Bacillus anthracis Thioredoxin Systems,Characterization and Role as Electron Donors for Ribonucleotide Reductase

Bacillus anthracis is the causative agent of anthrax, which is associated with a high mortality rate. Like several medically important bacteria, B. anthracis lacks glutathione but encodes many genes annotated as thioredoxins, thioredoxin reductases, and glutaredoxin-like proteins. We have cloned, expressed, and characterized three potential thioredoxins, two potential thioredoxin reductases, and three glutaredoxin-like proteins. Of these, thioredoxin 1 (Trx1) and NrdH reduced insulin, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), and the manganese-containing type Ib ribonucleotide reductase (RNR) from B. anthracis in the presence of NADPH and thioredoxin reductase 1 (TR1), whereas thioredoxin 2 (Trx2) could only reduce DTNB. Potential TR2 was verified as an FAD-containing protein reducible by dithiothreitol but not by NAD(P)H. The recently discovered monothiol bacillithiol did not work as a reductant for RNR, either directly or via any of the redoxins. The catalytic efficiency of Trx1 was 3 and 20 times higher than that of Trx2 and NrdH, respectively, as substrates for TR1. Additionally, the catalytic efficiency of Trx1 as an electron donor for RNR was 7-fold higher than that of NrdH. In extracts of B. anthracis, Trx1 was responsible for almost all of the disulfide reductase activity, whereas Western blots showed that the level of Trx1 was 15 and 60 times higher than that of Trx2 and NrdH, respectively. Our findings demonstrate that the most important general disulfide reductase system in B. anthracis is TR1/Trx1 and that Trx1 is the physiologically relevant electron donor for RNR. This information may provide a basis for the development of novel antimicrobial therapies targeting this severe pathogen.     

The Thioredoxin-Thioredoxin Reductase System Can Function in Vivo as an Alternative System to Reduce Oxidized Glutathione in Saccharomyces cerevisiae

Cellular mechanisms that maintain redox homeostasis are crucial, providing buffering against oxidative stress. Glutathione, the most abundant low molecular weight thiol, is considered the major cellular redox buffer in most cells. To better understand how cells maintain glutathione redox homeostasis, cells of Saccharomyces cerevisiae were treated with extracellular oxidized glutathione (GSSG), and the effect on intracellular reduced glutathione (GSH) and GSSG were monitored over time. Intriguingly cells lacking GLR1 encoding the GSSG reductase in S. cerevisiae accumulated increased levels of GSH via a mechanism independent of the GSH biosynthetic pathway. Furthermore, residual NADPH-dependent GSSG reductase activity was found in lysate derived from glr1 cell. The cytosolic thioredoxin-thioredoxin reductase system and not the glutaredoxins (Grx1p, Grx2p, Grx6p, and Grx7p) contributes to the reduction of GSSG. Overexpression of the thioredoxins TRX1 or TRX2 in glr1 cells reduced GSSG accumulation, increased GSH levels, and reduced cellular glutathione E_h′. Conversely, deletion of TRX1 or TRX2 in the glr1 strain led to increased accumulation of GSSG, reduced GSH levels, and increased cellular E_h′. Furthermore, it was found that purified thioredoxins can reduce GSSG to GSH in the presence of thioredoxin reductase and NADPH in a reconstituted in vitro system. Collectively, these data indicate that the thioredoxin-thioredoxin reductase system can function as an alternative system to reduce GSSG in S. cerevisiae in vivo.     

Induction of Cell Death through Alteration of Oxidants and Antioxidants in Lung Epithelial Cells Exposed to High Energy Protons

Radiation affects several cellular and molecular processes, including double strand breakage and modifications of sugar moieties and bases. In outer space, protons are the primary radiation source that poses a range of potential health risks to astronauts. On the other hand, the use of proton irradiation for tumor radiation therapy is increasing, as it largely spares healthy tissues while killing tumor tissues. Although radiation-related research has been conducted extensively, the molecular toxicology and cellular mechanisms affected by proton irradiation remain poorly understood. Therefore, in this study, we irradiated rat lung epithelial cells with different doses of protons and investigated their effects on cell proliferation and death. Our data show an inhibition of cell proliferation in proton-irradiated cells with a significant dose-dependent activation and repression of reactive oxygen species and antioxidants glutathione and superoxide dismutase, respectively, compared with control cells. In addition, the activities of apoptosis-related genes such as caspase-3 and -8 were induced in a dose-dependent manner with corresponding increased levels of DNA fragmentation in proton-irradiated cells compared with control cells. Together, our results show that proton irradiation alters oxidant and antioxidant levels in cells to activate the apoptotic pathway for cell death.     

Both thioredoxin 2 and glutaredoxin 2 contribute to the reduction of the mitochondrial 2-Cys peroxiredoxin Prx3

The proteins from the thioredoxin family are crucial actors in redox signaling and the cellular response to oxidative stress. The major intracellular source for oxygen radicals are the components of the respiratory chain in mitochondria. Here, we show that the mitochondrial 2-Cys peroxiredoxin (Prx3) is not only substrate for thioredoxin 2 (Trx2), but can also be reduced by glutaredoxin 2 (Grx2) via the dithiol reaction mechanism. Grx2 reduces Prx3 exhibiting catalytic constants (K(m), 23.8 μmol·liter(-1); V(max), 1.2 μmol·(mg·min)(-1)) similar to Trx2 (K(m), 11.2 μmol·liter(-1); V(max), 1.1 μmol·(mg·min)(-1)). The reduction of the catalytic disulfide of the atypical 2-Cys Prx5 is limited to the Trx system. Silencing the expression of either Trx2 or Grx2 in HeLa cells using specific siRNAs did not change the monomer:dimer ratio of Prx3 detected by a specific 2-Cys Prx redox blot. Only combined silencing of the expression of both proteins led to an accumulation of oxidized protein. We further demonstrate that the distribution of Prx3 in different mouse tissues is either linked to the distribution of Trx2 or Grx2. These results introduce Grx2 as a novel electron donor for Prx3, providing further insights into pivotal cellular redox signaling mechanisms.     

Mitochondrial Dysfunction Enhances Susceptibility to Oxidative Stress by Down-Regulation of Thioredoxin in Human Neuroblastoma Cells

Increasing evidence suggests that Alzheimer’s disease is associated with mitochondrial dysfunction and oxidative damage. To develop a cellular model of Alzheimer’s disease, we investigated the effects of thioredoxin (Trx) expression in the response to mitochondrial dysfunction-enhanced oxidative stress in the SH-SY5Y human neuroblastoma cells. Treatment of SH-SY5Y cells with 15 mM of NaN₃, an inhibitor of cytochrome c oxidase (complex IV), led to alteration of mitochondrial membrane potential but no significant changes in cell viability. Therefore, cells were first treated with 15 mM NaN₃ to induce mitochondrial dysfunction, then, exposed to different concentrations of H₂O₂. Cell susceptibility was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay and morphological observation. Expressions of Trx mRNA and protein were determined by RT-PCR; and Western-blot analysis, respectively. It was found that the SH-SY5Y cells with mitochondrial impairment had lower levels of Trx mRNA and protein, and were significantly more vulnerable than the normal cells after exposure to H₂O₂ while no significant changes of Trx mRNA and protein in SH-SY5Y cells exposed to H₂O₂ but without mitochondrial complex IV inhibition. These results, together with our previous study in primary cultured neurons, demonstrated that the increased susceptibility to oxidative stress is induced at least in part by the down-regulation of Trx in SH-SY5Y human neuroblastoma cells with mitochondrial impairment and also suggest the mitochondrial dysfunction-enhanced oxidative stress could be used as a cellular model to study the mechanisms of Alzheimer’s disease and agents for prevention and treatment.     

Inhibition of Cellular Methyltransferases Promotes Endothelial Cell Activation by Suppressing Glutathione Peroxidase 1 Protein Expression

S-Adenosylhomocysteine (SAH) is a negative regulator of most methyltransferases and the precursor for the cardiovascular risk factor homocysteine. We have previously identified a link between the homocysteine-induced suppression of the selenoprotein glutathione peroxidase 1 (GPx-1) and endothelial dysfunction. Here we demonstrate a specific mechanism by which hypomethylation, promoted by the accumulation of the homocysteine precursor SAH, suppresses GPx-1 expression and leads to inflammatory activation of endothelial cells. The expression of GPx-1 and a subset of other selenoproteins is dependent on the methylation of the tRNA^Sec to the Um34 form. The formation of methylated tRNA^Sec facilitates translational incorporation of selenocysteine at a UGA codon. Our findings demonstrate that SAH accumulation in endothelial cells suppresses the expression of GPx-1 to promote oxidative stress. Hypomethylation stress, caused by SAH accumulation, inhibits the formation of the methylated isoform of the tRNA^Sec and reduces GPx-1 expression. In contrast, under these conditions, the expression and activity of thioredoxin reductase 1, another selenoprotein, is increased. Furthermore, SAH-induced oxidative stress creates a proinflammatory activation of endothelial cells characterized by up-regulation of adhesion molecules and an augmented capacity to bind leukocytes. Taken together, these data suggest that SAH accumulation in endothelial cells can induce tRNA^Sec hypomethylation, which alters the expression of selenoproteins such as GPx-1 to contribute to a proatherogenic endothelial phenotype.     

NAD(P)H Cytochrome b5 Oxidoreductase Deficiency in Leishmania major Results in Impaired Linoleate Synthesis Followed by Increased Oxidative Stress and Cell Death

NAD(P)H cytochrome b₅ oxidoreductase (Ncb5or), comprising cytochrome b₅ and cytochrome b₅ reductase domains, is widely distributed in eukaryotic organisms. Although Ncb5or plays a crucial role in lipid metabolism of mice, so far no Ncb5or gene has been reported in the unicellular parasitic protozoa Leishmania species. We have cloned, expressed, and characterized Ncb5or gene from Leishmania major. Steady state catalysis and spectral studies show that NADH can quickly reduce the ferric state of the enzyme to the ferrous state and is able to donate an electron(s) to external acceptors. To elucidate its exact physiological role in Leishmania, we attempted to create NAD(P)H cytochrome b₅ oxidoreductase from L. major (LmNcb5or) knock-out mutants by targeted gene replacement technique. A free fatty acid profile in knock-out (KO) cells reveals marked deficiency in linoleate and linolenate when compared with wild type (WT) or overexpressing cells. KO culture has a higher percentage of dead cells compared with both WT and overexpressing cells. Increased O₂ uptake, uncoupling and ATP synthesis, and loss of mitochondrial membrane potential are evident in KO cells. Flow cytometric analysis reveals the presence of a higher concentration of intracellular H₂O₂, indicative of increased oxidative stress in parasites lacking LmNcb5or. Cell death is significantly reduced when the KO cells are pretreated with BSA bound linoleate. Real time PCR studies demonstrate a higher Δ12 desaturase, superoxide dismutase, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA with a concomitant fall in Δ9 desaturase mRNA expression in LmNcb5or null cell line. Together these findings suggest that decreased linoleate synthesis, and increased oxidative stress and apoptosis are the major consequences of LmNcb5or deficiency in Leishmania.     

Protease Activity of Procaspase-8 Is Essential for Cell Survival by Inhibiting Both Apoptotic and Nonapoptotic Cell Death Dependent on Receptor-interacting Protein Kinase 1 (RIP1) and RIP3

Caspase-8 has an important role as an initiator caspase during death receptor-mediated apoptosis. Moreover, it has been reported to contribute to the regulation of cell fate in various types of cells including T-cells. In this report, we show that caspase-8 has an essential role in cell survival in mouse T-lymphoma-derived L5178Y cells. The knockdown of caspase-8 expression decreased the growth rate and increased cell death, both of which were induced by the absence of protease activity of procaspase-8. The cell death was associated with reactive oxygen species (ROS) accumulation, caspase activation, and autophagosome formation. The cell death was inhibited completely by treatment with ROS scavengers, but only partly by treatment with caspase inhibitors, expression of Bcl-xL, and knockdown of caspase-3 or Atg-7 which completely inhibits apoptosis or autophagosome formation, respectively, indicating that apoptosis and autophagy-associated cell death are induced simultaneously by the knockdown of caspase-8 expression. Further analysis indicated that RIP1 and RIP3 regulate this multiple cell death, because the cell death as well as ROS production was completely inhibited by not only treatment with the RIP1 inhibitor necrostatin-1, but also by knockdown of RIP3. Thus, in the absence of protease activity of procaspase-8, RIP1 and RIP3 simultaneously induce not only nonapoptotic cell death conceivably including autophagic cell death and necroptosis but also apoptosis through ROS production in mouse T-lymphoma cells.     

4-Hydroxynonenal Induces G2/M Phase Cell Cycle Arrest by Activation of the Ataxia Telangiectasia Mutated and Rad3-related Protein (ATR)/Checkpoint Kinase 1 (Chk1) Signaling Pathway

4-Hydroxynonenal (HNE) has been widely implicated in the mechanisms of oxidant-induced toxicity, but the detrimental effects of HNE associated with DNA damage or cell cycle arrest have not been thoroughly studied. Here we demonstrate for the first time that HNE caused G₂/M cell cycle arrest of hepatocellular carcinoma HepG2 (p53 wild type) and Hep3B (p53 null) cells that was accompanied with decreased expression of CDK1 and cyclin B1 and activation of p21 in a p53-independent manner. HNE treatment suppressed the Cdc25C level, which led to inactivation of CDK1. HNE-induced phosphorylation of Cdc25C at Ser-216 resulted in its translocation from nucleus to cytoplasm, thereby facilitating its degradation via the ubiquitin-mediated proteasomal pathway. This phosphorylation of Cdc25C was regulated by activation of the ataxia telangiectasia and Rad3-related protein (ATR)/checkpoint kinase 1 (Chk1) pathway. The role of HNE in the DNA double strand break was strongly suggested by a remarkable increase in comet tail formation and H2A.X phosphorylation in HNE-treated cells in vitro. This was supported by increased in vivo phosphorylation of H2A.X in mGsta4 null mice that have impaired HNE metabolism and increased HNE levels in tissues. HNE-mediated ATR/Chk1 signaling was inhibited by ATR kinase inhibitor (caffeine). Additionally, most of the signaling effects of HNE on cell cycle arrest were attenuated in hGSTA4 transfected cells, thereby indicating the involvement of HNE in these events. A novel role of GSTA4-4 in the maintenance of genomic integrity is also suggested.     

Induction of a Tumor-associated Activating Mutation in Protein Tyrosine Phosphatase Ptpn11 (Shp2) Enhances Mitochondrial Metabolism,Leading to Oxidative Stress and Senescence

Activating mutations in Ptpn11 (Shp2), a protein tyrosine phosphatase involved in diverse cell signaling pathways, are associated with pediatric leukemias and solid tumors. However, the pathogenic effects of these mutations have not been fully characterized. Here, we report that induction of the Ptpn11^E76K/+ mutation, the most common and active Ptpn11 mutation found in leukemias and solid tumors, in primary mouse embryonic fibroblasts resulted in proliferative arrest and premature senescence. As a result, apoptosis was markedly increased. These cellular responses were accompanied and mediated by up-regulation of p53 and p21. Moreover, intracellular levels of reactive oxygen species (ROS), byproducts of mitochondrial oxidative phosphorylation, were elevated in Ptpn11^E76K/+ cells. Since Shp2 is also distributed to the mitochondria (in addition to the cytosol), the impact of the Ptpn11^E76K/+ mutation on mitochondrial function was analyzed. These analyses revealed that oxygen consumption of Ptpn11^E76K/+ cells and the respiratory function of Ptpn11^E76K/+ mitochondria were significantly increased. Furthermore, we found that phosphorylation of mitochondrial Stat3, one of the substrates of Shp2 phosphatase, was greatly decreased in the mutant cells with the activating mutation Ptpn11^E76K/+. This study provides novel insights into the initial effects of tumor-associated Ptpn11 mutations.     

Caspase 1 Activation Is Protective against Hepatocyte Cell Death by Up-regulating Beclin 1 Protein and Mitochondrial Autophagy in the Setting of Redox Stress

Caspase 1 activation can be induced by oxidative stress, which leads to the release of the proinflammatory cytokines IL1β and IL18 in myeloid cells and a potentially damaging inflammatory response. However, little is known about the role of caspase 1 in non-immune cells, such as hepatocytes, that express and activate the inflammasome but do not produce a significant amount of IL1β/IL18. Here we demonstrate that caspase 1 activation protects against cell death after redox stress induced by hypoxia/reoxygenation in hepatocytes. Mechanistically, we show that caspase 1 reduces mitochondrial respiration and reactive oxygen species by increasing mitochondrial autophagy and subsequent clearance of mitochondria in hepatocytes after hypoxia/reoxygenation. Caspase 1 increases autophagic flux through up-regulating autophagy initiator beclin 1 during redox stress and is an important cell survival factor in hepatocytes. We find that during hemorrhagic shock with resuscitation, an in vivo mouse model associated with severe hepatic redox stress, caspase 1 activation is also protective against liver injury and excessive oxidative stress through the up-regulation of beclin 1. Our findings suggest an alternative role for caspase 1 activation in promoting adaptive responses to oxidative stress and, more specifically, in limiting reactive oxygen species production and damage in cells and tissues where IL1β/IL18 are not highly expressed.     

Activation of AMP-activated protein kinase by temozolomide contributes to apoptosis in glioblastoma cells via p53 activation and mTORC1 inhibition

Methylating drugs such as temozolomide (TMZ) are widely used in the treatment of brain tumors including malignant glioblastoma. The mechanism of TMZ-induced glioblastoma cell death and apoptosis, however, is not fully understood. Here, we tested the potential involvement of AMP-activated protein kinase (AMPK) in this process. We found that methylating agents TMZ and N-methyl-N'-nitro-N-nitrosoguanidine induce AMPK activation in primary cultured human glioblastoma and glioblastoma cell lines. TMZ-induced O(6)-methylguanine production is involved in AMPK activation. O(6)-benzylguanine, an O(6)-methylguanine-DNA methyltransferase inhibitor, enhances TMZ-induced O(6)-methylguanine production, leading to enhanced reactive oxygen species production, which serves as an upstream signal for AMPK activation. Activation of AMPK is involved in TMZ-induced glioblastoma cell death and apoptosis. AMPK inhibitor (Compound C) or AMPKα siRNA knockdown inhibits TMZ-induced glioblastoma cell death and apoptosis, whereas AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside enhances it. In further studies, we found that activation of AMPK is involved in TMZ-induced p53 activation and subsequent p21, Noxa, and Bax up-regulation. Activation of AMPK by TMZ also inhibits mTOR complex 1 (mTORC1) signaling and promotes anti-apoptosis protein Bcl-2 down-regulation, which together mediate TMZ-induced pro-cell apoptosis effects. Our study suggests that activation of AMPK by TMZ contributes to glioblastoma cell apoptosis, probably by promoting p53 activation and inhibiting mTORC1 signaling.     

Facilitation of Mitochondrial Outer and Inner Membrane Permeabilization and Cell Death in Oxidative Stress by a Novel Bcl-2 Homology 3 Domain Protein

We identified a sequence homologous to the Bcl-2 homology 3 (BH3) domain of Bcl-2 proteins in SOUL. Tissues expressed the protein to different extents. It was predominantly located in the cytoplasm, although a fraction of SOUL was associated with the mitochondria that increased upon oxidative stress. Recombinant SOUL protein facilitated mitochondrial permeability transition and collapse of mitochondrial membrane potential (MMP) and facilitated the release of proapoptotic mitochondrial intermembrane proteins (PMIP) at low calcium and phosphate concentrations in a cyclosporine A-dependent manner in vitro in isolated mitochondria. Suppression of endogenous SOUL by diced small interfering RNA in HeLa cells increased their viability in oxidative stress. Overexpression of SOUL in NIH3T3 cells promoted hydrogen peroxide-induced cell death and stimulated the release of PMIP but did not enhance caspase-3 activation. Despite the release of PMIP, SOUL facilitated predominantly necrotic cell death, as revealed by annexin V and propidium iodide staining. This necrotic death could be the result of SOUL-facilitated collapse of MMP demonstrated by JC-1 fluorescence. Deletion of the putative BH3 domain sequence prevented all of these effects of SOUL. Suppression of cyclophilin D prevented these effects too, indicating that SOUL facilitated mitochondrial permeability transition in vivo. Overexpression of Bcl-2 and Bcl-x_L, which can counteract the mitochondria-permeabilizing effect of BH3 domain proteins, also prevented SOUL-facilitated collapse of MMP and cell death. These data indicate that SOUL can be a novel member of the BH3 domain-only proteins that cannot induce cell death alone but can facilitate both outer and inner mitochondrial membrane permeabilization and predominantly necrotic cell death in oxidative stress.