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1.
Shruti Chakraborty Sayak Ganguli Aritra Chowdhury Michael Ibba Rajat Banerjee 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(8):1801-1809
Background
Under oxidative stress cytoplasmic aminoacyl-tRNA synthetase (aaRSs) substrate specificity can be compromised, leading to tRNA mischarging and mistranslation of the proteome. Whether similar processes occur in mitochondria, which are major cellular sources of reactive oxygen species (ROS), is unknown. However, relaxed substrate specificity in yeast mitochondrial phenylalanyl-tRNA synthetase (ScmitPheRS) has been reported to increase tRNA mischarging and blocks mitochondrial biogenesis.Methods
Non-reducing denaturing PAGE, cysteine reactivity studies, MALDI-TOF mass spectrometry, enzyme assay, western blot, growth assay, circular dichroism, dynamic light scattering and fluorescence spectroscopy were used to study the effect of oxidative stress on ScmitPheRS activity.Results
ScmitPheRS is reversibly inactivated under oxidative stress. The targets for oxidative inactivation are two conserved cysteine residues resulting in reversible intra-molecular disulfide bridge formation. Replacement of either conserved cysteine residue increased viability during growth under oxidative stress.Conclusion
Formation of intra-molecular disulfide bridge under oxidative stress hinders the tRNAPhe binding of the enzyme, thus inactivating ScmitPheRS reversibly.General significance
The ScmitPheRS activity is compromised under oxidative stress due to formation of intra-molecular disulfide bridge. The sensitivity of ScmitPheRS to oxidation may provide a protective mechanism against error-prone translation under oxidative stress. 相似文献2.
Shuping Li Fei Gao Jiaqiang Huang Yuanyuan Wu Sen Wu Xin Gen Lei 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(11):2473-2479
Background
Selenium (Se) is an essential micronutrient required by avian species. Dietary Se/vitamin E deficiency induces three classical diseases in chicks: exudative diathesis, nutritional pancreatic atrophy, and nutritional muscular dystrophy.Scope of review
This review is to summarize and analyze the evolution, regulation, and function of avian selenogenome and selenoproteome and their relationship with the three classical Se/vitamin E deficiency diseases.Major conclusions
There are 24 selenoproteins confirmed in chicks, with two avian-specific members (SELENOU and SELENOP2) and two missing mammalian members (GPX6 and SELENOV). There are two forms of SELENOP containing 1 or 13 selenocysteine residues. In addition, a Gallus gallus gene was conjectured to be the counterpart of the human SEPHS2. Expression of selenoprotein genes in the liver, pancreas, and muscle of chicks seemed to be highly responsive to dietary Se changes. Pathogeneses of the Se/vitamin E deficient diseases in the chicks were likely produced by missing functions of selected selenoproteins in regulating cellular and tissue redox balance and inhibiting oxidative/reductive stress-induced cell death.General significance
Gene knockout models, similar to those of rodents, will help characterize the precise functions of avian selenoproteins and their comparisons with those of mammalian species. 相似文献3.
Johannes Knuesting Christophe Riondet Carlos Maria Inga Kruse No?lle Bécuwe Nicolas K?nig Carsten Berndt Sébastien Tourrette Jocelyne Guilleminot-Montoya Enrique Herrero Frédéric Gaymard Janneke Balk Gemma Belli Renate Scheibe Jean-Philippe Reichheld Nicolas Rouhier Pascal Rey 《Plant physiology》2015,167(4):1643-1658
4.
Radosveta Gencheva Qing Cheng Elias S.J. Arnér 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(11):2511-2517
Background
Toxoflavin (1,6-dimethylpyrimido[5,4-e][1,2,4]triazine-5,7-dione; xanthothricin) is a well-known natural toxin of the pyrimidinetriazinedione type that redox cycles with oxygen under reducing conditions. In mammalian systems, toxoflavin is an inhibitor of Wnt signaling as well as of SIRT1 and SIRT2 activities, but other molecular targets in mammalian cells have been scarcely studied. Interestingly, in a library of nearly 400,000 compounds (PubChem assay ID 588456), toxoflavin was identified as one out of only 56 potential substrates of the mammalian selenoprotein thioredoxin reductase 1 (TrxR1, TXNRD1). This activity was here examined in further detail.Methods
Kinetic parameters in interactions of toxoflavin with rat or human TrxR isoenzymes were determined and compared with those of juglone (5-Hydroxy-1,4-naphthoquinone; walnut toxin) and 9,10-phenanthrene quinone. Selenocysteine dependence was examined using Sec-to-Cys and Sec-to-Ser substituted variants of recombinant rat TrxR1.Results
Toxoflavin was confirmed as an efficient substrate for TrxR. Rat and human cytosolic TrxR1 supported NADPH-dependent redox cycling coupled to toxoflavin reduction, accompanied by H2O2 production under aerobic conditions. Apparent kinetic parameters for the initial rates of reduction showed that rat TrxR1 displayed higher apparent turnover (kcat?=?1700?±?330?min?1) than human TrxR1 (kcat?=?1100?±?82?min?1) but also a higher Km (Km?=?24?±?4.3?μM for human TrxR1 versus Km?=?54?±?18?μM for rat TrxR1). Human TrxR2 (TXNRD2) was less efficient in reduction of toxoflavin (Km?=?280?±?110?μM and kcat?=?740?±?240?min?1). The activity was absolutely dependent upon selenocysteine (Sec). Toxoflavin was also a subversive substrate indirectly inhibiting reduction of other substrates of TrxR1.Conclusions
Our results identify toxoflavin as an efficient redox cycling substrate of mammalian TrxR enzymes, in a strict Sec-dependent manner.General significance
Тhe interactions of toxoflavin with mammalian TrxR isoenzymes can help to explain parts of the molecular mechanisms giving rise to the well-known toxicity as well as pro-oxidant properties of this toxin. 相似文献5.
Najeh Krayem Goetz Parsiegla Hélène Gaussier Hanen Louati Raida Jallouli Pascal Mansuelle Frédéric Carrière Youssef Gargouri 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(5):1247-1261
Background
Heterodimeric phospholipase A2 from venom glands of Tunisian scorpion Scorpio maurus (Sm-PLGV) had been purified. It contains long and short chains linked by a disulfide bridge. Sm-PLGV exhibits hemolytic activity towards human erythrocytes and interacts with phospholipid monolayers at high surface pressure. The investigation of structure-function relationships should provide new clues to understand its activity.Methods
Molecular cloning of Sm-PLGV and heterologous expression in Escherichia coli of three recombinant forms was used to determine the role of the short chain on enzymatic activity. Infrared spectroscopy assisted 3D model building of the three recombinant constructs (phospholipases with and without the penta-peptide and Long chain only) allowed us to propose an explanation of the differences in specific activities and their interaction with various phospholipids.Results
Nucleotide sequence of Sm-PLGV encodes 129 residues corresponding to the Long chain, the penta-peptide and the short chain. Although recombinant phospholipases without and with the penta-peptide have different specific activities, they display a similar substrate specificity on various phospholipid monolayers and similar bell-shaped activity profiles with maxima at high surface pressure. The absence of the short chain reduces significantly enzymatic and hemolytic activities. The 3D models pointed to an interaction of the short chain with the catalytic residues, what might explain the difference in activities of our constructs.Conclusion
Infrared spectroscopy data and 3D modeling confirm the experimental findings that highlight the importance of the short chain for the Sm-PLGV activity.General significance
New informations are given to further establish the structure-function relationships of the Sm-PLGV. 相似文献6.
Background
Reactive oxygen species (ROS) production is an early event in the immune response of plants. ROS production affects the redox-based modification of cysteine residues in redox proteins, which contribute to protein functions such as enzymatic activity, protein-protein interactions, oligomerization, and intracellular localization. Thus, the sensitivity of cysteine residues to changes in the cellular redox status is critical to the immune response of plants.Methods
We used disulfide proteomics to identify immune response-related redox proteins. Total protein was extracted from rice cultured cells expressing constitutively active or dominant-negative OsRacl, which is a key regulator of the immune response in rice, and from rice cultured cells that were treated with probenazole, which is an activator of the plant immune response, in the presence of the thiol group-specific fluorescent probe monobromobimane (mBBr), which was a tag for reduced proteins in a differential display two-dimensional gel electrophoresis. The mBBr fluorescence was detected by using a charge-coupled device system, and total protein spots were detected using Coomassie brilliant blue staining. Both of the protein spots were analyzed by gel image software and identified using MS spectrometry. The possible disulfide bonds were identified using the disulfide bond prediction software. Subcellular localization and bimolecular fluorescence complementation analysis were performed in one of the identified proteins: Oryza sativa cold shock protein 2 (OsCSP2).Results
We identified seven proteins carrying potential redox-sensitive cysteine residues. Two proteins of them were oxidized in cultured cells expressing DN-OsRac1, which indicates that these two proteins would be inactivated through the inhibition of OsRac1 signaling pathway. One of the two oxidized proteins, OsCSP2, contains 197 amino acid residues and six cysteine residues. Site-directed mutagenesis of these cysteine residues revealed that a Cys140 mutation causes mislocalization of a green fluorescent protein fusion protein in the root cells of rice. Bimolecular fluorescence complementation analysis revealed that OsCSP2 is localized in the nucleus as a homo dimer in rice root cells.Conclusions
The findings of the study indicate that redox-sensitive cysteine modification would contribute to the immune response in rice.7.
8.
Caroline Vindry Théophile Ohlmann Laurent Chavatte 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(11):2480-2492
Background
Interest in selenium research has considerably grown over the last decades owing to the association of selenium deficiencies with an increased risk of several human diseases, including cancers, cardiovascular disorders and infectious diseases. The discovery of a genetically encoded 21st amino acid, selenocysteine, is a fascinating breakthrough in molecular biology as it is the first addition to the genetic code deciphered in the 1960s. Selenocysteine is a structural and functional analog of cysteine, where selenium replaces sulfur, and its presence is critical for the catalytic activity of selenoproteins.Scope of review
The insertion of selenocysteine is a non-canonical translational event, based on the recoding of a UGA codon in selenoprotein mRNAs, normally used as a stop codon in other cellular mRNAs. Two RNA molecules and associated partners are crucial components of the selenocysteine insertion machinery, the Sec-tRNA[Ser]Sec devoted to UGA codon recognition and the SECIS elements located in the 3′UTR of selenoprotein mRNAs.Major conclusions
The translational UGA recoding event is a limiting stage of selenoprotein expression and its efficiency is regulated by several factors.General significance
The control of selenoproteome expression is crucial for redox homeostasis and antioxidant defense of mammalian organisms. In this review, we summarize current knowledge on the co-translational insertion of selenocysteine into selenoproteins, and its layers of regulation. 相似文献9.
Zahia Touat-Hamici Anne-Laure Bulteau Juliusz Bianga Hélène Jean-Jacques Joanna Szpunar Ryszard Lobinski Laurent Chavatte 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(11):2493-2505
Background
Selenoproteins (25 genes in human) co-translationally incorporate selenocysteine using a UGA codon, normally used as a stop signal. The human selenoproteome is primarily regulated by selenium bioavailability with a tissue-specific hierarchy.Methods
We investigated the hierarchy of selenoprotein expression in response to selenium concentration variation in four cell lines originating from kidney (HEK293, immortalized), prostate (LNCaP, cancer), skin (HaCaT, immortalized) and liver (HepG2, cancer), using complementary analytical methods. We performed (i) enzymatic activity, (ii) RT-qPCR, (iii) immuno-detection, (iv) selenium-specific mass spectrometric detection after non-radioactive 76Se labeling of selenoproteins, and (v) luciferase-based reporter constructs in various cell extracts.Results
We characterized cell-line specific alterations of the selenoproteome in response to selenium variation that, in most of the cases, resulted from a translational control of gene expression. We established that UGA-selenocysteine recoding efficiency, which depends on the nature of the SECIS element, dictates the response to selenium variation.Conclusions
We characterized that selenoprotein hierarchy is cell-line specific with conserved features. This analysis should be done prior to any experiments in a novel cell line.General significance
We reported a strategy based on complementary methods to evaluate selenoproteome regulation in human cells in culture. 相似文献10.
Gildeíde Aparecida Costa Sávio Bastos de Souza Layz Ribeiro da Silva Teixeira Lev A. Okorokov Andrea Cristina Vetö Arnholdt Anna L. Okorokova-Façanha Arnoldo Rocha Façanha 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(3):684-691
Background
V-ATPase interactions with cholesterol enriched membrane microdomains have been related to metastasis in a variety of cancers, but the underlying mechanism remains at its beginnings. It has recently been reported that the inhibition of this H+ pump affects cholesterol mobilization to the plasma membrane.Methods
Inhibition of melanoma cell migration and invasiveness was assessed by wound healing and Transwell assays in murine cell lines (B16F10 and Melan-A). V-ATPase activity was measured in vitro by ATP hydrolysis and H+ transport in membrane vesicles, and intact cell H+ fluxes were measured by using a non-invasive Scanning Ion-selective Electrode Technique (SIET).Results
Cholesterol depletion by 5 mM MβCD was found to be inhibitory to the hydrolytic and H+ pumping activities of the V-ATPase of melanoma cell lines, as well as to the migration and invasiveness capacities of these cells. Nearly the same effects were obtained using concanamycin A, a specific inhibitor of V-ATPase, which also promoted a decrease of the H+ efflux in live cells at the same extent of MβCD.Conclusions
We found that cholesterol depletion significantly affects the V-ATPase activity and the initial metastatic processes following a profile similar to those observed in the presence of the V-ATPase specific inhibitor, concanamycin.General significance
The results shed new light on the functional role of the interactions between V-ATPases and cholesterol-enriched microdomains of cell membranes that contribute with malignant phenotypes in melanoma. 相似文献11.
Brunna Xavier Martins Raul Ferraz Arruda Gildeíde Aparecida Costa Hassan Jerdy Sávio Bastos de Souza Julianna Maria Santos William Rodrigues de Freitas Milton Masahiko Kanashiro Eulógio Carlos Queiroz de Carvalho Nadir Francisca Sant&#x;Anna Fernanda Antunes Raul Martinez-Zaguilan Sennoune Souad Anna Lvovna Okorokova-Fa?anha Arnoldo Rocha Fa?anha 《Biochimica et Biophysica Acta (BBA)/General Subjects》2019,1863(1):1-12
Background
Metastatic tumor cells have acidic extracellular pH and differential electrochemical H+ gradients generated across their cell membranes by V-type H+-ATPases. This study shows that inhibition of the V-ATPases by the plant-derived monoterpene Myrtenal results in tumor cell death and decreased metastatic dissemination in mice.Methods
The Myrtenal anticancer toxicity was evaluated in vitro using murine (B16F0 and B16F10) and human (SkMel-5) melanoma cell lines, and in in vivo mouse metastatic dissemination model. Proton flux and extracellular acidification were directly evaluated at the surface of living cells using a non-invasive selective ion electrode approach.Results
The inhibition of V-ATPases by 100?μM Myrtenal disrupted the electrochemical H+ gradient across the cell membranes, strongly induced cell death (4–5 fold), and decreased tumor cells migration and invasion in vitro. Myrtenal (15?mg/kg) also significantly reduced metastasis induced by B16F10 in vivo, further reinforcing that V-ATPase is a molecular target to halt the progression of cancers.Conclusions
These data revealed the therapeutic potential of Myrtenal as inhibitor of melanoma progression proposing a mechanism of action by which once inhibited by this monoterpene the proton pumps fail to activate cancer-related differential electrochemical gradients and H+ fluxes across the tumor cell membranes, disrupting pH signatures inherent in tumor progression, resulting in reprogrammed cell death and metastasis inhibition.General significance
The work represents a new mechanistic strategy for contention of melanoma, the most aggressive and deadly form of cutaneous neoplasm, and highlights Myrtenal, other related monoterpenes and derivatives as promising proton pump inhibitors with high chemotherapeutic potential. 相似文献12.
Alex Ferrandi Federica Castani Mauro Pitaro Sara Tagliaferri Claire Bouthier de la Tour Rosa Alduina Suzanne Sommer Mauro Fasano Paola Barbieri Monica Mancini Ian Marc Bonapace 《Biochimica et Biophysica Acta (BBA)/General Subjects》2019,1863(1):118-129
Background
Deinococcus radiodurans R1 (DR) survives conditions of extreme desiccation, irradiation and exposure to genotoxic chemicals, due to efficient DNA breaks repair, also through Mn2+ protection of DNA repair enzymes.Methods
Possible annotated domains of the DR1533 locus protein (Shp) were searched by bioinformatic analysis. The gene was cloned and expressed as fusion protein. Band-shift assays of Shp or the SRA and HNH domains were performed on oligonucleotides, genomic DNA from E. coli and DR. shp knock-out mutant was generated by homologous recombination with a kanamycin resistance cassette.Results
DR1533 contains an N-terminal SRA domain and a C-terminal HNH motif (SRA-HNH Protein, Shp). Through its SRA domain, Shp binds double-strand oligonucleotides containing 5mC and 5hmC, but also unmethylated and mismatched cytosines in presence of Mn2+. Shp also binds to Escherichia coli dcm+ genomic DNA, and to cytosine unmethylated DR and E. coli dcm? genomic DNAs, but only in presence of Mn2+. Under these binding conditions, Shp displays DNAse activity through its HNH domain. Shp KO enhanced >100 fold the number of spontaneous mutants, whilst the treatment with DNA double strand break inducing agents enhanced up to 3-log the number of survivors.Conclusions
The SRA-HNH containing protein Shp binds to and cuts 5mC DNA, and unmethylated DNA in a Mn2+ dependent manner, and might be involved in faithful genome inheritance maintenance following DNA damage.General significance
Our results provide evidence for a potential role of DR Shp protein for genome integrity maintenance, following DNA double strand breaks induced by genotoxic agents. 相似文献13.
Dunja Urbančič Anita Kotar Alenka Šmid Marko Jukič Stanislav Gobec Lars-Göran Mårtensson Janez Plavec Irena Mlinarič-Raščan 《Biochimica et Biophysica Acta (BBA)/General Subjects》2019,1863(1):182-190
Background
Methylation driven by thiopurine S-methylatransferase (TPMT) is crucial for deactivation of cytostatic and immunosuppressant thiopurines. Despite its remarkable integration into clinical practice, the endogenous function of TPMT is unknown.Methods
To address the role of TPMT in methylation of selenium compounds, we established the research on saturation transfer difference (STD) and 77Se NMR spectroscopy, fluorescence measurements, as well as computational molecular docking simulations.Results
Using STD NMR spectroscopy and fluorescence measurements of tryptophan residues in TPMT, we determined the binding of selenocysteine (Sec) to human recombinant TPMT. By comparing binding characteristics of Sec in the absence and in the presence of methyl donor, we confirmed S-adenosylmethionine (SAM)-induced conformational changes in TPMT. Molecular docking analysis positioned Sec into the active site of TPMT with orientation relevant for methylation reaction. Se-methylselenocysteine (MeSec), produced in the enzymatic reaction, was detected by 77Se NMR spectroscopy. A direct interaction between Sec and SAM in the active site of rTPMT and the formation of both products, MeSec and S-adenosylhomocysteine, was demonstrated using NMR spectroscopy.Conclusions
The present study provides evidence on in vitro methylation of Sec by rTPMT in a SAM-dependant manner.General significance
Our results suggest novel role of TPMT and demonstrate new insights into enzymatic modifications of the 21st amino acid. 相似文献14.
Stella A. Child Vanessa P. Rossi Stephen G. Bell 《Biochimica et Biophysica Acta (BBA)/General Subjects》2019,1863(2):408-417
Background
Cyp147G1 is one of 47 cytochrome P450 encoding genes in Mycobacterium marinum M, a pathogenic bacterium with a high degree of sequence similarity to Mycobacterium tuberculosis and Mycobacterium ulcerans. Cyp147G1 is one of only two of these cyp genes which are closely associated with a complete electron transfer system.Methods
The substrate range of the enzyme was tested in vitro and the activity of CYP147G1 was reconstituted in vivo by co-producing the P450 with the ferredoxin and ferredoxin reductase.Results
Substrates of CYP147G1 include fatty acids ranging from octanoic to hexadecanoic acid. CYP147G1 catalysed the selective hydroxylation of linear and ω-2 methyl branched fatty acids at the ω-1 position (≥ 98%). Oxidation of ω-1 methyl branched fatty acids generated the ω and ω-1 hydroxylation products in almost equal proportions, indicating altered position of hydrogen abstraction.Conclusions
This selectivity of fatty acid hydroxylation inferred that linear species must bind in the active site of the enzyme with the terminal methyl group sequestered so that abstraction at the CH bonds of the ω-1 position is favoured. With branched substrates, one of the methyl groups must be close to the compound I oxygen atom and enable hydroxylation at the terminal methyl group to compete with the reaction at the ω-1CH bond.General significance
Hydroxy fatty acids are widely used for industrial, food and medical purposes. CYP147G1 demonstrates high regioselectivity for hydroxylation at a sub-terminal position on a broad range of linear fatty acids, not seen in other CYP enzymes. 相似文献15.
Ryuta Tobe Hisaaki Mihara 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(11):2433-2440
Background
Selenophosphate, the key selenium donor for the synthesis of selenoprotein and selenium-modified tRNA, is produced by selenophosphate synthetase (SPS) from ATP, selenide, and H2O. Although free selenide can be used as the in vitro selenium substrate for selenophosphate synthesis, the precise physiological system that donates in vivo selenium substrate to SPS has not yet been characterized completely.Scope of review
In this review, we discuss selenium metabolism with respect to the delivery of selenium to SPS in selenoprotein biosynthesis.Major conclusions
Glutathione, selenocysteine lyase, cysteine desulfurase, and selenium-binding proteins are the candidates of selenium delivery system to SPS. The thioredoxin system is also implicated in the selenium delivery to SPS in Escherichia coli.General significance
Selenium delivered via a protein-bound selenopersulfide intermediate emerges as a central element not only in achieving specific selenoprotein biosynthesis but also in preventing the occurrence of toxic free selenide in the cell. This article is part of a Special Issue entitled “Selenium research in biochemistry and biophysics – 200 year anniversary”. 相似文献16.
Eduard V. Bocharov Dmitry M. Lesovoy Olga V. Bocharova Anatoly S. Urban Konstantin V. Pavlov Pavel E. Volynsky Roman G. Efremov Alexander S. Arseniev 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(6):1410-1420
Background
Prior studies of the human growth hormone receptor (GHR) revealed a distinct role of spatial rearrangements of its dimeric transmembrane domain in signal transduction across membrane. Detailed structural information obtained in the present study allowed elucidating the bases of such rearrangement and provided novel insights into receptor functioning.Methods
We investigated the dimerization of recombinant TMD fragment GHR254–294 by means of high-resolution NMR in DPC micelles and molecular dynamics in explicit POPC membrane.Results
We resolved two distinct dimeric structures of GHR TMD coexisting in membrane-mimicking micellar environment and providing left- and right-handed helix-helix association via different dimerization motifs. Based on the available mutagenesis data, the conformations correspond to the dormant and active receptor states and are distinguished by cis-trans isomerization of Phe-Pro266 bond in the transmembrane helix entry. Molecular dynamic relaxations of the structures in lipid bilayer revealed the role of the proline residue in functionally significant rearrangements of the adjacent juxtamembrane region supporting alternation between protein-protein and protein-lipid interactions of this region that can be triggered by ligand binding. Also, the importance of juxtamembrane SS bonding for signal persistency, and somewhat unusual aspects of transmembrane region interaction with water molecules were demonstrated.Conclusions
Two alternative dimeric structures of GHR TMD attributed to dormant and active receptor states interchange via allosteric rearrangements of transmembrane helices and extracellular juxtamembrane regions that support coordination between protein-protein and protein-lipid interactions.General significance
This study provides a holistic vision of GHR signal transduction across the membrane emphasizing the role of protein-lipid interactions. 相似文献17.
Wataru Yoshida Mizuki Terasaka Saowalak Laddachote Isao Karube 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(9):1933-1937
Background
DNA methylation at the 5-position of cytosine is an epigenetic modification of CpG dinucleotides. In addition to CpG methylation, the G-quadruplex (G4) structure has been reported as a regulator of gene expression. The identification of G4 forming sequences in CpG islands suggests an involvement of CpG-methylated G4 structures in biological processes; however, few reports have addressed the effects of CpG methylation on G4 structure.Methods
The thermostability of a methylated, 21-mer G4 structure located on the vascular endothelial growth factor (VEGF) gene promoter containing four CpG sites (C1, C6, C11, and C17) were investigated using circular dichroism (CD) spectral analysis.Results
CD melting analysis revealed that VEGF G4 was stabilized by a single CpG methylation on C11 in the presence of Na+ and Mg2+. However, either C1 or C11 methylation enhanced VEGF G4 thermal stability in the presence of K+.Conclusions
Single CpG methylation appears to enhance VEGF G4 thermostability in a manner dependent on both the CpG methylation site and cation type.General significance
These results are expected to contribute to the elucidation of the roles of CpG methylation-stabilized G4 structures in biological processes. 相似文献18.
Rita Petrucci Giuseppe Zollo Antonella Curulli Giancarlo Marrosu 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(8):1781-1789
Background
Antioxidant properties have been recently suggested for caffeine that seems showing protective effects against damages caused by oxidative stress. In particular, a HO scavenging activity has been ascribed to caffeine. Even if the oxidation of caffeine has been widely studied, the antioxidant mechanism is still far to be understood.Methods
The electrochemical behavior of caffeine, theobromine and theophylline was studied in aprotic medium by cyclic voltammetry and electrolysis in UV–vis cell; a computational analysis of the molecular structures based on the Density Functional Theory was performed; the reactivity of all substrates towards lead dioxide, superoxide and galvinoxyl radical was followed by UV–vis spectrophotometry.Results
Results supported the mono-electronic oxidation of the C4C5 bond for all substrates at high oxidation potentials, the electron-transfer process leading to a radical cation or a neutral radical according to the starting methylxanthine N7-substituted (caffeine and theobromine) or N7-unsubstituted (theophylline), respectively. A different following chemical fate might be predicted for the radical cation or the neutral radical. No interaction was evidenced towards the tested reactive oxygen species.Conclusions
No reactivity via H-atom transfer was evidenced for all studied compounds, suggesting that an antiradical activity should be excluded. Some reactivity only with strong oxidants could be predicted via electron-transfer. The acclaimed HO scavenging activity should be interpreted in these terms. The study suggested that CAF might be hardly considered an antioxidant.General significance
Beyond the experimental methods used, the discussion of the present results might provide food for thought to the wide audience working on antioxidants. 相似文献19.
Nathalie Lejal Sandrine Truchet Edna Bechor Edwige Bouguyon Vijay Khedkar Nicolas Bertho Jasmina Vidic Pierre Adenot Stéphanie Solier Edgar Pick Anny Slama-Schwok 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(6):1263-1275
Background
Targeting cells of the host immune system is a promising approach to fight against Influenza A virus (IAV) infection. Macrophage cells use the NADPH oxidase-2 (NOX2) enzymatic complex as a first line of defense against pathogens by generating superoxide ions O2– and releasing H2O2. Herein, we investigated whether targeting membrane -embedded NOX2 decreased IAV entry via raft domains and reduced inflammation in infected macrophages.Methods
Confocal microscopy and western blots monitored levels of the viral nucleoprotein NP and p67phox, NOX2 activator subunit, Elisa assays quantified TNF-α levels in LPS or IAV-activated mouse or porcine alveolar macrophages pretreated with a fluorescent NOX inhibitor, called nanoshutter NS1.Results
IAV infection in macrophages promoted p67phox translocation to the membrane, rafts clustering and activation of the NOX2 complex at early times. Disrupting rafts reduced intracellular viral NP. NS1 markedly reduced raft clustering and viral entry by binding to the C-terminal of NOX2 also characterized in vitro. NS1 decrease of TNF-α release depended on the cell type.Conclusion
NOX2 participated in IAV entry and raft-mediated endocytosis. NOX2 inhibition by NS1 reduced viral entry. NS1 competition with p67phox for NOX2 binding shown by in silico models and cell-free assays was in agreement with NS1 inhibiting p67phox translocation to membrane-embedded NOX2 in mouse and porcine macrophages.General significance
We introduce NS1 as a compound targeting NOX2, a critical enzyme controlling viral levels and inflammation in macrophages and discuss the therapeutic relevance of targeting the C-terminal of NADPH oxidases by probes like NS1 in viral infections. 相似文献20.
Prashanth Kumar Koochana Abhinav Mohanty Suman Das Biswamaitree Subhadarshanee Suresh Satpati Anshuman Dixit Surendra Chandra Sabat Rabindra K. Behera 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(5):1190-1198