnerable to oxidative stress.     

Metabolic derangement and cardiac injury early after reperfusion following intermittent cross-clamp fibrillation in patients undergoing coronary artery bypass graft surgery using conventional or miniaturized cardiopulmonary bypass

Reactive oxygen species (ROS)-induced oxidative stress increases in skeletal muscle with aging and decreases the viability of implanted cells. Type 1 insulin-like growth factor (IGF-1) promotes the survival of skeletal muscle cells under oxidative stress. It is unknown whether IGF-1 protects muscle-derived stem cells (MDSCs) from oxidative stress. In this study, we genetically engineered rat MDSCs to overexpress IGF-1 and determined cell viability, apoptosis, and VEGF secretion under oxidative stress. Overexpression of IGF-1 prevented MDSCs from H₂O₂-induced caspase-dependent apoptotic cell death by upregulating the PI3K/AKT pathway, accompanied with an increase of NF-κB, p-NF-κB, Bcl-2, and VEGF, as well as a decrease of Bax. In contrast, pre-administration of picropodophyllinb, wortmannin, 1L-6-hydroxymethyl-chiro-inositol-2-((R)-2-O-methyl-3-O-octadecylcarbonate), or pyrrolidine-dithiocarbamate, specific inhibitors of IGF-1R, PI3K, AKT, and NF-κB, respectively, followed by treatment with H₂O₂, resulted in cell death of MDSCs. Our data indicated that IGF-1 suppresses apoptosis and enhances the paracrine function of MDSCs under oxidative stress via enhancing IGF-1R/PI3K/AKT signaling. Thus, IGF-1 gene-modified MDSCs present a potential application in the treatment of muscle wasting, such as urethra intrinsic sphincter deficiency.     

The tyrosine phosphatase, SHP-1, is involved in bronchial mucin production during oxidative stress

Mucus hypersecretion is a clinically important manifestation of chronic inflammatory airway diseases, such as asthma and Chronic obstructive pulmonary disease (COPD). Mucin production in airway epithelia is increased under conditions of oxidative stress. Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 suppression is related to the development of airway inflammation and increased ROS levels. In this study, we investigated the role of SHP-1 in mucin secretion triggered by oxidative stress. Human lung mucoepidermoid H292 carcinoma cells were transfected with specific siRNA to eliminate SHP-1 gene expression. Cultured cells were treated with hydrogen peroxide (H₂O₂), and Mucin 5AC(MUC5AC) gene expression and mucin production were determined. Activation of p38 mitogen activated protein kinase (MAPK) in association with MUC5AC production was evaluated. N-acetylcysteine (NAC) was employed to determine whether antioxidants could block MUC5AC production. To establish the precise role of p38, mucin expression was observed after pre-treatment of SHP-1-depleted H292 cells with the p38 chemical blocker. We investigated the in vivo effects of oxidative stress on airway mucus production in SHP-1-deficient heterozygous (mev/+) mice. MUC5AC expression was enhanced in SHP-1 knockdown H292 cells exposed to H₂O₂, compared to that in control cells. The ratio between phosphorylated and total p38 was significantly increased in SHP-1-deficient cells under oxidative stress. Pre-treatment with NAC suppressed both MUC5AC production and p38 activation. Blockage of p38 MAPK led to suppression of MUC5AC mRNA expression. Notably, mucin production was enhanced in the airway epithelia of mev/+ mice exposed to oxidative stress. Our results clearly indicate that SHP-1 plays an important role in airway mucin production through regulating oxidative stress.     

Effects of N-acetyl-l-cysteine on bleomycin induced oxidative stress in malignant testicular germ cell tumors

Testicular cancer is a very common cancer in males aged 15–44 years. Bleomycin is used in chemotherapy regimens in the treatment of patients having testicular germ-cell tumor. Bleomycin generates oxygen radicals, induces oxidative cleavage of DNA strand and induces apoptosis in cancer cells. There is no study in the literature investigating effects of N-Acetyl-l-Cysteine (NAC) on bleomycin-induced oxidative stress in testicular germ cell tumors. For this reason, we studied effects of NAC on oxidative stress produced in wild-type NTera-2 and p53-mutant NCCIT testis cancer cells incubated with bleomycin and compared the results with H₂O₂ which directly produces oxidative stress. We determined protein carbonyl content, thiobarbituric acid reactive substances (TBARS), glutathione (GSH), 8-isoprostane, lipid hydroperoxide levels and total antioxidant capacity in both testicular cancer cells. Bleomycin and H₂O₂ significantly increased 8-isoprostane, TBARS, protein carbonyl and lipid hydroperoxide levels in NTera-2 and NCCIT cells. Bleomycin and H₂O₂ significantly decreased antioxidant capacity and GSH levels in both cell lines. Co-incubation with NAC significantly decreased lipid hydroperoxide, 8-isoprostane, protein carbonyl content and TBARS levels increased by bleomycin and H₂O₂. NAC enhanced GSH levels and antioxidant capacity in the NTera-2 and NCCIT cells. It can be concluded that NAC diminishes oxidative stress in human testicular cancer cells induced by bleomycin and H₂O₂.     

Radioprotection by short-term oxidative preconditioning: Role of manganese superoxide dismutase

Manganese superoxide dismutase (MnSOD) is vital to the protection of mitochondria and cells against oxidative stress. Earlier, we demonstrated that catalytically active homo-tetramer of MnSOD can be stabilized by oxidative cross-linking. Here we report that this effect may be translated into increased radioresistance of mouse embryonic cells (MECs) by pre-exposure to oxidative stress. Pre-treatment of MECs with antimycin A, rotenone or H₂O₂ increased their survival after irradiation. Using MnSOD siRNA, we show that MECs with decreased MnSOD levels displayed a lowered ability to preconditioning. Thus oxidative preconditioning may be used for targeted regulation of MnSOD.

Structured summary

MINT-7288408: MnSOD (uniprotkb:P04179) and MnSOD (uniprotkb:P04179) physically interact (MI:0915) by zymography (MI:0512)