Resveratrol-Induced Xenophagy Promotes Intracellular Bacteria Clearance in Intestinal Epithelial Cells and Macrophages

Autophagy is a lysosomal degradation process that contributes to host immunity by eliminating invasive pathogens and the modulating inflammatory response. Several infectious and immune disorders are associated with autophagy defects, suggesting that stimulation of autophagy in these diseases should be beneficial. Here, we show that resveratrol is able to boost xenophagy, a selective form of autophagy that target invasive bacteria. We demonstrated that resveratrol promotes in vitro autophagy-dependent clearance of intracellular bacteria in intestinal epithelial cells and macrophages. These results were validated in vivo using infection in a transgenic GFP-LC3 zebrafish model. We also compared the ability of resveratrol derivatives, designed to improve the bioavailability of the parent molecule, to stimulate autophagy and to induce intracellular bacteria clearance. Together, our data demonstrate the ability of resveratrol to stimulate xenophagy, and thereby enhance the clearance of two invasive bacteria involved life-threatening diseases, Salmonella Typhimurium and Crohn's disease-associated Adherent-Invasive Escherichia coli. These findings encourage the further development of pro-autophagic nutrients to strengthen intestinal homeostasis in basal and infectious states

Étude des interactions de la sélénoproteine P et de la vasopressine diséléniée en présence d’auranofine et de cisplatine

Selenium is known to be an essential trace element with antioxidant activities. Its physiological role is mainly due to its co-translational incorporation into selenoproteins as selenocysteine (SeCys), referred to as the 21st amino acid. selenoprotein P (SELENOP) is the major selenoprotein in plasma with up to 10 SeCys in its sequence. Because of the chemical complexity of the serum matrix, the low abundance of selenoprotein P, and the occurrence of its putative multiple isoforms and post-translational modifications (e.g., glycosylation, Se-S and Se-Se bridges), the characterization of selenoprotein P requires the protein selective pre-concentration and custom-designed optimization of analytical methodology. Containing 10 SeCys residues, SELENOP is a potential preferential target for metallodrugs such as auranofin and cisplatin. The interactions have not been studied yet at the molecular level, despite considerable evidence that free selenols are biding sites for auranofin in another selenoprotein, thioredoxin reductase. The lack of studies of selenoprotein P is likely due to the fact that the protein cannot be heterologously expressed and needs to be purified from serum where it is present at ng/g concentration (as Se). This thesis presents the development of the method of purification of SELENOP from human serum using two-dimensional affinity chromatography. The two chromatographic steps using immobilized metal (cobalt) affinity and heparin affinity chromatography allowed the purification of SELENOP with excellent selectivity. The subsequent characterization of the purified SELENOP by nanoHPLC - electrospray ESI MS/MS allowed accounting for almost all selenocysteine-containing tryptic SELENOP peptides and the elucidation of some glycosylation sites. In addition, the recovery of selenium incorporated in SELENOP was quantitatively monitored, for the first time, at each step of the purification procedure. The recovery of SELENOP was 14% after all the purification steps. Selenium present in SELENOP was found to account for 35% of total selenium in serum.The purification of SELENOP allowed the first study of its interactions with auranofin and cisplatin containing, Au(I) and Pt(II), respectively. The protein metalation was observed after incubation of SELENOP with both metallodrugs. After digestion with trypsin, Au and Pt modifications were observed on the resulting peptides. Either in the case of auranofin and cisplatin, two SELENOP peptides were found to form Au or Pt adducts by Cy and SeCys binding (MS/MS characterization). These four peptides, specific to SELENOP, displayed different sequences.Moreover, to comparatively study the interactions of the Se-Se and S-S bridges with metallodrugs, vasopressin and its di-selenide analogues were used as model peptides. Their reactivity with auranofin and its strict analogues was investigated by LC-electrospray MS/MS. Evidence was obtained of the possible cleavage of the S-S and Se-Se bridges induced by Au(I) in the absence of reducing agents, in contrast to the previous studies requiring a prior reduction of the Se-Se bond to make it react with Au(I) compounds. In addition, we found that at high temperatures (70 °C), the sulfur and selenium atoms were metallated with the preferential binding of gold to selenium, the reaction not taking place under physiological conditions (37 °C).L'un des objectifs de cette thèse a été de revisiter les schémas d'isolement de SELENOP rapportés ailleurs afin de développer une méthode capable de produire des quantités suffisantes de SELENOP pour produire des signaux spécifiques aux espèces de Se mesurables pour des études par spectrométrie de masse des interactions de SELENOP avec des composés à base de Au et Pt. Pour cela, différents types de chromatographie d'affinité, tels que l'IMAC ou l'héparine, seuls ou en séquence, ont été revisités. L'accent a été mis sur l'optimisation des étapes de concentration inter-chromatographie et le suivi de la récupération de SELENOP. Différentes conditions chromatographiques ont été testées pour obtenir la meilleure résolution et séparation à chaque étape et augmenter le recouvrement à chaque étape de la purification.Un élément inhérent à la thèse a été une caractérisation approfondie de la sélénoprotéine isolée par spectrométrie de masse haute résolution et haute précision. Aussi étrange que cela puisse paraître, très peu de preuves de spectrométrie de masse pour les données de séquence d'acides aminés de SELENOP existent dans la littérature. Par conséquent, une méthode basée sur la digestion trypsique et la spectrométrie HPLC MS/MS a dû être développée afin de démontrer l'identité chimique de la protéine isolée et purifiée et d'acquérir un support de bases pour l'interprétation des données sur les interactions de SELENOP avec les composés à base de Pt et d’Au.L'objectif ultime relatif à SELENOP a été d'étudier sa liaison avec l'auranofine et le cisplatine, en sondant la formation des composés métalliques SELENOP et en acquérant des preuves moléculaires sur les sites de liaison avec l'Au et le Pt.La propension des résidus de SeCys à s’oxyder pour former des liaisons Se-Se ou Se-S soulève une question importante sur leur réactivité avec les métallomédicaments. Alors qu'il est maintenant bien documenté que l'auranofine et ses analogues se lient rapidement et étroitement aux protéines portant des cystéines ou des sélénocystéines libres, la capacité de ces composés à base d'or d’interagir directement avec les ponts disulfure et diséléniure et à les cliver est beaucoup moins comprise. Cela nous a incité à étudier en détail les réactions de l'auranofine et de ses analogues avec des molécules modèles contenant des motifs disulfures et diséléniure.Pour cela, nous avons décidé d'utiliser, en tant que peptides modèles, l’hormone vasopressine (AVP), connue pour son action antidiurétique et vasopressive, et son analogue séléniée, la selenovasopressine ([Se-Se]-AVP), dans lequel le pont disulfure a été remplacé par un pont diselenide tout en gardant l’affinité de AVP envers le récepteur de la vasopressine

Studies of interaction of selenoprotein P and selenized vasopressin in presence of auranofin and cisplatin

L'un des objectifs de cette thèse a été de revisiter les schémas d'isolement de SELENOP rapportés ailleurs afin de développer une méthode capable de produire des quantités suffisantes de SELENOP pour produire des signaux spécifiques aux espèces de Se mesurables pour des études par spectrométrie de masse des interactions de SELENOP avec des composés à base de Au et Pt. Pour cela, différents types de chromatographie d'affinité, tels que l'IMAC ou l'héparine, seuls ou en séquence, ont été revisités. L'accent a été mis sur l'optimisation des étapes de concentration inter-chromatographie et le suivi de la récupération de SELENOP. Différentes conditions chromatographiques ont été testées pour obtenir la meilleure résolution et séparation à chaque étape et augmenter le recouvrement à chaque étape de la purification.Un élément inhérent à la thèse a été une caractérisation approfondie de la sélénoprotéine isolée par spectrométrie de masse haute résolution et haute précision. Aussi étrange que cela puisse paraître, très peu de preuves de spectrométrie de masse pour les données de séquence d'acides aminés de SELENOP existent dans la littérature. Par conséquent, une méthode basée sur la digestion trypsique et la spectrométrie HPLC MS/MS a dû être développée afin de démontrer l'identité chimique de la protéine isolée et purifiée et d'acquérir un support de bases pour l'interprétation des données sur les interactions de SELENOP avec les composés à base de Pt et d’Au.L'objectif ultime relatif à SELENOP a été d'étudier sa liaison avec l'auranofine et le cisplatine, en sondant la formation des composés métalliques SELENOP et en acquérant des preuves moléculaires sur les sites de liaison avec l'Au et le Pt.La propension des résidus de SeCys à s’oxyder pour former des liaisons Se-Se ou Se-S soulève une question importante sur leur réactivité avec les métallomédicaments. Alors qu'il est maintenant bien documenté que l'auranofine et ses analogues se lient rapidement et étroitement aux protéines portant des cystéines ou des sélénocystéines libres, la capacité de ces composés à base d'or d’interagir directement avec les ponts disulfure et diséléniure et à les cliver est beaucoup moins comprise. Cela nous a incité à étudier en détail les réactions de l'auranofine et de ses analogues avec des molécules modèles contenant des motifs disulfures et diséléniure.Pour cela, nous avons décidé d'utiliser, en tant que peptides modèles, l’hormone vasopressine (AVP), connue pour son action antidiurétique et vasopressive, et son analogue séléniée, la selenovasopressine ([Se-Se]-AVP), dans lequel le pont disulfure a été remplacé par un pont diselenide tout en gardant l’affinité de AVP envers le récepteur de la vasopressine.Selenium is known to be an essential trace element with antioxidant activities. Its physiological role is mainly due to its co-translational incorporation into selenoproteins as selenocysteine (SeCys), referred to as the 21st amino acid. selenoprotein P (SELENOP) is the major selenoprotein in plasma with up to 10 SeCys in its sequence. Because of the chemical complexity of the serum matrix, the low abundance of selenoprotein P, and the occurrence of its putative multiple isoforms and post-translational modifications (e.g., glycosylation, Se-S and Se-Se bridges), the characterization of selenoprotein P requires the protein selective pre-concentration and custom-designed optimization of analytical methodology. Containing 10 SeCys residues, SELENOP is a potential preferential target for metallodrugs such as auranofin and cisplatin. The interactions have not been studied yet at the molecular level, despite considerable evidence that free selenols are biding sites for auranofin in another selenoprotein, thioredoxin reductase. The lack of studies of selenoprotein P is likely due to the fact that the protein cannot be heterologously expressed and needs to be purified from serum where it is present at ng/g concentration (as Se). This thesis presents the development of the method of purification of SELENOP from human serum using two-dimensional affinity chromatography. The two chromatographic steps using immobilized metal (cobalt) affinity and heparin affinity chromatography allowed the purification of SELENOP with excellent selectivity. The subsequent characterization of the purified SELENOP by nanoHPLC - electrospray ESI MS/MS allowed accounting for almost all selenocysteine-containing tryptic SELENOP peptides and the elucidation of some glycosylation sites. In addition, the recovery of selenium incorporated in SELENOP was quantitatively monitored, for the first time, at each step of the purification procedure. The recovery of SELENOP was 14% after all the purification steps. Selenium present in SELENOP was found to account for 35% of total selenium in serum.The purification of SELENOP allowed the first study of its interactions with auranofin and cisplatin containing, Au(I) and Pt(II), respectively. The protein metalation was observed after incubation of SELENOP with both metallodrugs. After digestion with trypsin, Au and Pt modifications were observed on the resulting peptides. Either in the case of auranofin and cisplatin, two SELENOP peptides were found to form Au or Pt adducts by Cy and SeCys binding (MS/MS characterization). These four peptides, specific to SELENOP, displayed different sequences.Moreover, to comparatively study the interactions of the Se-Se and S-S bridges with metallodrugs, vasopressin and its di-selenide analogues were used as model peptides. Their reactivity with auranofin and its strict analogues was investigated by LC-electrospray MS/MS. Evidence was obtained of the possible cleavage of the S-S and Se-Se bridges induced by Au(I) in the absence of reducing agents, in contrast to the previous studies requiring a prior reduction of the Se-Se bond to make it react with Au(I) compounds. In addition, we found that at high temperatures (70 °C), the sulfur and selenium atoms were metallated with the preferential binding of gold to selenium, the reaction not taking place under physiological conditions (37 °C)

Resveratrol-Induced Xenophagy Promotes Intracellular Bacteria Clearance in Intestinal Epithelial Cells and Macrophages

International audienceAutophagy is a lysosomal degradation process that contributes to host immunity by eliminating invasive pathogens and the modulating inflammatory response. Several infectious and immune disorders are associated with autophagy defects, suggesting that stimulation of autophagy in these diseases should be bene ficial. Here, we show that resveratrol is able to boost xenophagy, a selective form of autophagy that target invasive bacteria. We demonstrated that resveratrol promotes in vitro autophagy-dependent clearance of intracellular bacteria in intestinal epithelial cells and macrophages. These results were validated in vivo using infection in a transgenic GFP-LC3 zebra fish model. We also compared the ability of resveratrol derivatives, designed to improve the bioavailability of the parent molecule, to stimulate autophagy and to induce intracellular bacteria clearance. Together, our data demonstrate the ability of resveratrol to stimulate xenophagy, and thereby enhance the clearance of two invasive bacteria involved life-threatening diseases, Salmonella Typhimurium and Crohn's disease-associated Adherent-Invasive Escherichia coli. These findings encourage the further development of pro-autophagic nutrients to strengthen intestinal homeostasis in basal and infectious states

Identification of Three-Way DNA Junction Ligands through Screening of Chemical Libraries and Validation by Complementary in Vitro Assays.

International audienceThe human genome is replete with repetitive DNA sequences that can fold into thermodynamically stable secondary structures such as hairpins and quadruplexes. Cellular enzymes exist to cope with these structures whose stable accumulation would result in DNA damage through interference with DNA transactions such as transcription and replication. Therefore, the chemical stabilization of secondary DNA structures offers an attractive way to foster DNA transaction-associated damages to trigger cell death in proliferating cancer cells. While much emphasis has been recently given to DNA quadruplexes, we focused here on three-way DNA junctions (TWJ) and report on a strategy to identify TWJ-targeting agents through a combination of in vitro techniques (TWJ-screen, polyacrylamide gel electrophoresis, fluorescence resonance energy transfer-melting, electrospray ionization mass spectrometry, dialysis equilibrium, and sulforhodamine B assays). We designed a complete workflow and screened 1200 compounds to identify promising TWJ ligands selected on stringent criteria in terms of TWJ-folding ability, affinity, and selectivity