Molecular basis for reduced estrone sulfate transport and altered modulator sensitivity of transmembrane helix (TM) 6 and TM17 mutants of multidrug resistance protein 1 (ABCC1)

Multidrug resistance protein 1 (MRP1) confers drug resistance and also mediates cellular efflux of many organic anions. MRP1 also transports glutathione (GSH); furthermore, this tripeptide stimulates transport of several substrates, including estrone 3-sulfate. We have previously shown that mutations of Lys(332) in transmembrane helix (TM) 6 and Trp(1246) in TM17 cause different substrate-selective losses in MRP1 transport activity. Here we have extended our characterization of mutants K332L and W1246C to further define the different roles these two residues play in determining the substrate and inhibitor specificity of MRP1. Thus, we have shown that TM17-Trp(1246) is crucial for conferring drug resistance and for binding and transport of methotrexate, estradiol glucuronide, and estrone 3-sulfate, as well as for binding of the tricyclic isoxazole inhibitor N-[3-(9-chloro-3-methyl-4-oxo-4H-isoxazolo-[4,3-c]quinolin-5-yl)-cyclohexylmethyl]-benzamide (LY465803). In contrast, TM6-Lys(332) is important for enabling GSH and GSH-containing compounds to serve as substrates (e.g., leukotriene C(4)) or modulators (e.g., S-decyl-GSH, GSH disulfide) of MRP1 and, further, for enabling GSH (or S-methyl-GSH) to enhance the transport of estrone 3-sulfate and increase the inhibitory potency of LY465803. On the other hand, both mutants are as sensitive as wild-type MRP1 to the non-GSH-containing inhibitors (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK571), 1-[2-hydroxy-3-propyl-4-[4-(1H-tetrazol-5-yl)butoxy]phenyl]-ethanone (LY171883), and highly potent 6-[4'-carboxyphenylthio]-5[S]-hydroxy-7[E], 11[Z]14[Z]-eicosatetrenoic acid (BAY u9773). Finally, the differing abilities of the cysteinyl leukotriene derivatives leukotriene C(4), D(4), and F(4) to inhibit estradiol glucuronide transport by wild-type and K332L mutant MRP1 provide further evidence that TM6-Lys(332) is involved in the recognition of the gamma-Glu portion of substrates and modulators containing GSH or GSH-like moieties.

Nrf2 activation supports cell survival during hypoxia and hypoxia/reoxygenation in cardiomyoblasts; the roles of reactive oxygen and nitrogen species

Adaptive mechanisms involving upregulation of cytoprotective genes under the control of transcription factors such as Nrf2 exist to protect cells from permanent damage and dysfunction under stress conditions. Here we explore of the hypothesis that Nrf2 activation by reactive oxygen and nitrogen species modulates cytotoxicity during hypoxia (H) with and without reoxygenation (H/R) in H9C2 cardiomyoblasts. Using MnTBap as a cell permeable superoxide dismutase (SOD) mimetic and peroxynitrite scavenger and L-NAME as an inhibitor of nitric oxide synthase (NOS), we have shown that MnTBap inhibited the cytotoxic effects of hypoxic stress with and without reoxygenation. However, L-NAME only afforded protection during H. Under reoxygenation, conditions, cytotoxicity was increased by the presence of L-NAME. Nrf2 activation was inhibited independently by MnTBap and L-NAME under H and H/R. The increased cytotoxicity and inhibition of Nrf2 activation by the presence of L-NAME during reoxygenation suggests that NOS activity plays an important role in cell survival at least in part via Nrf2-independent pathways. In contrast, O2 -• scavenging by MnTBap prevented both toxicity and Nrf2 activation during H and H/R implying that toxicity is largely dependent on O2 -.To confirm the importance of Nrf2 for myoblast metabolism, Nrf2 knockdown with siRNA reduced cell survival by 50% during 4h hypoxia with and without 2h of reoxygenation and although cellular glutathione (GSH) was depleted during H and H/R, GSH loss was not exacerbated by Nrf2 knockdown. These data support distinctive roles for ROS and RNS during H and H/R for Nrf2 induction which are important for survival independently of GSH salvage. © 2013 The Authors.

Development of a neurotoxicity test-system, using human post-mitotic, astrocytic and neuronal cell lines in co-culture

Astrocytes are essential for neuronal function and survival, so both cell types were included in a human neurotoxicity test-system to assess the protective effects of astrocytes on neurons, compared with a culture of neurons alone. The human NT2.D1 cell line was differentiated to form either a co-culture of post-mitotic NT2.N neuronal (TUJ1, NF68 and NSE positive) and NT2.A astrocytic (GFAP positive) cells (∼2:1 NT2.A:NT2.N), or an NT2.N mono-culture. Cultures were exposed to human toxins, for 4 h at sub-cytotoxic concentrations, in order to compare levels of compromised cell function and thus evidence of an astrocytic protective effect. Functional endpoints examined included assays for cellular energy (ATP) and glutathione (GSH) levels, generation of hydrogen peroxide (H2O2) and caspase-3 activation. Generally, the NT2.N/A co-culture was more resistant to toxicity, maintaining superior ATP and GSH levels and sustaining smaller significant increases in H2O2 levels compared with neurons alone. However, the pure neuronal culture showed a significantly lower level of caspase activation. These data suggest that besides their support for neurons through maintenance of ATP and GSH and control of H2O2 levels, following exposure to some substances, astrocytes may promote an apoptotic mode of cell death. Thus, it appears the use of astrocytes in an in vitro predictive neurotoxicity test-system may be more relevant to human CNS structure and function than neuronal cells alone. © 2007 Elsevier Ltd. All rights reserved.

Clinical oxidative stress during leprosy multidrug therapy:impact of dapsone oxidation

This study aims to assess the oxidative stress in leprosy patients under multidrug therapy (MDT; dapsone, clofazimine and rifampicin), evaluating the nitric oxide (NO) concentration, catalase (CAT) and superoxide dismutase (SOD) activities, glutathione (GSH) levels, total antioxidant capacity, lipid peroxidation, and methemoglobin formation. For this, we analyzed 23 leprosy patients and 20 healthy individuals from the Amazon region, Brazil, aged between 20 and 45 years. Blood sampling enabled the evaluation of leprosy patients prior to starting multidrug therapy (called MDT 0) and until the third month of multidrug therapy (MDT 3). With regard to dapsone (DDS) plasma levels, we showed that there was no statistical difference in drug plasma levels between multibacillary (0.518±0.029 μg/mL) and paucibacillary (0.662±0.123 μg/mL) patients. The methemoglobin levels and numbers of Heinz bodies were significantly enhanced after the third MDTsupervised dose, but this treatment did not significantly change the lipid peroxidation and NO levels in these leprosy patients. In addition, CAT activity was significantly reduced in MDT-treated leprosy patients, while GSH content was increased in these patients. However, SOD and Trolox equivalent antioxidant capacity levels were similar in patients with and without treatment. These data suggest that MDT can reduce the activity of some antioxidant enzyme and influence ROS accumulation, which may induce hematological changes, such as methemoglobinemia in patients with leprosy. We also explored some redox mechanisms associated with DDS and its main oxidative metabolite DDS-NHOH and we explored the possible binding of DDS to the active site of CYP2C19 with the aid of molecular modeling software. © 2014 Schalcher et al.

Modified lipids from LDL in the blood during mid-life increase blood brain barrier permeability

Low density lipoprotein levels (LDL) are consistently elevated in cardiovascular disease. It has been suggested that those with high circulating LDL levels in mid-life may be susceptible to develop neurodegenerative diseases in later life. In the circulation, high levels of LDL are associated with increased oxidative modification (oxLDL) and nitration. We have investigated the hypothesis that disruption of blood brain barrier function by oxLDL and their lipids may increase risk of neurodegeneration in later life and that statin intervention in mid-life can mitigate the neurodegenerative effects of hyperlipidaemia. Blood from statin-naïve, normo- and hyperlipidaemic subjects (n=10/group) was collected at baseline. Hyperlipidaemic subjects received statin-intervention whereas normolipidaemic subjects did not prior to a second blood sampling, taken after 3 months. The intervention will be completed in June 2013. Plasma was separated by centrifugation (200g, 30min) and LDL was isolated by potassium bromide density gradient ultracentrifugation. Total homocysteine, LDL cholesterol, 8-isoprostane F2α levels were measured in plasma using commercial kits. LDL were analysed by agarose gel electrophoresis. LDL-lipids were extracted by partitioning in 1:1 chloroform:methanol (v/v) and conjugated to fatty acid free-BSA in serum-free EGM-2 medium (4hrs, 370C) for co-culture with human microvascular endothelial cells (HMVEC). HMVEC were maintained on polycarbonate inserts for two weeks to create a microvascular barrier. Change in barrier permeability was measured by trans-endothelial electrical resistance (TER), FITC-dextran permeability and immunohistochemistry. HMVEC glutathione (GSH) levels were measured after 2 hours by GSH-glo assay. LDL isolated from statin-naïve hyperlipidaemic subjects had higher mobility by agarose gel electrophoresis (Rf;0.53±0.06) and plasma 8-isoprostane F2α (43.5±8.42 pg/ml) compared to control subjects (0.46±0.05 and 24.2±5.37 pg/ml; p<0.05). Compared to HMVEC treatment with the LDL-lipids (5μM) from normolipidaemic subjects, LDL-lipids from hyperlipidaemic subjects increased barrier permeability (103.4±12.5 Ωcm2 v 66.7±7.3 Ωcm2,P<0.01) and decreased GSH (18.5 nmol/mg v 12.3 nmol/mg; untreated cells 26.2±3.6 nmol/mg).

Synthesis and application of novel probes for the detection of cysteine sulphenic acids

Cysteine is a thiol containing amino acid that readily undergoes oxidation by reactive oxygen species (ROS) to form sulphenic (R-SOH) sulphinic (RSO2H) and sulphonic (RSO3H) acids. Thiol modifications of cysteine have been implicated as modulators of cellular processes and represent significant biological modifications that occur during oxidative stress and cell signalling. However, the different oxidation states are difficult to monitor in a physiological setting due to the limited availability of experimental tools. Therefore it is of interest to synthesise and use a chemical probe that selectively recognises the reversible oxidation state of cysteine sulphenic acid to understand more about oxidative signalling. The aim of this thesis was to investigate a synthetic approach for novel fluorescent probe synthesis, for the specific detection of cysteine sulphenic acids by fluorescence spectroscopy and confocal microscopy. N-[2-(Anthracen-2-ylamino)-2-oxoethyl]-3,5-dioxocyclohexanecarboxamide was synthesised in a multistep synthesis and characterised by nuclear magnetic resonance spectroscopy. The optimisation of conditions needed for sulphenic acid formation in a purified protein using human serum albumin (HSA) and the commercially available biotin tagged probe 3-(2,4-dioxocyclohexyl)propyl-5-((3aR,6S,6aS)-hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-6-yl)pentanoate (DCP-Bio1) were identified. This approach was extended to detect sulphenic acids in Jurkat T cells and CD4+ T cells pre- and post-stimulus. Buthionine sulfoximine (BSO) was used to manipulate the endogenous antioxidant glutathione (GSH) in human CD4+ T cells. Then the surface protein thiol levels and sulphenic acid formation was examined. T cells were also activated by the lectin phytohaemagglutinin-L (PHA-L) and formation of sulphenic acid was investigated using SDS-PAGE, western blotting and confocal microscopy. Resting Jurkat cells have two prominent protein bands that have sulphenic acid modifications whereas resting CD4+ T cells have an additional band present. When cells were treated with BSO the number of bands increased whereas activation reduced the number of proteins that were modified. The identities of the protein bands containing sulphenic acids were explored by mass spectrometry. Cysteine oxidation was observed in redox, metabolic and cytoskeletal proteins. In summary, a novel fluorescent probe for detection of cysteine sulphenic acids has been synthesised alongside a model system that introduces cysteine sulphenic acid in primary T cells. This probe has potential application in the subcellular localisation of cysteine oxidation during T cell signalling.

Endothelial cell redox regulation of ischemic angiogenesis

The endothelium produces and responds to reactive oxygen and nitrogen species (RONS), providing important redox regulation to the cardiovascular system in physiology and disease. In no other situation are RONS more critical than in the response to tissue ischemia. Here, tissue healing requires growth factor-mediated angiogenesis that is in part dependent on low levels of RONS, which paradoxically must overcome the damaging effects of high levels of RONS generated as a result of ischemia. While generation of endothelial cell RONS in hypoxia/reoxygenation is acknowledged, the mechanism for their role in angiogenesis is still poorly understood. During ischemia, the major low molecular weight thiol glutathione (GSH) reacts with RONS and protein cysteines, producing GSH-protein adducts. Recent data indicate that GSH adducts on certain proteins are essential to growth factor responses in endothelial cells. Genetic deletion of the enzyme glutaredoxin-1, which selectively removes GSH protein adducts, improves, while its overexpression impairs, revascularization of the ischemic hindlimb of mice. Ischemia-induced GSH adducts on specific cysteine residues of several proteins, including p65 NFkB and the sarcoplasmic reticulum calcium ATPase-2 (SERCA2), appear to promote ischemic angiogenesis. Identifying the specific proteins in the redox response to ischemia has provided therapeutic opportunities to improve clinical outcomes of ischemia.

The Nrf2-ARE activation protect neurones against oxidative stress

Oxidative stress has been implicated in the pathogenesis of many neurodegenerative diseases including Alzheimer’s disease. The transcription factor, Nrf2 (nuclear factor E2-related factor 2) that binds to the antioxidant responsive element (ARE) activates a battery of genes encoding enzymes and factors essential for neuronal survival. We have investigated the hypothesis that a downstream product of cyclooxygenase(COX-2), 15-deoxy-delta (12, 14)-prostagland in J2 (15d-PGJ2) has protective effects by activating the Nrf2 pathway during oxidative stress.Human neuroblastoma cells (SHSY5Y) were differentiated intoneuronal-like cells as described previously (Gimenez-Cassina et al.,2006). SHSY5Y cells were co-treated with 10 mM buthionine sulfoximine (BSO) 7 10 mM 15d-PGJ2. Cell viability was measured by MTT assay and cellular glutathione (GSH) levels were measured after treating cells for0.5-24 hours by GSH recycling assay. Cellular Nrf2 levels were determined by immunoblotting. IL-6 levels were measured by ELISA.15d-PGJ2 alone lowered GSH levels 30min after the treatment(12.870.64 nmol/mg protein) and returned to untreated control levels at 16hours (28.173.6 nmol/mg protein; Po0.01). Compared to intracellular GSH levels in untreated cells (27.871.8 nmol/mg protein) BSO treatment alone significantly decreased GSH (9.672.1 nmol/mg protein;Po0.001) but co-incubation with 15d-PGJ2 for 24 hours prevented the depletion elicited by BSO(21.372.7 nmol/mg protein). Compared to untreated cells BSO treatment decrease dIL-6 secretion (from 0.941.6ng/ml to 0.6971.3ng/ml) and total Nrf2 protein levels (by21%). Co-incubation with15d-PGJ2 for 24 hours with BSO did not change IL-6(0.6771.4ng/ml) or total Nrf2 level at any time point. This study suggests that neuronal toxicity resulting from glutathione depletion canbere stored by the induction of Nrf2-ARE pathway and the role of the Nrf2 signalling merits further investigation in neurodegenerative diseases.

Interactions of different arsenic species with thols: Chemical and biological implications

Arsenic has been classified as a group I carcinogen. It has been ranked number one in the CERCLA priority list of hazardous substances due to its frequency, toxicity and potential for human exposure. Paradoxically, arsenic has been employed as a successful chemotherapeutic agent for acute promyelocytic leukemia and has found some success in multiple myeloma. Since arsenic toxicity and efficacy is species dependent, a speciation method, based on the complementary use of reverse phase and cation exchange chromatography, was developed. Inductively coupled plasma mass spectrometer (ICP-MS), as an element specific detector, and electrospray ionization mass spectrometer (ESI-MS), as a molecule specific detector, were employed. Low detection limits in the µg. L−1 range on the ICP-MS and mg. L−1 range on the ESI-MS were obtained. The developed methods were validated against each other through the use of a Deming plot. With the developed speciation method, the effects of both pH on the stability of As species and reduced glutathione (GSH) concentration on the formation and stability of arsenic glutathione complexes were studied. To identify arsenicals in multiple myeloma (MM) cell lines post arsenic trioxide (ATO) and darinaparsin (DAR) incubation, an extraction method based on the use of ultrasonic probe was developed. Extraction tools and solvents were evaluated and the effect of GSH concentration on the quantitation of arsenic glutathione (As-GSH) complexes in MM cell extracts was studied. The developed method was employed for the identification of metabolites in DAR incubated cell lines where the effect of extraction pH, DAR incubation concentration and incubation time on the relative distribution of the As metabolites was assessed. A new arsenic species, dimethyarsinothioyl glutathione (DMMTA V-GS), a pentavalent thiolated arsenical, was identified in the cell extracts through the use of liquid chromatography tandem mass spectrometry. The formation of the new metabolite in the extracts was dependent on the decomposition of s-dimethylarsino glutathione (DMA(GS)). These results have major implications in both the medical and toxicological fields of As because they involve the metabolism of a chemotherapeutic agent and the role sulfur compounds play in this mechanism.

Preservation of nitric oxide availability as nitrite and nitrosothiols

Nitric Oxide (NO) has been known for long to regulate vessel tone. However, the close proximity of the site of NO production to "sinks" of NO such as hemoglobin (Hb) in blood suggest that blood will scavenge most of the NO produced. Therefore, it is unclear how NO is able to play its physiological roles. The current study deals with means by which this could be understood. Towards studying the role of nitrosothiols and nitrite in preserving NO availability, a study of the kinetics of glutathione (GSH) nitrosation by NO donors in aerated buffered solutions was undertaken first. Results suggest an increase in the rate of the corresponding nitrosothiol (GSNO) formation with an increase in GSH with a half-maximum constant EC50 that depends on NO concentration, thus indicating a significant contribution of NO2 mediated nitrosation in the production of GSNO. Next, the ability of nitrite to be reduced to NO in the smooth muscle cells was evaluated. The NO formed was inhibited by sGC inhibitors and accelerated by activators and was independent of O2 concentration. Nitrite transport mechanisms and effects of exogenous nitrate on transport and reduction of nitrite were examined. The results showed that sGC can mediate nitrite reduction to NO and nitrite is transported across the smooth muscle cell membrane via anion channels, both of which can be attenuated by nitrate. Finally, a 2-D axisymmetric diffusion model was constructed to test the accumulation of NO in the smooth muscle layer from reduction of nitrite. It was observed that at the end of the simulation period with physiological concentrations of nitrite in the smooth muscle cells (SMC), a low sustained NO generated from nitrite reduction could maintain significant sGC activity and might affect vessel tone. The major nitrosating mechanism in the circulation at reduced O2 levels was found to be anaerobic and a Cu+ dependent GSNO reduction activity was found to deliver minor amounts of NO from physiological GSNO levels in the tissue.

Efeitos da azilsartana sobre a doença periodontal em um modelo experimental com ratos wistar

Periodontal diseases, highly prevalent disease in worldwide population, manifest primarily in two distinct entities: plaque-induced gingivitis and periodontitis. Periodontitis is a chronic inflammatory disease characterized of different levels of collagen, cementum, and alveolar bone destruction. Recent experimental studies demonstrated anti-inflammatory and antirreabsortive effect of antihypertensive agents of the angiotensin II receptor blockers class on periodontal disease. The aim of this study was to evaluate the effects of azilsartan (AZT), a potent inhibitor of the angiotensin II receptor which has minimal adverse effects on bone loss, inflammation, and the expression of matrix metallo proteinases (MMPs), receptor activator of nuclear factor kB ligand (RANKL), receptor activator of nuclear factor kB (RANK), osteoprotegerin (OPG), cyclooxygenase-2 (COX-2), and cathepsin K in periodontal tissue in a rat model of ligature-induced periodontitis. Male Wistar albino rats were randomly divided into 5 groups of 20 rats each: (1) nonligated, water; (2) ligated, water; (3) ligated, 1 mg/kg AZT; (4) ligated, 5 mg/kg AZT; and (5) ligated, 10 mg/kg AZT. All groups were treated with water or AZT for 10 days. Periodontal tissues were analyzed by morphometric exam, histopathology and immunohistochemical detection of MMP-2, MMP-9, COX-2, RANKL, RANK, OPG, and cathepsin K. Levels of IL-1b, IL-10, TNF-a, myeloperoxidase (MPO), and glutathione (GSH) were determined by ELISA. Treatment with 5 mg/kg AZT resulted in reduced MPO (p˂0.05) and IL-1b (p˂0.05) levels and increased in Il-10 levels (p˂0.05). It was observed a reduced expression of MMP-2, MMP-9, COX-2, RANK, RANKL, cathepsin K, and a increased expression of OPG in the animals subjected to experimental periodontitis and threated with AZT (5 mg/kg). Conclusions: These findings suggest an anti-inflammatory and anti-reabsortive effects of AZT on ligature-induced periodontitis in rats.

Efeitos da terapia fotodinâmica com aluminio – cloro ftalocianina sobre a peroxidação lipídica e produtos antioxidantes em tecidos inflamados animais

Photodynamic therapy (PDT) consists of a non-toxic photosensitizing agent (FS) administration followed by a laser source resulting in a sequence of photochemical and photobiological processes that generate reactive oxygen species (ROS) that damaging cells. The present work evaluated the effects of PDT nanoemulsion-aluminum chloride phthalocyanine (AlClFc) mediated on malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels, which represent indicators involved in oxidative stress and antioxidant defenses. For this purpose, this study used 120 female rats of the Rattus norvegicus species, Wistar race, divided into 5 groups: Healthy (H), with periodontal disease (PD), with periodontal disease and treatment with FS (F), with periodontal disease and treatment with the laser (L); and periodontal disease and treatment with PDT (FL). An experimental model for represent periodontal disease (PD) was induced by ligature (split-mouth). Seven days later the induction of PD, the treatments were instituted according to the groups. In the group treated with PDT was applied 40μl FS (5μM) followed by laser irradiation diode InGaAlP (660nm, 100J / cm2). The rats were sacrificed on the 7th and 28th day after treatment and tissue specimens were removed and subjected to histological, immunohistochemical methods and enzymatic colorimetric measurements with detection by UV / VIS spectroscopy. Inflammatory changes, connective tissue disorganization and alveolar bone loss were displaying in groups with PD induced. The enzyme dosages showed that MDA levels were higher in PD induced groups, with no statistically significant differences (p> 0.05). High levels of GSH were found in groups L (p = 0.028) and FL (p = 0.028) compared with PD group, with statistically significant differences. Immunohistochemistry for SOD showed higher immunostaining in L and FL groups, compared to the PD group without statistically significant differences (p> 0.05). GPx showed lower immunoreactivity in the DP group when compared to the other groups and statistically significant differences were observed between the DPxL groups (p <0.05). TFD administered in this experiment did not induce elevation of MDA levels significantly increased the GSH levels and showed intense immunostaining pada SOD and GPx, showing that this therapy does not accentuated lipid peroxidation, however, it was able to induce effects on the antioxidant defenses processes. The LBI therapy appeared to show photomodulatory promoting effects reduction of the MDA levels, increasing GSH levels and with intense immunostaining for SOD and GPx, demonstrating that laser therapy induced antioxidant effects.

Biochemical and Structural Studies on PrfA, the Transcriptional Regulator of Virulence in Listeria monocytogenes

Abstract

Listeria monocytogenes is a gram-positive soil saprophytic bacterium that is capable of causing fatal infection in humans. The main virulence regulator PrfA, a member of the Crp/FNR family of transcriptional regulators, activates the expression of essential proteins required for host cell invasion and cell-to-cell spread. The mechanism of PrfA activation and the identity of its small molecule coactivator have remained a mystery for more than 20 years, but it is hypothesized that PrfA shares mechanistic similarity to the E. coli cAMP binding protein, Crp. Crp activates gene expression by binding cAMP, increasing the DNA binding affinity of the protein and causing a significant DNA bend that facilitates RNA polymerase binding and downstream gene activation. Our data suggests PrfA activates virulence protein expression through a mechanism distinct from the canonical Crp activation mechanism that involves a combination of cysteine residue reduction and glutathione (GSH) binding.

Listeria lacking glutathione synthase (ΔgshF) is avirulent in mice; however virulence is rescued when the bacterium expresses the constitutively active PrfA mutant G145S. Interestingly, Listeria expressing a PrfA mutant in which its four cysteines are mutated to alanine (Quad PrfA), demonstrate a 30-fold decrease in virulence. The Quad and ΔgshF double mutant strains are avirulent. DNA-binding affinity, measured through fluorescence polarization assays, indicate reduction of the cysteine side chains is sufficient to allow PrfA to binds its physiological promoters Phly and PactA with low nanomolar affinity. Oxidized PrfA binds the promoters poorly.

Unexpectedly, Quad also binds promoter DNA with nanomolar affinity, suggesting that the cysteines play a role in transcription efficiency in addition to DNA binding. Both PrfA and Quad bind GSH at physiologically relevant and comparable affinities, however GSH did not affect DNA binding in either case. Thermal denaturation assays suggest that Quad and wild-type PrfA differ structurally upon binding GSH, which supports the in vivo difference in infection between the regulator and its mutant.

Structures of PrfA in complex with cognate DNA, determined through X-ray crystallography, further support the disparity between PrfA and Crp activation mechanisms as two structures of reduced PrfA bound to Phly (PrfA-Phly30 and PrfA-Phly24) suggest the DNA adopts a less bent DNA conformation when compared to Crp-cAMP- DNA. The structure of Quad-Phly30 confirms the DNA-binding data as the protein-DNA complex adopts the same overall conformation as PrfA-Phly.

From these results, we hypothesize a two-step activation mechanism wherein PrfA, oxidized upon cell entry and unable to bind DNA, is reduced upon its intracellular release and binds DNA, causing a slight bend in the promoter and small increase in transcription of PrfA-regulated genes. The structures of PrfA-Phly30 and PrfA-Phly24 likely visualize this intermediate complex. Increasing concentrations of GSH shift the protein to a (PrfA-GSH)-DNA complex which is fully active transcriptionally and is hypothesized to resemble closely the transcriptionally active structure of the cAMP-(Crp)-DNA complex. Thermal denaturation results suggest Quad PrfA is deficient in this second step, which explains the decrease in virulence and implicates the cysteine residues as critical for transcription efficiency. Further structural and biochemical studies are on-going to clarify this mechanism of activation.

A novel mechanism of action of HER2 targeted immunotherapy is explained by inhibition of NRF2 function in ovarian cancer cells

Nuclear erythroid related factor-2 (NRF2) is known to promote cancer therapeutic detoxification and crosstalk with growth promoting pathways. HER2 receptor tyrosine kinase is frequently overexpressed in cancers leading to uncontrolled receptor activation and signaling. A combination of HER2 targeting monoclonal antibodies shows greater anticancer efficacy than the single targeting antibodies, however, its mechanism of action is largely unclear. Here we report novel actions of anti-HER2 drugs, Trastuzumab and Pertuzumab, involving NRF2. HER2 targeting by antibodies inhibited growth in association with persistent generation of reactive oxygen species (ROS), glutathione (GSH) depletion, reduction in NRF2 levels and inhibition of NRF2 function in ovarian cancer cell lines. The combination of antibodies produced more potent effects than single alone; downregulated NRF2 substrates by repressing the Antioxidant Response (AR) pathway with concomitant transcriptional inhibition of NRF2. We showed the antibody combination produced increased methylation at the NRF2 promoter consistent with repression of NRF2 antioxidant function, as HDAC and methylation inhibitors reversed such produced transcriptional effects. These findings demonstrate a novel mechanism and role for NRF2 in mediating the response of cancer cells to the combination of Trastuzumab and Pertuzumab and reinforce the importance of NRF2 in drug resistance and as a key anticancer target.

Exposure to toxic Alexandrium minutum activates the detoxifying and antioxidant systems in gills of the oyster Crassostrea gigas

Harmful algal blooms of Alexandrium spp. dinoflagellates regularly occur in French coastal waters contaminating shellfish. Studies have demonstrated that toxic Alexandrium spp. disrupt behavioural and physiological processes in marine filter-feeders, but molecular modifications triggered by phycotoxins are less well understood. This study analyzed the mRNA levels of 7 genes encoding antioxidant/detoxifying enzymes in gills of Pacific oysters (Crassostrea gigas) exposed to a cultured, toxic strain of A. minutum, a producer of paralytic shellfish toxins (PST) or fed Tisochrysis lutea (T. lutea, formerly Isochrysis sp., clone Tahitian (T. iso)), a non-toxic control diet, in four repeated experiments. Transcript levels of sigma-class glutathione S-transferase (GST), glutathione reductase (GR) and ferritin (Fer) were significantly higher in oysters exposed to A. minutum compared to oysters fed T. lutea. The detoxification pathway based upon glutathione (GSH)-conjugation of toxic compounds (phase II) is likely activated, and catalyzed by GST. This system appeared to be activated in gills probably for the detoxification of PST and/or extra-cellular compounds, produced by A. minutum. GST, GR and Fer can also contribute to antioxidant functions to prevent cellular damage from increased reactive oxygen species (ROS) originating either from A. minutum cells directly, from oyster hemocytes during immune response, or from other gill cells as by-products of detoxification.