Acid challenge exacerbates activation of matrix metalloproteinases in permanent teeth undergoing radiotherapy.

The aim of this study was to investigate the effect of acid challenge on the activation of matrix metalloproteinases (MMPs) in the Dentinoenamel junction of primary and permanent teeth submitted to radiotherapy. For this purpose, a total of 178 dental fragments obtained from molars were used, and randomly divided into 2 groups (primary and permanent teeth) / 4 experimental subgroups (irradiated and non-irradiated, demineralized and non-demineralized). The fragments were exposed to radiation, with a dose fraction of 2 Gy, for 5 consecutive days, until a total dose of 60 Gy was reached, with a total of 30 cycles, for 6 weeks. To determine the activity of MMPs on the dentinoenamel junction (DEJ), in situ zymography assays on 0.6mm dental fragments were performed. To assess whether MMP activity would be impacted by an acidic environment, the fragments were placed in a demineralizing solution (pH of 4.8). The finding was that irradiation activated MMPs in DEJ and these effects were more evident in permanent when compared with primary teeth. When the effect of an acid challenge on MMPs activity was investigated, demineralization was observed not to increase MMPs activity in non-irradiated teeth, but it did increase MMPs activity in irradiated teeth. In conclusion, an acid challenge was found to exacerbate activation of MMPs in DEJ of permanent teeth submitted to irradiation, but not in primary teeth.

Structural basis of human NOX5 activation.

NADPH oxidase 5 (NOX5) catalyzes the production of superoxide free radicals and regulates physiological processes from sperm motility to cardiac rhythm. Overexpression of NOX5 leads to cancers, diabetes, and cardiovascular diseases. NOX5 is activated by intracellular calcium signaling, but the underlying molecular mechanism of which - in particular, how calcium triggers electron transfer from NADPH to FAD - is still unclear. Here we capture motions of full-length human NOX5 upon calcium binding using single-particle cryogenic electron microscopy (cryo-EM). By combining biochemistry, mutagenesis analyses, and molecular dynamics (MD) simulations, we decode the molecular basis of NOX5 activation and electron transfer. We find that calcium binding to the EF-hand domain increases NADPH dynamics, permitting electron transfer between NADPH and FAD and superoxide production. Our structural findings also uncover a zinc-binding motif that is important for NOX5 stability and enzymatic activity, revealing modulation mechanisms of reactive oxygen species (ROS) production.

Activation of heme oxygenase-1 by laminar shear stress ameliorates high glucose-induced endothelial cell and smooth muscle cell dysfunction.

High glucose (HG)-induced endothelial cell (EC) and smooth muscle cell (SMC) dysfunction is critical in diabetes-associated atherosclerosis. However, the roles of heme oxygenase-1 (HO-1), a stress-response protein, in hemodynamic force-generated shear stress and HG-induced metabolic stress remain unclear. This investigation examined the cellular effects and mechanisms of HO-1 under physiologically high shear stress (HSS) in HG-treated ECs and adjacent SMCs. We found that exposure of human aortic ECs to HSS significantly increased HO-1 expression; however, this upregulation appeared to be independent of adenosine monophosphate-activated protein kinase, a regulator of HO-1. Furthermore, HSS inhibited the expression of HG-induced intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and reactive oxygen species (ROS) production in ECs. In an EC/SMC co-culture, compared with static conditions, subjecting ECs close to SMCs to HSS and HG significantly suppressed SMC proliferation while increasing the expression of physiological contractile phenotype markers, such as α-smooth muscle actin and serum response factor. Moreover, HSS and HG decreased the expression of vimentin, an atherogenic synthetic phenotypic marker, in SMCs. Transfecting ECs with HO-1-specific small interfering (si)RNA reversed HSS inhibition on HG-induced inflammation and ROS production in ECs. Similarly, reversed HSS inhibition on HG-induced proliferation and synthetic phenotype formation were observed in co-cultured SMCs. Our findings provide insights into the mechanisms underlying EC-SMC interplay during HG-induced metabolic stress. Strategies to promote HSS in the vessel wall, such as continuous exercise, or the development of HO-1 analogs and mimics of the HSS effect, could provide an effective approach for preventing and treating diabetes-related atherosclerotic vascular complications.

Optogenetically engineered calcium oscillations promote autophagy-mediated cell death via AMPK activation.

Autophagy is a double-edged sword for cells; it can lead to both cell survival and death. Calcium (Ca2+) signalling plays a crucial role in regulating various cellular behaviours, including cell migration, proliferation and death. In this study, we investigated the effects of modulating cytosolic Ca2+ levels on autophagy using chemical and optogenetic methods. Our findings revealed that ionomycin and thapsigargin induce Ca2+ influx to promote autophagy, whereas the Ca2+ chelator BAPTA-AM induces Ca2+ depletion and inhibits autophagy. Furthermore, the optogenetic platform allows the manipulation of illumination parameters, including density, frequency, duty cycle and duration, to create different patterns of Ca2+ oscillations. We used the optogenetic tool Ca2+-translocating channelrhodopsin, which is activated and opened by 470 nm blue light to induce Ca2+ influx. These results demonstrated that high-frequency Ca2+ oscillations induce autophagy. In addition, autophagy induction may involve Ca2+-activated adenosine monophosphate (AMP)-activated protein kinases. In conclusion, high-frequency optogenetic Ca2+ oscillations led to cell death mediated by AMP-activated protein kinase-induced autophagy.

Computational Analysis of Activation of Dimerized Epidermal Growth Factor Receptor Kinase Using the String Method and Markov State Model.

Epidermal growth factor receptor (EGFR) activation is accompanied by dimerization. During the activation of the intracellular kinase domain, two EGFR kinases form an asymmetric dimer, and one side of the dimer (receiver) is activated. Using the string method and Markov state model (MSM), we performed a computational analysis of the structural changes in the activation of the EGFR dimer in this study. The string method reveals the minimum free-energy pathway (MFEP) from the inactive to active structure. The MSM was constructed from numerous trajectories of molecular dynamics simulations around the MFEP, which revealed the free-energy map of structural changes. In the activation of the receiver kinase, the unfolding of the activation loop (A-loop) is followed by the rearrangement of the C-helix, as observed in other kinases. However, unlike other kinases, the free-energy map of EGFR at the asymmetric dimer showed that the active state yielded the highest stability and revealed how interactions at the dimer interface induced receiver activation. As the H-helix of the activator approaches the C-helix of the receiver during activation, the A-loop unfolds. Subsequently, L782 of the receiver enters the pocket between the G- and H-helices of the activator, leading to a rearrangement of the hydrophobic residues around L782 of the receiver, which constitutes a structural rearrangement of the C-helix of the receiver from an outward to an inner position. The MSM analysis revealed long-time scale trajectories via kinetic Monte Carlo.

Structural determinants for activation of the Tau kinase CDK5 by the serotonin receptor 5-HT7R.

BACKGROUND: Multiple neurodegenerative diseases are induced by the formation and deposition of protein aggregates. In particular, the microtubule-associated protein Tau leads to the development of so-called tauopathies characterized by the aggregation of hyperphosphorylated Tau within neurons. We recently showed that the constitutive activity of the serotonin receptor 7 (5-HT7R) is required for Tau hyperphosphorylation and aggregation through activation of the cyclin-dependent kinase 5 (CDK5). We also demonstrated physical interaction between 5-HT7R and CDK5 at the plasma membrane suggesting that the 5-HT7R/CDK5 complex is an integral part of the signaling network involved in Tau-mediated pathology. METHODS: Using biochemical, microscopic, molecular biological, computational and AI-based approaches, we investigated structural requirements for the formation of 5-HT7R/CDK5 complex. RESULTS: We demonstrated that 5-HT7R domains responsible for coupling to Gs proteins are not involved in receptor interaction with CDK5. We also created a structural model of the 5-HT7R/CDK5 complex and refined the interaction interface. The model predicted two conserved phenylalanine residues, F278 and F281, within the third intracellular loop of 5-HT7R to be potentially important for complex formation. While site-directed mutagenesis of these residues did not influence Gs protein-mediated receptor signaling, replacement of both phenylalanines by alanine residues significantly reduced 5-HT7R/CDK5 interaction and receptor-mediated CDK5 activation, leading to reduced Tau hyperphosphorylation and aggregation. Molecular dynamics simulations of 5-HT7R/CDK5 complex for wild-type and receptor mutants confirmed binding interface stability of the initial model. CONCLUSIONS: Our results provide a structural basis for the development of novel drugs targeting the 5-HT7R/CDK5 interaction interface for the selective treatment of Tau-related disorders, including frontotemporal dementia and Alzheimer's disease.

Cannabidiol induces ERK activation and ROS production to promote autophagy and ferroptosis in glioblastoma cells.

Small molecule-driven ERK activation is known to induce autophagy and ferroptosis in cancer cells. Herein the effect of cannabidiol (CBD), a phytochemical derived from Cannabis sativa, on ERK-driven autophagy and ferroptosis has been demonstrated in glioblastoma (GBM) cells (U87 and U373 cells). CBD imparted significant cytotoxicity in GBM cells, induced activation of ERK (not JNK and p38), and increased intracellular reactive oxygen species (ROS) levels. It increased the autophagy-related proteins such as LC3 II, Atg7, and Beclin-1 and modulated the expression of ferroptosis-related proteins such as glutathione peroxidase 4 (GPX4), SLC7A11, and TFRC. CBD significantly elevated the endoplasmic reticulum stress, ROS, and iron load, and decreased GSH levels. Inhibitors of autophagy (3-MA) and ferroptosis (Fer-1) had a marginal effect on CBD-induced autophagy/ferroptosis. Treatment with N-acetyl-cysteine (antioxidant) or PD98059 (ERK inhibitor) partly reverted the CBD-induced autophagy/ferroptosis by decreasing the activation of ERK and the production of ROS. Overall, CBD induced autophagy and ferroptosis through the activation of ERK and generation of ROS in GBM cells.

Proteasome activation is critical for cell death induced by inhibitors of polo-like kinase 1 (PLK1) in multiple cancers.

Inhibitors of polo-like kinase (PLK) are currently being evaluated as anticancer drugs. However, the molecular mechanism of PLK inhibitor-induced cell death is not fully understood. In this study, we found that GW843682X and BI2536, two inhibitors of PLK1, significantly induced cell death in multiple type cells. The induction of cell death was related to the preferring expression of PLK1. However, in human umbilical vascular endothelial cells (HUVEC) and human colorectal carcinoma cells, which expressed higher levels of both PLK1 and PLK2, PLK1 inhibitors induced very low levels of cell death. Clinical analysis reveals PLK1 presence in 26 of 30 NPC tumor tissues. In in vivo NPC lung metastasis nude mouse models, PLK1 inhibitors decreased NPC progress. Mechanistically, the PLK1 inhibitor did not activate p53, and the cell death was not reversed by p53 inhibition. Moreover, PLK1 inhibitor-induced cell death was PARP- and caspase-independent. Although PLK1 inhibitors induced down-regulation of calpain inhibitor calpastatin and calpain was activated by PLK1 inhibition, calpain blocking did not reverse cell death induced by PLK1 inhibitors, suggesting the non-involvement of calpain. Surprisingly, we found that PLK1 inhibitors induced the activation of proteasome, and the treatment of cells with PLK1 inhibitors reduced the levels of ubiquitinated proteins. And proteasome inhibitors reversed cell death induced by PLK1 inhibitors in various cell types in which PLK1 was preferentially expressed. Moreover, PLK1 inhibition reversed the degradation of proteins including p53, caspase 8, PARP and calpastatin. These results suggest that the activation of proteasome is critical for cell death induced by PLK1 inhibition.

Metabolic rewiring promotes anti-inflammatory effects of glucocorticoids.

Glucocorticoids represent the mainstay of therapy for a broad spectrum of immune-mediated inflammatory diseases. However, the molecular mechanisms underlying their anti-inflammatory mode of action have remained incompletely understood1. Here we show that the anti-inflammatory properties of glucocorticoids involve reprogramming of the mitochondrial metabolism of macrophages, resulting in increased and sustained production of the anti-inflammatory metabolite itaconate and consequent inhibition of the inflammatory response. The glucocorticoid receptor interacts with parts of the pyruvate dehydrogenase complex whereby glucocorticoids provoke an increase in activity and enable an accelerated and paradoxical flux of the tricarboxylic acid (TCA) cycle in otherwise pro-inflammatory macrophages. This glucocorticoid-mediated rewiring of mitochondrial metabolism potentiates TCA-cycle-dependent production of itaconate throughout the inflammatory response, thereby interfering with the production of pro-inflammatory cytokines. By contrast, artificial blocking of the TCA cycle or genetic deficiency in aconitate decarboxylase 1, the rate-limiting enzyme of itaconate synthesis, interferes with the anti-inflammatory effects of glucocorticoids and, accordingly, abrogates their beneficial effects during a diverse range of preclinical models of immune-mediated inflammatory diseases. Our findings provide important insights into the anti-inflammatory properties of glucocorticoids and have substantial implications for the design of new classes of anti-inflammatory drugs.

AMP-activated protein kinase can be allosterically activated by ADP but AMP remains the key activating ligand.

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. When activated by increases in ADP:ATP and/or AMP:ATP ratios (signalling energy deficit), AMPK acts to restore energy balance. Binding of AMP to one or more of three CBS repeats (CBS1, CBS3, CBS4) on the AMPK-γ subunit activates the kinase complex by three complementary mechanisms: (i) promoting α-subunit Thr172 phosphorylation by the upstream kinase LKB1; (ii) protecting against Thr172 dephosphorylation; (iii) allosteric activation. Surprisingly, binding of ADP has been reported to mimic the first two effects, but not the third. We now show that at physiologically relevant concentrations of Mg.ATP2- (above those used in the standard assay) ADP binding does cause allosteric activation. However, ADP causes only a modest activation because (unlike AMP), at concentrations just above those where activation becomes evident, ADP starts to cause competitive inhibition at the catalytic site. Our results cast doubt on the physiological relevance of the effects of ADP and suggest that AMP is the primary activator in vivo. We have also made mutations to hydrophobic residues involved in binding adenine nucleotides at each of the three γ subunit CBS repeats of the human α2ß2γ1 complex and examined their effects on regulation by AMP and ADP. Mutation of the CBS3 site has the largest effects on all three mechanisms of AMP activation, especially at lower ATP concentrations, while mutation of CBS4 reduces the sensitivity to AMP. All three sites appear to be required for allosteric activation by ADP.

A common protein C inhibitor exosite partially controls the heparin induced activation and inhibition of serine proteases.

Protein C inhibitor (PCI) maintains hemostasis by inhibiting both procoagulant and anticoagulant serine proteases, and plays important roles in coagulation, fibrinolysis, reproduction, and anti-angiogenesis. The reactive site loop of PCI traps and irreversibly inhibits the proteases like APC (activating protein C), thrombin (FIIa) and factor Xa (FXa). Previous studies on antithrombin (ATIII) had identified Tyr253 and Glu255 as functional exosites that interact and aid in the inhibition of factor IXa and FXa. Presence of exosite in PCI is not known, however a sequence comparison with the PCI from different vertebrate species and ATIII identified Glu239 to be absolutely conserved. PCI residues analogous to ATIII exosite residues were mutated to R238A and E239A. Purified variant PCI in the presence of heparin (10 µg/ml) showed a 2-4 fold decrease in the rate of inhibition of the proteases. However, the stoichiometry of inhibition of FIIa, APC, and FXa by native PCI, R238A and E239A variants were found to be close to 1.0, which also indicated the formation of stable complexes based on SDS-PAGE and western blot analysis with thrombin and APC. Our findings revealed the possible presence of an exosite in PCI that influences the protease inhibition rates.

Mechanism of GTPase activation of a prokaryotic small Ras-like GTPase MglA by an asymmetrically interacting MglB dimer.

Cell polarity oscillations in Myxococcus xanthus motility are driven by a prokaryotic small Ras-like GTPase, mutual gliding protein A (MglA), which switches from one cell pole to the other in response to extracellular signals. MglA dynamics is regulated by MglB, which functions both as a GTPase activating protein (GAP) and a guanine nucleotide exchange factor (GEF) for MglA. With an aim to dissect the asymmetric role of the two MglB protomers in the dual GAP and GEF activities, we generated a functional MglAB complex by coexpressing MglB with a linked construct of MglA and MglB. This strategy enabled us to generate mutations of individual MglB protomers (MglB1 or MglB2 linked to MglA) and delineate their role in GEF and GAP activities. We establish that the C-terminal helix of MglB1, but not MglB2, stimulates nucleotide exchange through a site away from the nucleotide-binding pocket, confirming an allosteric mechanism. Interaction between the N-terminal ß-strand of MglB1 and ß0 of MglA is essential for the optimal GEF activity of MglB. Specific residues of MglB2, which interact with Switch-I of MglA, partially contribute to its GAP activity. Thus, the role of the MglB2 protomer in the GAP activity of MglB is limited to restricting the conformation of MglA active site loops. The direct demonstration of the allosteric mechanism of GEF action provides us new insights into the regulation of small Ras-like GTPases, a feature potentially present in many uncharacterized GEFs.

Distinctive activation of ß-galactosidase by carboxymethylated ß-glucan in vitro and mechanism study: Critical role of hydrophobic and electrostatic interactions.

ß-galactosidase (lactase) is commercially important as a dietary supplement to alleviate the symptoms of lactose intolerance. This work investigated a unique activation of CMP (carboxymethylated (1 â†’ 3)-ß-d-glucan) on lactase and its mechanism by comparing it with carboxymethyl chitosan (CMCS), an inhibitor of lactase. The results illustrated that the secondary and tertiary structures of lactase were altered and its active sites exposed after complexation with CMP, and dissociation of lactase aggregates was also observed. These changes favored better accessibility of the substrate to the active sites of lactase, resulting in a maximum increase of 60.5 % in lactase activity. Furthermore, the hydrophobic and electrostatic interactions with lactase caused by the carboxymethyl group of CMP were shown to be crucial for its activation ability. Thus, the improvement of lactase activity and stability by CMP shown here is important for the development of new products in the food and pharmaceutical industries.

Nitrite Attenuates the In Vitro Inflammatory Response of Immune Cells to the SARS-CoV-2 S Protein without Interfering in the Antioxidant Enzyme Activation.

SARS-CoV-2 induces a hyperinflammatory reaction due to the excessive release of cytokines during the immune response. The bacterial endotoxin lipopolysaccharide (LPS) contributes to the low-grade inflammation associated with the metabolic syndrome, enhancing the hyperinflammatory reaction induced by the SARS-CoV-2 infection. The intake of sodium nitrate, a precursor of nitrite and nitric oxide, influences the antioxidant and pro-inflammatory gene expression profile after immune stimulation with LPS in peripheral blood mononuclear cells from metabolic syndrome patients. We aimed to assess the inflammatory and antioxidant responses of immune cells from metabolic syndrome patients to exposure to the SARS-CoV-2 spike protein (S protein) together with LPS and the effect of nitrite in these responses. Whole blood samples obtained from six metabolic syndrome patients were cultured for 16 h at 37 °C with four different media: control medium, control medium plus LPS (100 ng/mL), control medium plus LPS (100 ng/mL) plus S protein (10 ng/mL), and control medium plus LPS (100 ng/mL) plus S protein (10 ng/mL) plus nitrite (5 µM). Immune stimulation with the LPS/S protein enhanced nitrate biosynthesis from nitrite oxidation and probably from additional organic precursors. In vitro incubations with the LPS/S protein enhanced the expression and/or release of pro-inflammatory TNFα, IL-6, IL-1ß, and TLR4, as well as the expression of the anti-inflammatory IL-1ra and IL-10 and antioxidant enzymes. Nitrite attenuated the pro- and anti-inflammatory response induced by the S protein without interfering with the activation of TLR4 and antioxidant enzyme expression, raising the possibility that nitrite could have potential as a coadjutant in the treatment of COVID-19.

Determinants of CRISPR Cas12a nuclease activation by DNA and RNA targets.

The RNA-guided CRISPR-associated (Cas) enzyme Cas12a cleaves specific double-stranded (ds-) or single-stranded (ss-) DNA targets (in cis), unleashing non-specific ssDNA cleavage (in trans). Though this trans-activity is widely coopted for diagnostics, little is known about target determinants promoting optimal enzyme performance. Using quantitative kinetics, we show formation of activated nuclease proceeds via two steps whereby rapid binding of Cas12a ribonucleoprotein to target is followed by a slower allosteric transition. Activation does not require a canonical protospacer-adjacent motif (PAM), nor is utilization of such PAMs predictive of high trans-activity. We identify several target determinants that can profoundly impact activation times, including bases within the PAM (for ds- but not ssDNA targets) and sequences within and outside those complementary to the spacer, DNA topology, target length, presence of non-specific DNA, and ribose backbone itself, uncovering previously uncharacterized cleavage of and activation by RNA targets. The results provide insight into the mechanism of Cas12a activation, with direct implications on the role of Cas12a in bacterial immunity and for Cas-based diagnostics.

Darkness inhibits autokinase activity of bacterial bathy phytochromes.

Bathy phytochromes are a subclass of bacterial biliprotein photoreceptors that carry a biliverdin IXα chromophore. In contrast to prototypical phytochromes that adopt a red-light-absorbing Pr ground state, the far-red light-absorbing Pfr-form is the thermally stable ground state of bathy phytochromes. Although the photobiology of bacterial phytochromes has been extensively studied since their discovery in the late 1990s, our understanding of the signal transduction process to the connected transmitter domains, which are often histidine kinases, remains insufficient. Initiated by the analysis of the bathy phytochrome PaBphP from Pseudomonas aeruginosa, we performed a systematic analysis of five different bathy phytochromes with the aim to derive a general statement on the correlation of photostate and autokinase output. While all proteins adopt different Pr/Pfr-fractions in response to red, blue, and far-red light, only darkness leads to a pure or highly enriched Pfr-form, directly correlated with the lowest level of autokinase activity. Using this information, we developed a method to quantitatively correlate the autokinase activity of phytochrome samples with well-defined stationary Pr/Pfr-fractions. We demonstrate that the off-state of the phytochromes is the Pfr-form and that different Pr/Pfr-fractions enable the organisms to fine-tune their kinase output in response to a certain light environment. Furthermore, the output response is regulated by the rate of dark reversion, which differs significantly from 5 s to 50 min half-life. Overall, our study indicates that bathy phytochromes function as sensors of light and darkness, rather than red and far-red light, as originally postulated.

DNA hypomethylation activates Cdk4/6 and Atr to induce DNA replication and cell cycle arrest to constrain liver outgrowth in zebrafish.

Coordinating epigenomic inheritance and cell cycle progression is essential for organogenesis. UHRF1 connects these functions during development by facilitating maintenance of DNA methylation and cell cycle progression. Here, we provide evidence resolving the paradoxical phenotype of uhrf1 mutant zebrafish embryos which have activation of pro-proliferative genes and increased number of hepatocytes in S-phase, but the liver fails to grow. We uncover decreased Cdkn2a/b and persistent Cdk4/6 activation as the mechanism driving uhrf1 mutant hepatocytes into S-phase. This induces replication stress, DNA damage and Atr activation. Palbociclib treatment of uhrf1 mutants prevented aberrant S-phase entry, reduced DNA damage, and rescued most cellular and developmental phenotypes, but it did not rescue DNA hypomethylation, transposon expression or the interferon response. Inhibiting Atr reduced DNA replication and increased liver size in uhrf1 mutants, suggesting that Atr activation leads to dormant origin firing and prevents hepatocyte proliferation. Cdkn2a/b was downregulated pro-proliferative genes were also induced in a Cdk4/6 dependent fashion in the liver of dnmt1 mutants, suggesting DNA hypomethylation as a mechanism of Cdk4/6 activation during development. This shows that the developmental defects caused by DNA hypomethylation are attributed to persistent Cdk4/6 activation, DNA replication stress, dormant origin firing and cell cycle inhibition.

Resection of DNA double-strand breaks activates Mre11-Rad50-Nbs1- and Rad9-Hus1-Rad1-dependent mechanisms that redundantly promote ATR checkpoint activation and end processing in Xenopus egg extracts.

Sensing and processing of DNA double-strand breaks (DSBs) are vital to genome stability. DSBs are primarily detected by the ATM checkpoint pathway, where the Mre11-Rad50-Nbs1 (MRN) complex serves as the DSB sensor. Subsequent DSB end resection activates the ATR checkpoint pathway, where replication protein A, MRN, and the Rad9-Hus1-Rad1 (9-1-1) clamp serve as the DNA structure sensors. ATR activation depends also on Topbp1, which is loaded onto DNA through multiple mechanisms. While different DNA structures elicit specific ATR-activation subpathways, the regulation and mechanisms of the ATR-activation subpathways are not fully understood. Using DNA substrates that mimic extensively resected DSBs, we show here that MRN and 9-1-1 redundantly stimulate Dna2-dependent long-range end resection and ATR activation in Xenopus egg extracts. MRN serves as the loading platform for ATM, which, in turn, stimulates Dna2- and Topbp1-loading. Nevertheless, MRN promotes Dna2-mediated end processing largely independently of ATM. 9-1-1 is dispensable for bulk Dna2 loading, and Topbp1 loading is interdependent with 9-1-1. ATR facilitates Mre11 phosphorylation and ATM dissociation. These data uncover that long-range end resection activates two redundant pathways that facilitate ATR checkpoint signaling and DNA processing in a vertebrate system.

Structure of human phagocyte NADPH oxidase in the activated state.

Phagocyte NADPH oxidase, a protein complex with a core made up of NOX2 and p22 subunits, is responsible for transferring electrons from intracellular NADPH to extracellular oxygen1. This process generates superoxide anions that are vital for killing pathogens1. The activation of phagocyte NADPH oxidase requires membrane translocation and the binding of several cytosolic factors2. However, the exact mechanism by which cytosolic factors bind to and activate NOX2 is not well understood. Here we present the structure of the human NOX2-p22 complex activated by fragments of three cytosolic factors: p47, p67 and Rac1. The structure reveals that the p67-Rac1 complex clamps onto the dehydrogenase domain of NOX2 and induces its contraction, which stabilizes the binding of NADPH and results in a reduction of the distance between the NADPH-binding domain and the flavin adenine dinucleotide (FAD)-binding domain. Furthermore, the dehydrogenase domain docks onto the bottom of the transmembrane domain of NOX2, which reduces the distance between FAD and the inner haem. These structural rearrangements might facilitate the efficient transfer of electrons between the redox centres in NOX2 and lead to the activation of phagocyte NADPH oxidase.

RAGE induces physiological activation of NADPH oxidase in neurons and astrocytes and neuroprotection.

The transmembrane receptor for advanced glycation end products (RAGE) is a signaling receptor for many damage- and pathogen-associated molecules. Activation of RAGE is associated with inflammation and an increase in reactive oxygen species (ROS) production. Although several sources of ROS have been previously suggested, how RAGE induces ROS production is still unclear, considering the multiple targets of pathogen-associated molecules. Here, using acute brain slices and primary co-culture of cortical neurons and astrocytes, we investigated the effects of a range of synthetic peptides corresponding to the fragments of the RAGE V-domain on redox signaling. We found that the synthetic fragment (60-76) of the RAGE V-domain induces activation of ROS production in astrocytes and neurons from the primary co-culture and acute brain slices. This effect occurred through activation of RAGE and could be blocked by a RAGE inhibitor. Activation of RAGE by the synthetic fragment stimulates ROS production in NADPH oxidase (NOX). This RAGE-induced NOX activation produced only minor decreases in glutathione levels and increased the rate of lipid peroxidation, although it also reduced basal and ß-amyloid induced cell death in neurons and astrocytes. Thus, specific activation of RAGE induces redox signaling through NOX, which can be a part of a cell protective mechanism.