Antioxid Redox Signal Research Articles

Peroxiredoxin-1 is an H2O2 safe-guard antioxidant and signalling enzyme in M1 macrophages

Macrophages are characterised by their high plasticity and ability to adapt their phenotype and functionality in response to environmental cues, resulting in a spectrum of activation states the two extremes of which are M1 and M2. Reactive oxygen species, such as hydrogen peroxide, are among the cues that impact macrophage polarisation. Moreover, high levels of hydrogen peroxide play a role in the phagocytic response executed by M1 macrophages. Therefore, macrophages must balance the need to shield themselves from the harmful effects of hydrogen peroxide bursts with the ability to interpret hydrogen peroxide signals from the surroundings and initiate a cellular response. Peroxiredoxins (PRDX) are proteins capable of performing both roles. Specifically, PRDX1 and PRDX5 have been demonstrated to safeguard macrophages against reactive oxygen species while also impacting their polarisation status. Previously conducted studies did not differentiate between the polarisation state of macrophages or investigate the signalling events triggered by PRDXs. In this study, we utilised bone marrow-derived murine macrophages polarised to the M1 and M2 states. Our findings revealed that the expression of PRDX1 was significantly higher in M1 macrophages than in M2 and unpolarised macrophages. Moreover, we present evidence that in M1 macrophages, PRDX1 interacts with ASK1, its established interaction partner, and also binds to other proteins that regulate the cellular antioxidant response. Interestingly, we found that pharmacological elevation of hydrogen peroxide levels leads to an increase in PRDX1 expression on the mRNA level, but not in the highly related PRDX2 expression. Taken together, our findings suggest that PRDX1 plays a critical role in macrophage antioxidant defence and redox signalling, and provide scope for exploring redox-signalling proteins as highly sought-after candidates for macrophage repolarisation.

Selenoprotein: Potential Player in Redox Regulation in Chlamydomonas reinhardtii.

Selenium (Se) is an essential micro-element for many organisms, including Chlamydomonas reinhardtii, and is required in trace amounts. It is obtained from the 21st amino acid selenocysteine (Sec, U), genetically encoded by the UGA codon. Proteins containing Sec are known as selenoproteins. In eukaryotes, selenoproteins are present in animals and algae, whereas fungi and higher plants lack them. The human genome contains 25 selenoproteins, most of which are involved in antioxidant defense activity, redox regulation, and redox signaling. In algae, 42 selenoprotein families were identified using various bioinformatics approaches, out of which C. reinhardtii is known to have 10 selenoprotein genes. However, the role of selenoproteins in Chlamydomonas is yet to be reported. Chlamydomonas selenoproteins contain conserved domains such as CVNVGC and GCUG, in the case of thioredoxin reductase, and CXXU in other selenoproteins. Interestingly, Sec amino acid residue is present in a catalytically active domain in Chlamydomonas selenoproteins, similar to human selenoproteins. Based on catalytical active sites and conserved domains present in Chlamydomonas selenoproteins, we suggest that Chlamydomonas selenoproteins could have a role in redox regulation and defense by acting as antioxidants in various physiological conditions.

Long noncoding RNA MAGI2-AS3 regulates the H2O2 level and cell senescence via HSPA8

The redox homeostasis system regulates many biological processes, intracellular antioxidant production and redox signaling. However, long noncoding RNAs (lncRNAs) involved in redox regulation have rarely been reported. Herein, we reported that downregulation of MAGI2-AS3 decreased the superoxide level in Human fibroblasts (Fbs), a replicative aging model, as detected by the fluorescent probes dihydroethidium (DHE) and MitoSOX™ Red. RNA pulldown combined with mass spectrometry showed that HSPA8 is a novel interacting protein of MAGI2-AS3, which was further confirmed by photoactivatable ribonucleoside–enhanced crosslinking and immunoprecipitation (PAR-CLIP). Downregulation of MAGI2-AS3 decreased the hydrogen peroxide (H2O2) content by stabilizing the HSPA8 protein level via inhibiting the protesome degradation of HSPA8. Further evidence showed that MAGI2-AS3 interacted with the C-terminal domain (CTD) of HSPA8. Downregulation of MAGI2-AS3 delayed cell senescence, while this antiaging effect was abolished by HSPA8 knockdown. The underlying molecular mechanism by which MAGI2-AS3 knockdown inhibited cell senescence was mediated via suppression of the ROS/MAP2K6/p38 signaling pathway. Taken together, these findings revealed that downregulation of lncRNA MAGI2-AS3 decreased the H2O2 content and delayed cell senescence by stabilizing the HSPA8 protein level, identifying a potential antiaging application.

Mitochondrial TOM70 in Hyperoxia‐induced Acute Lung Injury

Study objectiveHyperoxia‐induced acute lung injury (HALI) leads to respiratory failure and mitochondrial dysfunction is a critical mediator of HALI. We previously reported Toll‐Like Receptor (TLR)4 prevents oxidant‐induced ALI by maintaining mitochondrial bioenergetics in mammalian lung endothelium (Ec). Translocase of Outer Mitochondrial Membrane (TOM) have recently been identified to play important roles in mitochondrial quality control, primarily in yeast [1]. TOM70 is an essential subunit of TOM and mediates uptake of newly synthesized proteins to mitochondria as well has putative anti‐oxidant and anti‐apoptotic properties. TOM70 appeared to parallel many of the cytoprotective signaling properties of TLR4 [2‐4]. Therefore, our hypothesis is TLR4 regulates TOM70 expression and TOM70 has mitochondrial and lung protective functions in HALI.MethodsC57BL/6J mice (6‐8 wks, both sex) were purchased from Jackson Laboratory, and Tlr4‐/‐ mice were bred at Duke Animal facilities (IACUC A160‐19‐07) [5]. For hyperoxia exposure, mice were placed in a Plexiglas chamber with 5L/min of 100% oxygen continuous flow with food and water ad libitum. We previously reported 72 h of continuous hyperoxia as a time of maximal lung injury [6], which we used as a representative time point. Bodyweight and survival were monitored daily. Bronchoalveolar lavage (BAL) were collected and BAL protein were determined by a BCA assay. Protein and RNA were extracted from lung tissues and expression was determined by western blot and qPCR, respectively. Mouse lung Ec (MLEC) were isolated from lung tissues and treated with control or Tom70siRNA for 48 h. Statistical differences were analyzed by a Student‐t test.ResultsTlr4‐/‐ MLEC decreased TOM70 and increased Parkin, PINK1, and MFN2 protein expression compared to WT. Tom70 siRNA effectively silenced Tom70 with >75% efficiency at 48 h after transfection. Tom70 siRNA‐treated MLEC showed increased Mfn1, Mff, and Fis1mRNA expression, which are markers of mitochondrial fusion‐fission. Hyperoxia‐exposed mice showed marked weight loss and higher levels of BAL proteins compared to control, indicating increased permeability of the alveolar‐capillary membrane. Moreover, TOM70 protein expression was significantly decreased by hyperoxia in both male and female mice.ConclusionTLR4 deficiency decreased TOM70 expression and induced mitophagy and mitochondrial fusion, indicating that TOM70 is regulated by TLR4. Silencing TOM70 resulted in increased mitochondrial fusion and fission in MLEC, suggesting that TOM70 impacts mitochondrial quality control in mammalian, primary cells (MLEC) that are critical for lung airspace barrier function. Hyperoxia decreased TOM70 protein expression in mouse lungs, which may be a potential mechanism whereby hyperoxia leads to respiratory failure. Given that TOM70 maintains mitochondrial health, our data suggest that TOM70 may play a key role in the pathological mechanisms of HALI.1. Liu et al. (2021) bioRxiv, 2. Zhang et al. (2019) Antioxid Redox Signal, 3. Zhang et al. (2016) Antioxid Redox Signal, 4. Takyar et al. (2015) FASEB J. 5. Kim et al. (2019) Aging Cell. 6. Zhang et al. (2021) AJP Lung Cell Mol Physiol.

Interplay between Selenium, Selenoproteins and HIV-1 Replication in Human CD4 T-Lymphocytes.

The infection of CD4 T-lymphocytes with human immunodeficiency virus (HIV), the etiological agent of acquired immunodeficiency syndrome (AIDS), disrupts cellular homeostasis, increases oxidative stress and interferes with micronutrient metabolism. Viral replication simultaneously increases the demand for micronutrients and causes their loss, as for selenium (Se). In HIV-infected patients, selenium deficiency was associated with a lower CD4 T-cell count and a shorter life expectancy. Selenium has an important role in antioxidant defense, redox signaling and redox homeostasis, and most of these biological activities are mediated by its incorporation in an essential family of redox enzymes, namely the selenoproteins. Here, we have investigated how selenium and selenoproteins interplay with HIV infection in different cellular models of human CD4 T lymphocytes derived from established cell lines (Jurkat and SupT1) and isolated primary CD4 T cells. First, we characterized the expression of the selenoproteome in various human T-cell models and found it tightly regulated by the selenium level of the culture media, which was in agreement with reports from non-immune cells. Then, we showed that selenium had no significant effect on HIV-1 protein production nor on infectivity, but slightly reduced the percentage of infected cells in a Jurkat cell line and isolated primary CD4 T cells. Finally, in response to HIV-1 infection, the selenoproteome was slightly altered.

Nogo-a Pathway Contributes to Neural Injury and Pain in Sickle Cell Disease

Sickle cell disease (SCD) is characterized by chronic pain and bouts of extreme acute pain from vasoocclusive crises (VOC). Sickle pain has both neuropathic and inflammatory features (Tran et al., Blood 2017). Mechanisms underlying neural injury remain unknown in SCD. Neurite outgrowth inhibitor (NOGO-A/reticulon-4) and its receptor NGR1 contribute to pain, neuronal damage, and inhibition of neurite outgrowth (Hu et al., FASEB J 2019). We examined if NOGO-A pathway is activated in a sickle microenvironment and if its inhibition will ameliorate hyperalgesia in BERK sickle mice. We used Rho kinase activity (ROCK) downstream of NGR1 as a readout of activation of NOGO-A/NGR1 pathway. We observed increased expression of NOGO-A (~260%, p<0.05) and NGR1 (~180%, p<0.05) in the dorsal root ganglia, and increased NOGO-A and ROCK activity in spinal cords of sickle mice compared to control mice expressing normal human hemoglobin A. Earlier, we found that an endogenous cannabinoid, palmitoylethanolamide (PEA) inhibits spinal NOGO-A expression and ROCK activity in sickle mice (Argueta et al., ASH 2020 #225). We hypothesized that sickle microenvironment with cell-free heme and inflammation activates NOGO-A/NGR1-ROCK pathway leading to nerve injury and pain, and inhibition of this pathway will ameliorate hyperalgesia in sickle mice.Using terminally differentiated rat pheochromocytoma (PC12) cells, we simulated a sickle microenvironment with hemin (40 µM) and TNFα (1 ng/ml) (H+T). H+T elevated ROCK activity compared to vehicle (~40%, p<0.05). PEA (30 uM) and 2 µM NEP (1-40), a competitive antagonist of NGR1, attenuated H+T-induced ROCK activity (both p<0.01); co-treatment had no additive effect, indicating a common pathway. As well, siRNA (10 nM) knockdown of NGR1 reversed H+T-induced ROCK activity (p<0.001), which was equally effective with 30 µM PEA co-treatment. Functionally, treatment with 30 µM PEA or 2 µM NEP (1-40) enhanced neurite outgrowth in H+T-treated PC-12 cells (~120%, p<0.001). NEP (1-40) at 5 mg/kg reduced mechanical and cold (both ~50%, p<0.001) hyperalgesia in sickle mice compared to baseline (BL) and/or vehicle treatment. Together, these data demonstrate that NOGO-A/NGR1 pathway activation may underlie nerve injury, and inhibition of this pathway with a NGR1 antagonist or PEA promotes neurite outgrowth and reduces hyperalgesia in a sickle microenvironment.We next examined the contribution of exogenously administered and endogenously produced PEA in ameliorating hyperalgesia. Mass spectrometry revealed that spinal PEA is reduced in female (p<0.05) and male (p<0.001) sickle mice compared to age/sex-matched control mice. Exogenous PEA (i.p. 20 mg/kg/d) reduced cold avoidance over a 3-day treatment period, showing significantly more time in the cold chamber compared to BL or vehicle at 1 h, 24 h, and 72 h (p<0.05). The analgesic effect of PEA was maintained for 9 days of treatment without developing tolerance. We next increased endogenous PEA by inhibiting its degradative enzyme, N-acylethanolamine acid amidase, with ARN19702 (i.p. 3, 10, & 30 mg/kg/d), which reduced mechanical (~50%, p<0.001) and cold hyperalgesia (~40%, p<0.001) over 72 hours in a dose-dependent manner. Since hypoxia and ischemia reperfusion injury contribute to acute VOC pain, we incited hypoxia-reoxygenation (HR; 3 h @ 8% O 2, 92% N 2, followed by 1 h @ normoxia) to simulate acute VOC pain. We observed that 5-day pretreatment with PEA (i.p. 20 mg/kg/d) before HR prevented mechanical and cold hyperalgesia following HR in sickle mice. Moreover, treatment with PEA after HR incitement significantly reduced hyperalgesia for 24 h after HR compared to BL (~30%, p<0.001) and vehicle treated (~66%, p<0.001) sickle mice.NGR1 antagonism reduces spinal microglial injury/activation. Heme and TNFα have been shown to cause microglial injury in vitro, while spinal microglial activation has been demonstrated in sickle mice (Lei et al., Antioxid Redox Signal 2021). Thus, NGR1/ROCK cascade may contribute to both neuronal injury and inflammation in the central nervous system leading to neuropathic pain. Our data suggest that PEA and targeting NOGO-A pathway may prevent/reduce chronic and acute hyperalgesia in sickle mice. We speculate that interventions targeting NOGO-A pathway may prevent/reduce neuropathic pain and that PEA has the translational potential for the treatment of chronic and acute pain in SCD. DisclosuresGupta: Tautona Group: Consultancy, Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; 1910 Genetics: Other: Grantee; Grifols: Other: Grantee; SCDAA: Membership on an entity's Board of Directors or advisory committees; CSL Behring LLC: Honoraria; NIH: Other: Grantee; University of Minnestoa Foundation: Other: Philanthropic Funding; Southern California Institute for Research and Education Foundation: Other: Philanthropic Funding; Cyclerion: Research Funding; UCI Foundation: Other: Philanthropic Funding.

Obstructive sleep apnea predicts 10-year cardiovascular disease-related mortality in the Sleep Heart Health Study: a machine learning approach.

Obstructive sleep apnea (OSA) is considered to be an important risk factor for the development of cardiovascular disease (CVD). This study aimed to develop and evaluate a machine learning approach with a set of features for assessing the 10-year CVD mortality risk of the OSA population. This study included 2,464 patients with OSA who met study inclusion criteria and were selected from the Sleep Heart Health Study. We evaluated the importance of potential features by mutual information. The top 9 features were selected to develop a random forest model. We evaluated the model performance on a test set (n = 493) using the area under the receiver operating curve with 95% confidence interval and confusion matrix. A random forest model awarded the highest area under the receiver operating curve of 0.84 (95% confidence interval: 0.78-0.89). The specificity and sensitivity were 73.94% and 81.82%, respectively. Sixty-three years old was a threshold for increased risk of 10-year CVD mortality. Persons with severe OSA had higher risk than those with mild OSA. This study demonstrated that a random forest model can provide a quick assessment of the risk of 10-year CVD mortality. Our model may be more informative for patients with OSA in determining their future CVD mortality risk. Li A, Roveda JM, Powers LS, Quan SF. Obstructive sleep apnea predicts 10-year cardiovascular disease-related mortality in the Sleep Heart Health Study: a machine learning approach. J Clin Sleep Med. 2022;18(2):497-504.

Thiol-based redox-active proteins as cardioprotective therapeutic agents in cardiovascular diseases.

Thiol-based redox compounds, namely thioredoxins (Trxs), glutaredoxins (Grxs) and peroxiredoxins (Prxs), stand as a pivotal group of proteins involved in antioxidant processes and redox signaling. Glutaredoxins (Grxs) are considered as one of the major families of proteins involved in redox regulation by removal of S-glutathionylation and thereby reactivation of other enzymes with thiol-dependent activity. Grxs are also coupled to Trxs and Prxs recycling and thereby indirectly contribute to reactive oxygen species (ROS) detoxification. Peroxiredoxins (Prxs) are a ubiquitous family of peroxidases, which play an essential role in the detoxification of hydrogen peroxide, aliphatic and aromatic hydroperoxides, and peroxynitrite. The Trxs, Grxs and Prxs systems, which reversibly induce thiol modifications, regulate redox signaling involved in various biological events in the cardiovascular system. This review focuses on the current knowledge of the role of Trxs, Grxs and Prxs on cardiovascular pathologies and especially in cardiac hypertrophy, ischemia/reperfusion (I/R) injury and heart failure as well as in the presence of cardiovascular risk factors, such as hypertension, hyperlipidemia, hyperglycemia and metabolic syndrome. Further studies on the roles of thiol-dependent redox systems in the cardiovascular system will support the development of novel protective and therapeutic strategies against cardiovascular diseases.

Abstract 3065: Modeling common aspects of the metabolic response of cancer cells to ionizing radiation

Abstract Flexibility and reprogramming of cancer metabolism supports cancer progression and therapy resistance. In previous work we described opportunities for overcoming environment-induced resistance to ionizing radiation (IR) by pharmacologic inhibition of metabolic processes [1,2,3]. Here, we hypothesized that certain aspects of cancer metabolism will support the repair of radiation-induced DNA double-strand breaks (DSBs) and that the identification of pathways critical to the repair of radiation-induced DNA damage and cell survival will allow to enhance sensitivity of cancer cells to IR by interfering with such metabolic liabilities. Yet little is known about metabolic aspects of the cancer cell response to IR. We therefore systematically screened a panel of cancer cell lines with distinct genetic backgrounds for time-dependent changes in their acute metabolic response to IR and recorded the kinetics of IR-induced DSB in parallel. Mathematical modelling of the obtained metabolic and radiobiological data revealed a common metabolic response of irradiated cancer cells with impact on the kinetics of DSB repair. These were characterized by a rapid but transient downregulation of glycolysis and mitochondrial respiration, and gradual reactivation of glycolysis prior to mitochondrial recovery, presumably to compensate for disturbed metabolic functions of impaired mitochondria. Application of fractionated IR doses retarded mitochondrial recovery, highlighting the inhibitory effects of IR on mitochondrial functions. Furthermore, combining IR with inhibitors of compensatory glycolysis slowed mitochondrial recovery and DNA repair kinetics, supporting the functional relevance of compensatory glycolysis for DNA repair. Mathematical modeling of the metabolic response to IR allowed us to generate new hypotheses on the metabolic requirements of irradiated cancer cells and potential associated metabolic vulnerabilities. Further mechanistic studies will reveal the role of oncogenic drivers and co-mutations in metabolic enzymes for metabolic flexibility and metabolic liabilities of irradiated cancer cells in support of personalized approaches for combinatorial treatments. [1] Matschke J, et al., Antioxid Redox Signal 2016; 25: 89-107. [2] Hlouschek J et al., Front Oncol 2018; 8: 170. [3] Hlouschek J et al., Cancer Lett 2018; 439: 24-38. Supported by grants of the DFG (GRK1739/2 to V.J. and MA 8970/1-1 to J.M.), the Deutsche Krebshilfe/Mildred Scheel-Stiftung (70112711 to V.J.). Citation Format: Adam Krysztofiak, Klaudia A. Szymonowicz, Julian Hlouschek, Christoph Waterkamp, Kexu Xiang, Daniel Hoffmann, Verena Jendrossek, Johann Matschke. Modeling common aspects of the metabolic response of cancer cells to ionizing radiation [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 3065.

Oxidative Stress in the Early Stage of Psychosis.

In the past few decades, increasing evidence in the literature has appeared describing the role of the antioxidant defense system and redox signaling in the multifactorial pathophysiology of psychosis. It is of interest to clinicians and researchers alike that abnormalities of the antioxidant defense system are associated with alterations of cellular membranes, immune functions and neurotransmission, all of which have some clinical implications. This narrative review summarizes the evidence regarding oxidative stress in the early stages of psychosis. We included 136 peer-reviewed articles published from 2007 to 2020 on PubMed EMBASE, The Cochrane Library and Google Scholar. Patients affected by psychotic disorders show a decreased level of non-enzymatic antioxidants, an increased level of lipid peroxides, nitric oxides, and a homeostatic imbalance of purine catabolism. In particular, a significantly reduced antioxidant defense has been described in the early onset first episode of psychosis, including reduced levels of glutathione. Also, it has been shown that a decreased basal low-antioxidant capacity correlates with cognitive deficits and negative symptoms, mostly related to glutamate-receptor hypofunction. In addition, atypical antipsychotic drugs seem to show significant antioxidant activity. These factors are critical in order to treat cases of first-onset psychosis effectively. This systematic review indicates the importance that must be given to anti-oxidant defense systems.

Abstract PO-022: Inhibition of the citrate carrier SLC25A1 affects repair of radiation-induced DNA damage, increases radiosensitivity, and enhances lethality of IR in combination with DNA repair defects or DSB inhibitors

Abstract Introduction: Balancing redox-homeostasis and energy metabolism allows cancer cells exposed to chemotherapy or radiotherapy to ensure DNA repair and survival. Our work revealed that adaptive changes in antioxidant defense and cancer cell metabolism in adverse environments enhance cancer cell radioresistance and create exploitable therapeutic metabolic dependencies for overcoming adaptive radioresistance1-4. Here, we aimed to decipher the mechanisms behind enhances lethality of ionizing radiation (IR) upon SLC25A1-inhibition (SLC25A1i) beyond disturbance of mitochondrial metabolism. Therefore, we explored a potential impact of SLC25A1i on the suggested accumulation of D-2-hydroxyglutarate (D2HG) and the repair of IR-induced lethal DNA lesions with a focus on homologous recombination (HR) repair. We hypothesized that metabolic disturbance of HR by SLC25A1i might offer opportunities for context-dependent lethality approaches. Methods: We used genetic (SLC25A1 RNAi) or pharmacologic SLC25A1i (small molecule SLC25A1 inhibitor CTPi2) to determine the effects of SLC25A1i alone or in combination with IR on 2HG-accumulation, cellular function, radiosensitivity and DNA repair kinetics in lung (A549, NCI-H460) and glioblastoma (U87-MG, T98G) cell lines. Isogenic MEF cell lines with defects in HR repair or non-homologous end joining (NHEJ) DSB repair were used to explore potential differences in the radiosensitizing effects of SLC25A1i in cells with defects in specific DSB repair pathways. Finally, we analyzed the potential of SLC25A1i to induce context-dependent lethality of IR with clinically relevant inhibitors of poly(ADP-ribose)-polymerase (PARP) or of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in cancer cells. Results: Genetic and pharmacologic SLC25A1i increased D2HG-levels, and sensitized lung cancer and glioblastoma cells to the cytotoxic action of IR. Radiosensitization was abrogated in MEFs with a HR defect. Accumulation of D2HG was associated with increased DNA damage and delayed resolution of IR-induced gH2AX and Rad51 foci. Combining SLC25A1i with PARP- or DNA-PKcs-inhibitors further delayed DNA repair kinetics of IR-induced DNA damage and enhanced the radiation-induced eradication of clonogenic tumor cells. Conclusion: The metabolic effects of SLC25A1i involve disturbance of energy metabolism and inhibition of HR execution. SLC25A1i increases the efficacy of IR in cancer cells but not in MEFs with a defect in HR. SLC25A1i might offer opportunities for the metabolic induction of HRness and thus increased lethality of radiotherapy in cells with defects in end-joining pathways or in combination with clinically relevant DSB repair inhibitors in cancer cells without or weak HR defects. 1 Matschke et al., Antioxid Redox Signal 2016,25:89-107, 2 Matschke et al., Radiat Oncol 2016,11(1):75, 3 Hlouschek et al., Cancer Lett. 2018 Dec 28;439:24-38, 4 Hlouschek et al., Front Oncol. 2018 May 25;8:170 Supported by grants of the DFG (GRK1739/2, MA 8970/1-1) and Mildred Scheel-Stiftung (70112711). Citation Format: Christian Kalthoff, Kexu Xiang, Corinna Muench, Julian Hlouschek, Verena Jendrossek, Johann Matschke. Inhibition of the citrate carrier SLC25A1 affects repair of radiation-induced DNA damage, increases radiosensitivity, and enhances lethality of IR in combination with DNA repair defects or DSB inhibitors [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-022.

P0988THE PROTECTIVE EFFECT OF RED ORANGE AND LEMON EXTRACT (RLE) IN DIABETIC OBESE ZUCKER RATS IS RELATED TO ITS ACTION ON THE NADPH OXIDASE SUBUNIT

Abstract Background and Aims Type 2 diabetes mellitus is a pathological and chronical condition that induce a rearrangement in carbohydrate metabolism (Kaneto H et al. Antioxid Redox Signal. 2007). Even if the pathological mechanism of DN is not yet clear, NADPH oxidase (NOX) has been demonstrated to be involved in kidney ROS (Reactive Oxygen Species) generation during diabetic state (Palicz A. et al. J Biol Chem. 2001). Therefore, NOX-4, whose p22-phox subunit have shown a pivotal role in diabetes development, has been considered a potential pharmacological target in DN (Gill PS and Wilcox CS. Antioxid Redox Signal. 2006). In previous experiments performed on diabetic rat animal model with DN, we have found a beneficial effect of an extract rich in anthocyanins (ANT), members of flavonoids group (Shih PH. J Agric Food Chem. 2007). The aim of the present work was to investigate the molecular mechanisms involved in the DN and the protective effects of the new extract particularly abundant in ANT, the Red orange and Lemon Extract (RLE). Method Zucker diabetic fatty rats were divided into three groups and treated by gavage daily until 30 weeks of age, as follows: control group (ZF rats) received 1 mL of normal saline; ZDF Group (ZDF rats) received 1mL of normal saline; ZDF + RLE group (ZDF rats) treated with 90 mg/ kg of RLE dissolved in 1mL of normal saline. We examined the gene and protein expression of NOX4 catalytic subunit and its regulatory subunit p22-phox through Real Time PCR and western blot analysis. Results The expression gene levels of NOX4 significantly increased in diabetic rats ZDF, compared to control ZF (***p&amp;lt;0.001). Exposure of ZDF rats with RLE induces a decreased expression of NOX4 (Figure 1) compared to ZDF. On the contrary, there were not significant expression levels variations for regulatory subunit p22-phox. Western blot results are in agreement with Real Time PCR data, in fact an increase in NOX4 protein levels (*p&amp;lt;0.05) is evident, but no significant increase in p22-phox subunit was observed. Conclusion In conclusion, we hypothesize that the protective effect of RLE on the development of DN is related to its ability to inhibit NADPH oxidase activity and opens new perspectives for the treatment of DN.

Multi-targeted trehalose-6-phosphate phosphatase I harbors a novel peroxisomal targeting signal 1 and is essential for flowering and development

Main conclusionThis work reveals information about new peroxisomal targeting signals type 1 and identifies trehalose-6-phosphate phosphatase I as multitargeted and is implicated in plant development, reproduction, and stress response.A putative, non-canonical peroxisomal targeting signal type 1 (PTS1) Pro-Arg-Met > was identified in the extreme C-terminus of trehalose-6-phosphate phosphatase (TPP)I. TPP catalyzes the final step of trehalose synthesis, and the enzyme was previously characterized to be nuclear only (Krasensky et al. in Antioxid Redox Signal 21(9):1289–1304, 2014). Here we show that the TPPI C-terminal decapeptide ending with Pro-Arg-Met > or Pro-Lys-Met > can indeed function as a PTS1. Upon transient expression in two plant expression systems, the free C- or N-terminal end led to the full-length TPPI targeting to peroxisomes and plastids, respectively. The nucleus and nucleolus targeting of the full-length TPPI was observed in both cases. The homozygous T-DNA insertion line of TPPI showed a pleiotropic phenotype including smaller leaves, shorter roots, delayed flowering, hypersensitivity to salt, and a sucrose dependent seedling development. Our results identify novel PTS1s, and TPPI as a protein multi-targeted to peroxisomes, plastids, nucleus, and nucleolus. Altogether our findings implicate an essential role for TPPI in development, reproduction, and cell signaling.

A Novel Thioredoxin-Dependent Peroxiredoxin (TPx-Q) Plays an Important Role in Defense Against Oxidative Stress and Is a Possible Drug Target in Babesia microti.

Thioredoxin peroxidases (TPxs) are ubiquitous cysteine-based peroxidases that reduce peroxides as part of antioxidant defenses and redox signaling and are essential for Babesia microti protection against adverse environment agents like reactive oxygen species (ROS) and reactive nitrogen species (RNS). To better systematically understand TPxs, we identified a novel 2-Cys peroxiredoxin-Q (BmTPx-Q) of B. microti. The full-length BmTPx-Q gene is 653 bp that consists of an intact open reading frame of 594 bp that encodes a 197-amino acid protein. The predicted protein has a molecular weight of 22.3 kDa and an isoelectric point of 9.18. Moreover, BmTPx-Q showed low identity at the amino acid level to other peroxiredoxins (Prxs) among the currently known subfamilies. The recombinant BmTPx-Q protein (rBmTPx-Q) was expressed in Escherichia coli and purified with beads. The native protein BmTPx-Q was detected using mouse anti-BmTPx-Q polyclonal serum with western blotting and indirect immunofluorescence assay (IFA). In addition, enzyme activity was observed using nicotinamide adenine dinucleotide phosphate (NADPH) as substrate and triggered the NADPH-dependent reduction of the Trx/TrxR system. It was also discovered that BmTPx-Q mainly exists as a monomer whether under its native or functional states. In addition, when incubated with Chloroquine diphosphate salt for 24 h in vitro, the expression of BmTPx-Q showed a marked downward trend with the increase of drug concentration. These results suggest that B. microti uses BmTPx-Q to reduce and detoxify hydrogen peroxides to survive and proliferate inside the host. Furthermore, BmTPx-Q showed the lowest identity with host enzymes and could be a potential drug target for the development of novel strategies to control B. microti infection.

Direct Observation of Methylmercury and Auranofin Binding to Selenocysteine in Thioredoxin Reductase.

Selenoenzymes, containing a selenocysteine (Sec) residue, fulfill important roles in biology. The mammalian thioredoxin reductase selenoenzymes are key regulators of antioxidant defense and redox signaling and are inhibited by methylmercury species and by the gold-containing drug auranofin. It has been proposed that such inhibition is mediated by metal binding to Sec in the enzyme. However, direct structural observations of these classes of inhibitors binding to selenoenzymes have been few to date. Here we therefore have used extended X-ray absorption fine structure as a direct structural probe to investigate binding to the selenium site in recombinant rat thioredoxin reductase 1 (TrxR1). The results demonstrate for the first time the direct and complete binding of the metal atom of the inhibitors to the selenium atom in TrxR1 for both methylmercury and auranofin, indicating that TrxR1 inhibition indeed can be attributed to such direct metal-selenium binding.

Correction to: Redox-Driven Signaling: 2-Oxo Acid Dehydrogenase Complexes as Sensors and Transmitters of Metabolic Imbalance by Victoria I. Bunik. Antioxid Redox Signal 30:1911-1947, 2019. DOI: 10.1089/ars.2017.7311.

Antioxidants & Redox SignalingVol. 31, No. 9 CorrectionFree AccessCorrection to: Redox-Driven Signaling: 2-Oxo Acid Dehydrogenase Complexes as Sensors and Transmitters of Metabolic Imbalance by Victoria I. Bunik. Antioxid Redox Signal 30:1911–1947, 2019. DOI: 10.1089/ars.2017.7311is erratum ofRedox-Driven Signaling: 2-Oxo Acid Dehydrogenase Complexes as Sensors and Transmitters of Metabolic ImbalancePublished Online:25 Jul 2019https://doi.org/10.1089/ars.2017.7311.correxAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail In the June 1, 2019 issue of Antioxidants & Redox Signaling (vol. 30, no. 16; 1911–1947) the article entitled “Redox-Driven Signaling: 2-Oxo Acid Dehydrogenase Complexes as Sensors and Transmitters of Metabolic Imbalance” by Victoria I. Bunik requires correction.Dr. Schirris was not listed as a reviewing editor.The list of reviewing editors originally read:Reviewing Editors: Miguel A. Aon, Rui Benfeitas, Enrique Cadenas, Jose Manuel Cuezva, Arne Holmgren, Saijaliisa Kangasjärvi, John Mieyal, and Andras PerlDr. Schirris has now been added to the list:Reviewing Editors: Miguel A. Aon, Rui Benfeitas, Enrique Cadenas, Jose Manuel Cuezva, Arne Holmgren, Saijaliisa Kangasjärvi, John Mieyal, Andras Perl, and Tom J.J. SchirrisThe online version has been corrected to reflect this.FiguresReferencesRelatedDetailsRelated articlesRedox-Driven Signaling: 2-Oxo Acid Dehydrogenase Complexes as Sensors and Transmitters of Metabolic Imbalance11 Apr 2019Antioxidants & Redox Signaling Volume 31Issue 9Sep 2019 InformationCopyright 2019, Mary Ann Liebert, Inc., publishersTo cite this article:Correction to: Redox-Driven Signaling: 2-Oxo Acid Dehydrogenase Complexes as Sensors and Transmitters of Metabolic Imbalance by Victoria I. Bunik. Antioxid Redox Signal 30:1911–1947, 2019. DOI: 10.1089/ars.2017.7311.Antioxidants & Redox Signaling.Sep 2019.671-671.http://doi.org/10.1089/ars.2017.7311.correxPublished in Volume: 31 Issue 9: July 25, 2019PDF download

Selenium, Selenoproteins and Viral Infection.

Reactive oxygen species (ROS) are frequently produced during viral infections. Generation of these ROS can be both beneficial and detrimental for many cellular functions. When overwhelming the antioxidant defense system, the excess of ROS induces oxidative stress. Viral infections lead to diseases characterized by a broad spectrum of clinical symptoms, with oxidative stress being one of their hallmarks. In many cases, ROS can, in turn, enhance viral replication leading to an amplification loop. Another important parameter for viral replication and pathogenicity is the nutritional status of the host. Viral infection simultaneously increases the demand for micronutrients and causes their loss, which leads to a deficiency that can be compensated by micronutrient supplementation. Among the nutrients implicated in viral infection, selenium (Se) has an important role in antioxidant defense, redox signaling and redox homeostasis. Most of biological activities of selenium is performed through its incorporation as a rare amino acid selenocysteine in the essential family of selenoproteins. Selenium deficiency, which is the main regulator of selenoprotein expression, has been associated with the pathogenicity of several viruses. In addition, several selenoprotein members, including glutathione peroxidases (GPX), thioredoxin reductases (TXNRD) seemed important in different models of viral replication. Finally, the formal identification of viral selenoproteins in the genome of molluscum contagiosum and fowlpox viruses demonstrated the importance of selenoproteins in viral cycle.

Peroxiredoxin expression and redox status in neutrophils and HL-60 cells

Peroxiredoxins (Prxs) are thiol peroxidases with a key role in antioxidant defense and redox signaling. They could be important in neutrophils for handling the large amount of oxidants that these cells produce. We investigated the redox state of Prx1 and Prx2 in HL-60 promyelocytic cells differentiated to neutrophil-like cells (dHL-60) and in human neutrophils. HL-60 cell differentiation with dimethyl sulfoxide caused a large decrease in expression of both Prxs, and all-trans retinoic acid also decreased Prx1 expression. Prx1 was mostly reduced in dHL-60 cells. NADPH oxidase activation by phorbol myristate acetate (PMA) or ingestion of Staphylococcus aureus induced rapid oxidation to disulfide-linked dimers, and eventually hyperoxidation. The NADPH oxidase inhibitor, diphenyleneiodonium, prevented Prx1 dimerization in stimulated dHL-60 cells, and decreased the extent of oxidation under resting conditions. In contrast, Prx1 and Prx2 were present in neutrophils from human blood as disulfides, and PMA or S. aureus caused no further oxidation. They remained oxidized on incubation with diphenyleneiodonium in media. Although this suggests that Prx redox cycling could be deficient in neutrophils, thioredoxin expression and thioredoxin reductase activity were similar in neutrophils and dHL-60 cells. Additionally, neutrophil thioredoxin was initially reduced and underwent oxidation after PMA activation. Thus, although the Prxs respond to oxidant generation in dHL-60 cells, in neutrophils they appear “locked” as disulfides. On this basis we propose that neutrophil Prxs are inefficient antioxidants and contribute little to peroxide removal during the oxidative burst, and speculate that they might be involved in other cell processes.

Regulation of the Proton Leak in Mitochondria

Mechanisms of proton transport regulation in mitochondria Elena E. Pohl Institute of Physiology, Pathophysiology and Biophysics, University of Veterinary Medicine Vienna Protein-mediated proton transport contributes to the proton-motive force in mitochondria and is therefore important for the variety of voltage-dependent processes, including the production of ATP and heat. Several membrane proteins such as uncoupling proteins (UCP), ATP/ADP carrier (ANT), and phosphate carrier are associated with H+ transport. It is tightly regulated; however, the underlying molecular mechanisms are highly debated1,2. I discuss existing models of UCP activation and inhibition, as well as proposed regulators. References 1 Macher, G. et al. Inhibition of mitochondrial UCP1 and UCP3 by purine nucleotides and phosphate. Biochim Biophys Acta 1860, 664-672, https://doi.org/10.1016/j.bbamem.2017.12.001 (2018). 2 Jezek, P., Holendova, B., Garlid, K. D. & Jaburek, M. Mitochondrial uncoupling proteins: subtle regulators of cellular redox signaling. Antioxid Redox Signal, https://doi.org/10.1089/ars.2017.7225 (2018).

Correction to: Suppression of Lipogenesis via Reactive Oxygen Species-AMPK Signaling for Treating Malignant and Proliferative Disease, by Fan XX, Leung ELH, Xie Y, Liu ZQ, Zheng YF, Yao ZJ, Lu LL, Wu JL, He JX, Yuan ZW, Fu J, Wei CL, Huang J, Xiao DK, Luo LX, Jiang ZB, Zhou YL, Kam RKT, and Liu L. Antioxid Redox Signal 28:

Antioxidants & Redox SignalingVol. 30, No. 4 CorrectionFree AccessCorrection to: Suppression of Lipogenesis via Reactive Oxygen Species–AMPK Signaling for Treating Malignant and Proliferative Disease, by Fan XX, Leung ELH, Xie Y, Liu ZQ, Zheng YF, Yao ZJ, Lu LL, Wu JL, He JX, Yuan ZW, Fu J, Wei CL, Huang J, Xiao DK, Luo LX, Jiang ZB, Zhou YL, Kam RKT, and Liu L. Antioxid Redox Signal 28: 339–357, 2018. DOI: 10.1089/ars.2017.7090is erratum ofSuppression of Lipogenesis via Reactive Oxygen Species–AMPK Signaling for Treating Malignant and Proliferative DiseasesPublished Online:3 Jan 2019https://doi.org/10.1089/ars.2017.7090.correxAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail In the February 10, 2018 issue of Antioxidants & Redox Signaling (vol. 28, no. 5, pages 339–357), in the article entitled Suppression of Lipogenesis via Reactive Oxygen Species–AMPK Signaling for Treating Malignant and Proliferative Diseases by Xing-Xing Fan et al., Figures 2B and 7B appeared incorrectly.In Figure 2B the panels labeled “Model” and “MTX” mistakenly used the same image. A revised figure has been supplied with the correct panel for “MTX.”Original Figure 2BCorrected Figure 2BFor Figure 7B, an error in the labeling was discovered. “Anti-arthritis” was used where it should have read “Arthritis.”Original Figure 7BCorrected Figure 7BThe online version of both figures in the article have been corrected to reflect these changes.The authors wish to apologize for the error.FiguresReferencesRelatedDetailsRelated articlesSuppression of Lipogenesis via Reactive Oxygen Species–AMPK Signaling for Treating Malignant and Proliferative Diseases14 Dec 2018Antioxidants & Redox Signaling Volume 30Issue 4Feb 2019 InformationCopyright 2018, Mary Ann Liebert, Inc., publishersTo cite this article:Correction to: Suppression of Lipogenesis via Reactive Oxygen Species–AMPK Signaling for Treating Malignant and Proliferative Disease, by Fan XX, Leung ELH, Xie Y, Liu ZQ, Zheng YF, Yao ZJ, Lu LL, Wu JL, He JX, Yuan ZW, Fu J, Wei CL, Huang J, Xiao DK, Luo LX, Jiang ZB, Zhou YL, Kam RKT, and Liu L. Antioxid Redox Signal 28: 339–357, 2018. DOI: 10.1089/ars.2017.7090.Antioxidants & Redox Signaling.Feb 2019.710-710.http://doi.org/10.1089/ars.2017.7090.correxPublished in Volume: 30 Issue 4: January 3, 2019PDF download