Université PSL

Publications

RECHERCHER

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Performance evaluation of a MIP for the MISPE-LC determination of p-[18F]MPPF and a potential metabolite in human plasma
Laboratoire Spectrométrie de masse biologique et protéomique - F.Lecomte J.Aerts Plenevaux .Defraiteur. Chapuis-Hugonc. Rozetd. Chiape. Luxen. Pichon, Ph.Huberta C.Huberta
ELSEVIER - 180 113015 - https://doi.org/10.1016/j.jpba.2019.113015 - 2020
The mapping of post-translational modifications (PTMs) of proteins can be addressed by bottom-up proteomics strategy using proteases to achieve the enzymatic digestion of the biomolecule. Glycosylation is one of the most challenging PTM to characterize due to its large structural heterogeneity. In this work, two Immobilized Enzyme Reactors (IMERs) based on trypsin and pepsin protease were used for the first time to fasten and improve the reliability of the specific mapping of the N-glycosylation heterogeneity of glycoproteins. The performance of the supports was evaluated with the digestion of human Chorionic Gonadotropin hormone (hCG), a glycoprotein characterized by four N- and four O-glycosylation sites, prior to the analysis of the digests by nanoliquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). Firstly, the repeatability of the nanoLC-MS/MS was evaluated and a method to control the identification of the identified glycans was developed to validate them regarding the retention time of glycopeptides in reversed phase nanoLC separation. The repeatability of the digestion with trypsin-based IMER was evaluated on the same hCG batch and on three independent batches with common located glycans up to 75%. Then, the performance of the IMER digestions was compared to in-solution digestions to evaluate the qualitative mapping of the glycosylation. It has given rise to 42 out of 45 common glycans between both digestions modes. For the first time, the complementarity of trypsin and pepsin was illustrated for the glycosylation mapping as trypsin led to identifications on 2 out of 4 glycosylation site while pepsin was informative on the 4 glycosylation site. The potential of IMERs for the study of the glycosylation of a protein was illustrated with the comparison of two hCG-based drugs, Ovitrelle® and Pregnyl
Identification and semi-relative quantification of intact glycoforms by nano-LC–(Orbitrap)MS: application to the α-subunit of human chorionic gonadotropin and follicle-stimulating hormone
Laboratoire Spectrométrie de masse biologique et protéomique - Amira Al Matari, Audrey Combès, Julien Camperi, Thierry Fournier, Valérie Pichon & Nathalie Delaunay
ELSEVIER - 412 5729–5741 - , 10.1007/s00216-020-02794-3 - 2020
Human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH) belong to the family of glycoprotein polypeptide hormones called gonadotropins. They are heterodimers sharing the α-subunit structure that has 2 N-glycosylation sites. A method based on nano-reversed-phase liquid chromatography coupled to high-resolution mass spectrometry with an Orbitrap analyzer was developed for the first time to characterize the glycosylation state of the α-subunit at the intact level. A recombinant hCG-based drug, Ovitrelle®, was analyzed. This method combined with an appropriate data treatment allowed the detection of not only the major isoforms but also the minority ones with a high mass accuracy. More than 30 hCGα glycoforms were detected without overlapping of the isotopic patterns. The figures of merit of the method were assessed. The relative standard deviations (RSDs) of the retention time ranged between 0.1 and 6.08% (n = 3), with an average of 0.4%. The RSDs of the peak area measured on the extracted ion chromatogram of each glycoform are below 38% (n = 3), with an average of 16%, thus allowing semi-relative quantification. The ability to accurately profile glycosylated variants of hCGα was next demonstrated by comparing qualitatively and semi-quantitatively 3 batches of Ovitrelle®. The method was also used to analyze 3 batches of a recombinant FSH-based drug, Puregon®, and 30 FSHα glycoforms were detected and semi-quantified. This demonstrates the high potential of this method for fast quality control or comparison of the glycosylation of glycoprotein-based pharmaceutical preparations.
Identification and semi-relative quantification of intact glycoforms by nano-LC–(Orbitrap)MS: application to the α-subunit of human chorionic gonadotropin and follicle-stimulating hormone
Laboratoire Spectrométrie de masse biologique et protéomique - Julien Camperi, Audrey Combès, Thierry Fournier, Valerie Pichon & Nathalie Delaunay
Research Paper - 412 4423–4432 - 10.1007/s00216-020-02684-8 - 2020
In the present work, the human chorionic gonadotropin (hCG) hormone was characterized for the first time by hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution (HR) quadrupole/time-of-flight (qTOF) mass spectrometry (MS) at the intact level. This heterodimeric protein, consisting of two subunits (hCGα and hCGβ), possesses 8 potential glycosylation sites leading to a high number of glycoforms and has a molecular weight of about 35 kDa. The HILIC conditions optimized in a first paper but using UV detection were applied here with MS for the analysis of two hCG-based drugs, a recombinant hCG and a hCG isolated from the urine of pregnant women. An amide column (150 × 2.1 mm, 2.6 μm, 150 Å), a mobile phase composed of acetonitrile and water both containing 0.1% of trifluoroacetic acid, and a temperature of 60 °C were used. The gradient was from 85 to 40% ACN in 30 min. The use of TFA that had been shown to be necessary for the separation of glycoforms caused, as expected, an ion suppression effect in MS that was partially overcome by increasing the amount of protein injected (2 μL at 1 mg mL−1) and reducing the detection m/z range (from 1500 to 300). These conditions allowed the detection of different glycoforms of hCGα. The performance of the HILIC-HRMS method was compared with that previously obtained in RPLC-HRMS in terms of the number of detected glycoforms, selectivity, and sensitivity. The complementarity and orthogonality of the HILIC and RP modes for the analysis of hCG at the intact level were demonstrated.
Investigation of serum proteome homeostasis during radiation therapy by a quantitative proteomics approach
Laboratoire Spectrométrie de masse biologique et protéomique - Amira Ouerhani ; Giovanni Chiappetta ; Oussema Souiai ; Halima Mahjoubi ; Joelle Vinh
Biosci Rep - 39 (7) BSR20182319 - doi.org/10.1042/BSR20182319 - 2019
The purpose of the present study is to analyze the serum proteome of patients receiving Radiation Therapy (RT) at different stages of their treatment to discovery candidate biomarkers of the radiation-induced skin lesions and the molecular pathways underlying the radiation signatures. Six stages of RT treatment were monitored from patients treated because of brain cancer: before starting the treatment, during the treatment (four time points), and at 4 weeks from the last RT dose. Serum samples were analyzed by a proteomics approach based on the Data Independent Acquisition (DIA) mass spectrometry (MS). RT induced clear changes in the expression levels of 36 serum proteins. Among these, 25 proteins were down- or up-regulated significantly before the emergence of skin lesions. Some of these were still deregulated after the completion of the treatment. Few days before the appearance of the skin lesions, the levels of some proteins involved in the wound healing processes were down-regulated. The pathway analysis indicated that after partial body irradiation, the expression levels of proteins functionally involved in the acute inflammatory and immune response, lipoprotein process and blood coagulation, were deregulated.
Development of Immobilized Enzyme Reactors for the characterization of the glycosylation heterogeneity of a protein
Laboratoire Spectrométrie de masse biologique et protéomique - Stan Perchepied, Nicolas Eskenazi, Chiara Giangrande, Julien Camperi, Thierry Fournier, Joëlle Vinh, Nathalie Delaunay, Valérie Pichon
Biosci Rep - 206 120171 - doi.org/10.1016/j.talanta.2019.120171 - 2019
The mapping of post-translational modifications (PTMs) of proteins can be addressed by bottom-up proteomics strategy using proteases to achieve the enzymatic digestion of the biomolecule. Glycosylation is one of the most challenging PTM to characterize due to its large structural heterogeneity. In this work, two Immobilized Enzyme Reactors (IMERs) based on trypsin and pepsin protease were used for the first time to fasten and improve the reliability of the specific mapping of the N-glycosylation heterogeneity of glycoproteins. The performance of the supports was evaluated with the digestion of human Chorionic Gonadotropin hormone (hCG), a glycoprotein characterized by four N- and four O-glycosylation sites, prior to the analysis of the digests by nanoliquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). Firstly, the repeatability of the nanoLC-MS/MS was evaluated and a method to control the identification of the identified glycans was developed to validate them regarding the retention time of glycopeptides in reversed phase nanoLC separation. The repeatability of the digestion with trypsin-based IMER was evaluated on the same hCG batch and on three independent batches with common located glycans up to 75%. Then, the performance of the IMER digestions was compared to in-solution digestions to evaluate the qualitative mapping of the glycosylation. It has given rise to 42 out of 45 common glycans between both digestions modes. For the first time, the complementarity of trypsin and pepsin was illustrated for the glycosylation mapping as trypsin led to identifications on 2 out of 4 glycosylation site while pepsin was informative on the 4 glycosylation site. The potential of IMERs for the study of the glycosylation of a protein was illustrated with the comparison of two hCG-based drugs, Ovitrelle® and Pregnyl
Contribution of proteases and cellulases produced by solid-state fermentation to the improvement of corn ethanol production
Laboratoire Spectrométrie de masse biologique et protéomique - Anaïs Guillaume, Aurore Thorigné, Yoann Carré, Joëlle Vinh and Loïc Levavasseur
Biosci Rep - 6 7 - doi.org/10.1186/s40643-019-0241-0 - 2019
By cultivating a strain of Aspergillus tubingensis on agro-industrial by-products using solid-state fermentation technology, a biocatalyst containing more than 130 different enzymes was obtained. The enzymatic complex was composed mainly of hydrolases, among which a protease, an aspergillopepsin, accounted for more than half of the total proteins. Cell-wall-degrading enzymes such as pectinases, cellulases and hemicellulases were also highly represented. Adding the biocatalyst to corn mash at 1 kg/T corn allowed to significantly improve ethanol production performances. The final ethanol concentration was increased by 6.8% and the kinetics was accelerated by 14 h. The aim of this study was to identify the enzymes implicated in the effect on corn ethanol production. By fractionating the biocatalyst, the particular effect of the major enzymes was investigated. Experiments revealed that, together, the protease and two cellulolytic enzymes (an endoglucanase and a β-glucosidase) were responsible for 80% of the overall effect of the biocatalyst. Nevertheless, the crude extract of the biocatalyst showed greater impact than the combination of up to seven purified enzymes, demonstrating the complementary enzymatic complex obtained by solid-state fermentation. This technology could, therefore, be a relevant natural alternative to the use of GMO-derived enzymes in the ethanol industry.
Global host molecular perturbations upon in situ loss of bacterial endosymbionts in the deep-sea mussel Bathymodiolus azoricus assessed using proteomics and transcriptomics
Laboratoire Spectrométrie de masse biologique et protéomique - Détrée C1, Haddad I, Demey-Thomas E, Vinh J, Lallier FH, Tanguy A, Mary J
BMC Genomics - 20(1) 109 - doi: 10.1186/s12864-019-5456-0. - 2019
BACKGROUND:
Colonization of deep-sea hydrothermal vents by most invertebrates was made efficient through their adaptation to a symbiotic lifestyle with chemosynthetic bacteria, the primary producers in these ecosystems. Anatomical adaptations such as the establishment of specialized cells or organs have been evidenced in numerous deep-sea invertebrates. However, very few studies detailed global inter-dependencies between host and symbionts in these ecosystems. In this study, we proposed to describe, using a proteo-transcriptomic approach, the effects of symbionts loss on the deep-sea mussel Bathymodiolus azoricus' molecular biology. We induced an in situ depletion of symbionts and compared the proteo-transcriptome of the gills of mussels in three conditions: symbiotic mussels (natural population), symbiont-depleted mussels and aposymbiotic mussels.

RESULTS:
Global proteomic and transcriptomic results evidenced a global disruption of host machinery in aposymbiotic organisms. We observed that the total number of proteins identified decreased from 1118 in symbiotic mussels to 790 in partially depleted mussels and 761 in aposymbiotic mussels. Using microarrays we identified 4300 transcripts differentially expressed between symbiont-depleted and symbiotic mussels. Among these transcripts, 799 were found differentially expressed in aposymbiotic mussels and almost twice as many in symbiont-depleted mussels as compared to symbiotic mussels. Regarding apoptotic and immune system processes - known to be largely involved in symbiotic interactions - an overall up-regulation of associated proteins and transcripts was observed in symbiont-depleted mussels.

CONCLUSION:
Overall, our study showed a global impairment of host machinery and an activation of both the immune and apoptotic system following symbiont-depletion. One of the main assumptions is the involvement of symbiotic bacteria in the inhibition and regulation of immune and apoptotic systems. As such, symbiotic bacteria may increase their lifespan in gill cells while managing the defense of the holobiont against putative pathogens.
Development of Immobilized Enzyme Reactors for the characterization of the glycosylation heterogeneity of a protein
Laboratoire Spectrométrie de masse biologique et protéomique - Stan Perchepied, Nicolas Eskenazi, Chiara Giangrande, Julien Camperi, Thierry Fournier, Joëlle Vinh, Nathalie Delaunay, Valérie Pichon
ELSEVIER - 206 120171 - doi.org/10.1016/j.talanta.2019.120171 - 2019
The mapping of post-translational modifications (PTMs) of proteins can be addressed by bottom-up proteomics strategy using proteases to achieve the enzymatic digestion of the biomolecule. Glycosylation is one of the most challenging PTM to characterize due to its large structural heterogeneity. In this work, two Immobilized Enzyme Reactors (IMERs) based on trypsin and pepsin protease were used for the first time to fasten and improve the reliability of the specific mapping of the N-glycosylation heterogeneity of glycoproteins. The performance of the supports was evaluated with the digestion of human Chorionic Gonadotropin hormone (hCG), a glycoprotein characterized by four N- and four O-glycosylation sites, prior to the analysis of the digests by nanoliquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). Firstly, the repeatability of the nanoLC-MS/MS was evaluated and a method to control the identification of the identified glycans was developed to validate them regarding the retention time of glycopeptides in reversed phase nanoLC separation. The repeatability of the digestion with trypsin-based IMER was evaluated on the same hCG batch and on three independent batches with common located glycans up to 75%. Then, the performance of the IMER digestions was compared to in-solution digestions to evaluate the qualitative mapping of the glycosylation. It has given rise to 42 out of 45 common glycans between both digestions modes. For the first time, the complementarity of trypsin and pepsin was illustrated for the glycosylation mapping as trypsin led to identifications on 2 out of 4 glycosylation site while pepsin was informative on the 4 glycosylation site. The potential of IMERs for the study of the glycosylation of a protein was illustrated with the comparison of two hCG-based drugs, Ovitrelle® and Pregnyl
Conventional and Neo-Antigenic Peptides Presented by β Cells Are Targeted by Circulating Naïve CD8+ T Cells in Type 1 Diabetic and Healthy Donors.
Laboratoire Spectrométrie de masse biologique et protéomique - Sergio Gonzalez-Duque , Marie Eliane Azoury , Maikel L Colli , Georgia Afonso , Jean-Valery Turatsinze , Laura Nigi , Ana Ines Lalanne , Guido Sebastiani , Alexia Carré , Sheena Pinto, Slobodan Culina , Noémie Corcos , Marco Bugliani , Pier
Cell Metab - 28(6) 946-960.e6. - doi: 10.1016/j.cmet.2018.07.007. - 2018
Although CD8+ T-cell-mediated autoimmune β cell destruction occurs in type 1 diabetes (T1D), the target epitopes processed and presented by β cells are unknown. To identify them, we combined peptidomics and transcriptomics strategies. Inflammatory cytokines increased peptide presentation in vitro, paralleling upregulation of human leukocyte antigen (HLA) class I expression. Peptide sources featured several insulin granule proteins and all known β cell antigens, barring islet-specific glucose-6-phosphatase catalytic subunit-related protein. Preproinsulin yielded HLA-A2-restricted epitopes previously described. Secretogranin V and its mRNA splice isoform SCG5-009, proconvertase-2, urocortin-3, the insulin gene enhancer protein ISL-1, and an islet amyloid polypeptide transpeptidation product emerged as antigens processed into HLA-A2-restricted epitopes, which, as those already described, were recognized by circulating naive CD8+ T cells in T1D and healthy donors and by pancreas-infiltrating cells in T1D donors. This peptidome opens new avenues to understand antigen processing by β cells and for the development of T cell biomarkers and tolerogenic vaccination strategies.
Conventional and Neo-antigenic Peptides Presented by β Cells Are Targeted by Circulating Naïve CD8+ T Cells in Type 1 Diabetic and Healthy Donors
Laboratoire Spectrométrie de masse biologique et protéomique - Sergio Gonzalez-Duque , Marie Eliane Azoury , Maikel L Colli , Georgia Afonso , Jean-Valery Turatsinze , Laura Nigi , Ana Ines Lalanne , Guido Sebastiani , Alexia Carré , Sheena Pinto, Slobodan Culina , Noémie Corcos , Marco Bugliani , Pier
Cell Metab - 28(6) 946-960.e6. - doi: 10.1016/j.cmet.2018.07.007. - 2018
Although CD8+ T-cell-mediated autoimmune β cell destruction occurs in type 1 diabetes (T1D), the target epitopes processed and presented by β cells are unknown. To identify them, we combined peptidomics and transcriptomics strategies. Inflammatory cytokines increased peptide presentation in vitro, paralleling upregulation of human leukocyte antigen (HLA) class I expression. Peptide sources featured several insulin granule proteins and all known β cell antigens, barring islet-specific glucose-6-phosphatase catalytic subunit-related protein. Preproinsulin yielded HLA-A2-restricted epitopes previously described. Secretogranin V and its mRNA splice isoform SCG5-009, proconvertase-2, urocortin-3, the insulin gene enhancer protein ISL-1, and an islet amyloid polypeptide transpeptidation product emerged as antigens processed into HLA-A2-restricted epitopes, which, as those already described, were recognized by circulating naive CD8+ T cells in T1D and healthy donors and by pancreas-infiltrating cells in T1D donors. This peptidome opens new avenues to understand antigen processing by β cells and for the development of T cell biomarkers and tolerogenic vaccination strategies.
Quantitative Phosphoproteomic Analysis Reveals Shared and Specific Targets of Arabidopsis Mitogen-Activated Protein Kinases (MAPKs) MPK3, MPK4, and MPK6
Laboratoire Spectrométrie de masse biologique et protéomique - Naganand Rayapuram, Jean Bigeard, Hanna Alhoraibi, Ludovic Bonhomme, Anne-Marie Hesse, Joëlle Vinh, Heribert Hirt, Delphine Pflieger
Cell Metab - 17(1) 61-80 - DOI: 10.1074/mcp.RA117.000135 - 2018
In Arabidopsis, mitogen-activated protein kinases MPK3, MPK4, and MPK6 constitute essential relays for a variety of functions including cell division, development and innate immunity. Although some substrates of MPK3, MPK4 and MPK6 have been identified, the picture is still far from complete. To identify substrates of these MAPKs likely involved in cell division, growth and development we compared the phosphoproteomes of wild-type and mpk3, mpk4, and mpk6. To study the function of these MAPKs in innate immunity, we analyzed their phosphoproteomes following microbe-associated molecular pattern (MAMP) treatment. Partially overlapping substrates were retrieved for all three MAPKs, showing target specificity to one, two or all three MAPKs in different biological processes. More precisely, our results illustrate the fact that the entity to be defined as a specific or a shared substrate for MAPKs is not a phosphoprotein but a particular (S/T)P phosphorylation site in a given protein. One hundred fifty-two peptides were identified to be differentially phosphorylated in response to MAMP treatment and/or when compared between genotypes and 70 of them could be classified as putative MAPK targets. Biochemical analysis of a number of putative MAPK substrates by phosphorylation and interaction assays confirmed the global phosphoproteome approach. Our study also expands the set of MAPK substrates to involve other protein kinases, including calcium-dependent (CDPK) and sugar nonfermenting (SnRK) protein kinases
Identification of BAG3 target proteins in anaplastic thyroid cancer cells by proteomic analysis
Laboratoire Spectrométrie de masse biologique et protéomique - Galdiero F, Bello AM, Spina A, Capiluongo A, Liuu S, De Marco M, Rosati A, Capunzo M, Napolitano M, Vuttariello E, Monaco M, Califano D, Turco MC, Chiappetta G, Vinh J, Chiappetta G
Cell Metab - 9(8) 8016-8026 - DOI: 10.18632/oncotarget.23858 - 2018
BAG3 protein is an apoptosis inhibitor and is highly expressed in Anaplastic Thyroid Cancer. We investigated the entire set of proteins modulated by BAG3 silencing in the human anaplastic thyroid 8505C cancer cells by using the Stable-Isotope Labeling by Amino acids in Cell culture strategy combined with mass spectrometry analysis. By this approach we identified 37 up-regulated and 54 down-regulated proteins in BAG3-silenced cells. Many of these proteins are reportedly involved in tumor progression, invasiveness and resistance to therapies. We focused our attention on an oncogenic protein, CAV1, and a tumor suppressor protein, SERPINB2, that had not previously been reported to be modulated by BAG3. Their expression levels in BAG3-silenced cells were confirmed by qRT-PCR and western blot analyses, disclosing two novel targets of BAG3 pro-tumor activity. We also examined the dataset of proteins obtained by the quantitative proteomics analysis using two tools, Downstream Effect Analysis and Upstream Regulator Analysis of the Ingenuity Pathways Analysis software. Our analyses confirm the association of the proteome profile observed in BAG3-silenced cells with an increase in cell survival and a decrease in cell proliferation and invasion, and highlight the possible involvement of four tumor suppressor miRNAs and TP53/63 proteins in BAG3 activity.
Physiological effects caused by microcystin-producing and non-microcystin producing Microcystis aeruginosa on medaka fish: A proteomic and metabolomic study on liver
Laboratoire Spectrométrie de masse biologique et protéomique - Séverine Le Manach , Benoit Sotton , Hélène Huet , Charlotte Duval , Alain Paris , Arul Marie , Claude Yépremian , Arnaud Catherine , Lucrèce Mathéron , Joelle Vinh , Marc Edery , Benjamin Marie
Environmental pollution - 234 523-537 - doi: 10.1016/j.envpol.2017.11.011 - 2018
Cyanobacterial blooms have become a common phenomenon in eutrophic freshwater ecosystems worldwide. Microcystis is an important bloom-forming and toxin-producing genus in continental aquatic ecosystems, which poses a potential risk to Human populations as well as on aquatic organisms. Microcystis is known to produce along with various bioactive peptides, the microcystins (MCs) that have attracted more attention notably due to their high hepatotoxicity. To better understand the effects of cyanobacterial blooms on fish, medaka fish (Oryzias latipes) were sub-chronically exposed to either non-MC-producing or MC-producing living strains and, for this latter, to its subsequent MC-extract of Microcystis aeruginosa. Toxicological effects on liver have been evaluated through the combined approach of histopathology and 'omics' (i.e. proteomics and metabolomics). All treatments induce sex-dependent effects at both cellular and molecular levels. Moreover, the modalities of exposure appear to induce differential responses as the direct exposure to the cyanobacterial strains induce more acute effects than the MC-extract treatment. Our histopathological observations indicate that both non-MC-producing and MC-producing strains induce cellular impairments. Both proteomic and metabolomic analyses exhibit various biological disruptions in the liver of females and males exposed to strain and extract treatments. These results support the hypothesis that M. aeruginosa is able to produce bioactive peptides, other than MCs, which can induce toxicological effects in fish liver. Moreover, they highlight the importance of considering cyanobacterial cells as a whole to assess the realistic environmental risk of cyanobacteria on fish.
Metabolic changes in Medaka fish induced by cyanobacterial exposures in mesocosms: an integrative approach combining proteomic and metabolomic analyses OPEN
Laboratoire Spectrométrie de masse biologique et protéomique - Benoît Sotton, Alain Paris, Séverine Le Manach, Alain Blond, Gérard Lacroix, Alexis Millot, Charlotte Duval, Hélène Huet, Qin Qiao, Sophie Labrut, Giovanni Chiappetta, Joelle Vinh, Arnaud Catherine & Benjamin Marie
Scientific Reports - 7 4051 - DOI: 10.1038/s41598-017-04423-z - 2018
Cyanobacterial blooms pose serious threats to aquatic organisms and strongly impact the functioning of aquatic ecosystems. Due to their ability to produce a wide range of potentially bioactive secondary metabolites, so called cyanotoxins, cyanobacteria have been extensively studied in the past decades. Proteomic and metabolomic analyses provide a unique opportunity to evaluate the global response of hundreds of proteins and metabolites at a glance. In this study, we provide the first combined utilization of these methods targeted to identify the response of fish to bloom-forming cyanobacteria. Medaka fish (Oryzias latipes) were exposed for 96 hours either to a MC-producing or to a non-MC-producing strain of Microcystis aeruginosa and cellular, proteome and metabolome changes following exposure to cyanobacteria were characterized in the fish livers. The results suggest that a short-term exposure to cyanobacteria, producing or not MCs, induces sex-dependent molecular changes in medaka fish, without causing any cellular alterations. Globally, molecular entities involved in stress response, lipid metabolism and developmental processes exhibit the most contrasted changes following a cyanobacterial exposure. Moreover, it appears that proteomic and metabolomic analyses are useful tools to verify previous information and to additionally bring new horizons concerning molecular effects of cyanobacteria on fish.
Metabolic changes in Medaka fish induced by cyanobacterial exposures in mesocosms : an integrative approach combining proteomic and metabolomic analyses.
Laboratoire Spectrométrie de masse biologique et protéomique - Benoît Sotton, Alain Paris, Séverine Le Manach, Alain Blond, Gérard Lacroix, Alexis Millot, Charlotte Duval, Hélène Huet, Qin Qiao, Sophie Labrut, Giovanni Chiappetta, Joelle Vinh, Arnaud Catherine & Benjamin Marie
Scientific Reports - 4051 - doi: 10.1016/j.cmet.2018.07.007. - 2017
Cyanobacterial blooms pose serious threats to aquatic organisms and strongly impact the functioning of aquatic ecosystems. Due to their ability to produce a wide range of potentially bioactive secondary metabolites, so called cyanotoxins, cyanobacteria have been extensively studied in the past decades. Proteomic and metabolomic analyses provide a unique opportunity to evaluate the global response of hundreds of proteins and metabolites at a glance. In this study, we provide the first combined utilization of these methods targeted to identify the response of fish to bloom-forming cyanobacteria. Medaka fish (Oryzias latipes) were exposed for 96 hours either to a MC-producing or to a non-MC-producing strain of Microcystis aeruginosa and cellular, proteome and metabolome changes following exposure to cyanobacteria were characterized in the fish livers. The results suggest that a short-term exposure to cyanobacteria, producing or not MCs, induces sex-dependent molecular changes in medaka fish, without causing any cellular alterations. Globally, molecular entities involved in stress response, lipid metabolism and developmental processes exhibit the most contrasted changes following a cyanobacterial exposure. Moreover, it appears that proteomic and metabolomic analyses are useful tools to verify previous information and to additionally bring
RACK1 controls IRES-mediated translation of viruses
Laboratoire Spectrométrie de masse biologique et protéomique - Majzoub K., Hafirassou M.L, Meignin C., Goto A., Marzi S., Fedorova A., Verdier Y., Vinh J., Hoffmann J.A, Martin F., Baumert T.F, Schuster C., Imler J.L
Cell - 159(5) 1086-95 - DOI: 10.1016/j.cell.2014.10.041 - 2014
Fighting viral infections is hampered by the scarcity of viral targets and their variability, resulting in development of resistance. Viruses depend on cellular molecules-which are attractive alternative targets-for their life cycle, provided that they are dispensable for normal cell functions. Using the model organism Drosophila melanogaster, we identify the ribosomal protein RACK1 as a cellular factor required for infection by internal ribosome entry site (IRES)-containing viruses. We further show that RACK1 is an essential determinant for hepatitis C virus translation and infection, indicating that its function is conserved for distantly related human and fly viruses. Inhibition of RACK1 does not affect Drosophila or human cell viability and proliferation, and RACK1-silenced adult flies are viable, indicating that this protein is not essential for general translation. Our findings demonstrate a specific function for RACK1 in selective mRNA translation and uncover a target for the development of broad antiviral intervention.
Abnormal recruitment of extracellular matrix proteins by excess Notch3ECD: a new pathomechanism in CADASIL
Laboratoire Spectrométrie de masse biologique et protéomique - Monet-Leprêtre M, Haddad I, Baron-Menguy C, Fouillot-Panchal M, Riani M, Domenga-Denier V, Dussaule C, Cognat E, Vinh J and Joutel A
Brain Oxford - (Pt 6) 1830-45 - DOI: 10.1093/brain/awt092 - 2013
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, or CADASIL, one of the most common inherited small vessel diseases of the brain, is characterized by a progressive loss of vascular smooth muscle cells and extracellular matrix accumulation. The disease is caused by highly stereotyped mutations within the extracellular domain of the NOTCH3 receptor (Notch3(ECD)) that result in an odd number of cysteine residues. While CADASIL-associated NOTCH3 mutations differentially affect NOTCH3 receptor function and activity, they all are associated with early accumulation of Notch3(ECD)-containing aggregates in small vessels. We still lack mechanistic explanation to link NOTCH3 mutations with small vessel pathology. Herein, we hypothesized that excess Notch3(ECD) could recruit and sequester functionally important proteins within small vessels of the brain. We performed biochemical, nano-liquid chromatography-tandem mass spectrometry and immunohistochemical analyses, using cerebral and arterial tissue derived from patients with CADASIL and mouse models of CADASIL that exhibit vascular lesions in the end- and early-stage of the disease, respectively. Biochemical fractionation of brain and artery samples demonstrated that mutant Notch3(ECD) accumulates in disulphide cross-linked detergent-insoluble aggregates in mice and patients with CADASIL. Further proteomic and immunohistochemical analyses identified two functionally important extracellular matrix proteins, tissue inhibitor of metalloproteinases 3 (TIMP3) and vitronectin (VTN) that are sequestered into Notch3(ECD)-containing aggregates. Using cultured cells, we show that increased levels or aggregation of Notch3 enhances the formation of Notch3(ECD)-TIMP3 complex, promoting TIMP3 recruitment and accumulation. In turn, TIMP3 promotes complex formation including NOTCH3 and VTN. In vivo, brain vessels from mice and patients with CADASIL exhibit elevated levels of both insoluble cross-linked and soluble TIMP3 species. Moreover, reverse zymography assays show a significant elevation of TIMP3 activity in the brain vessels from mice and patients with CADASIL. Collectively, our findings lend support to a Notch3(ECD) cascade hypothesis in CADASIL disease pathology, which posits that aggregation/accumulation of Notch3(ECD) in the brain vessels is a central event, promoting the abnormal recruitment of functionally important extracellular matrix proteins that may ultimately cause multifactorial toxicity. Specifically, our results suggest a dysregulation of TIMP3 activity, which could contribute to mutant Notch3(ECD) toxicity by impairing extracellular matrix homeostasis in small vessels.
Landscape of protein–protein interactions in Drosophila immune deficiency signaling during bacterial challenge
Laboratoire Spectrométrie de masse biologique et protéomique - Hidehiro Fukuyamaa, Yann Verdierb, Yongsheng Guana, Chieko Makino-Okamuraa, Victoria Shilovaa, Xi Liua, E. Maksouda, J. Matsubayashia, I. Haddadb, K. Spirohne, K. Onof, C. Hetruc, J. Rossierb, Trey Idekerf, M. Boutrose, Joëlle Vinh and Jules A. Hoffmann
Proc. Nat. Acad. Sci. USA - vol.110(n°26) 10717–22 - DOI: 10.1073/pnas.1304380110 - 2013
The Drosophila defense against pathogens largely relies on the activation of two signaling pathways: immune deficiency (IMD) and Toll. The IMD pathway is triggered mainly by Gram-negative bacteria, whereas the Toll pathway responds predominantly to Gram-positive bacteria and fungi. The activation of these pathways leads to the rapid induction of numerous NF-κB–induced immune response genes, including antimicrobial peptide genes. The IMD pathway shows significant similarities with the TNF receptor pathway. Recent evidence indicates that the IMD pathway is also activated in response to various noninfectious stimuli (i.e., inflammatory-like reactions). To gain a better understanding of the molecular machinery underlying the pleiotropic functions of this pathway, we first performed a comprehensive proteomics analysis to identify the proteins interacting with the 11 canonical members of the pathway initially identified by genetic studies. We identified 369 interacting proteins (corresponding to 291 genes) in heat-killed Escherichia coli-stimulated Drosophila S2 cells, 92% of which have human orthologs. A comparative analysis of gene ontology from fly or human gene annotation databases points to four significant common categories: (i) the NuA4, nucleosome acetyltransferase of H4, histone acetyltransferase complex, (ii) the switching defective/sucrose nonfermenting-type chromatin remodeling complex, (iii) transcription coactivator activity, and (iv) translation factor activity. Here we demonstrate that sumoylation of the IκB kinase homolog immune response-deficient 5 plays an important role in the induction of antimicrobial peptide genes through a highly conserved sumoylation consensus site during bacterial challenge. Taken together, the proteomics data presented here provide a unique avenue for a comparative functional analysis of proteins involved in innate immune reactions in flies and mammals.
Tau Pathology modulates Pin1 post-translational modifications and may be relevant as biomarker
Laboratoire Spectrométrie de masse biologique et protéomique - Ando K, Dourlen P, Sambo AV, Bretteville A, Bélarbi K, Vingtdeux V, Eddarkaoui S, Drobecq H, Ghestem A, Bégard S, Demey-Thomas E, Melnyk P, Smet C, Lippens G, Maurage CA, Caillet-Boudin ML, Verdier Y, Vinh J, Landrieu I, Galas MC, Blum D, Hamdane M, Serg
Neurobiol Aging - 34(3) :757-69 - DOI:10.1016/j.neurobiolaging.2012.08.004 - 2012
A prerequisite to dephosphorylation at Ser-Pro or Thr-Pro motifs is the isomerization of the imidic peptide bond preceding the proline. The peptidyl-prolyl cis/trans isomerase named Pin1 catalyzes this mechanism. Through isomerization, Pin1 regulates the function of a growing number of targets including the microtubule-associated tau protein and is supposed to be deregulated Alzheimer's disease (AD). Using proteomics, we showed that Pin1 is posttranslationally modified on more than 5 residues, comprising phosphorylation, N-acetylation, and oxidation. Although Pin1 expression remained constant, Pin1 posttranslational two-dimensional pattern was modified by tau overexpression in a tau-inducible neuroblastoma cell line, in our THY-Tau22 mouse model of tauopathy as well as in AD. Interestingly, in all of these systems, Pin1 modifications were very similar. In AD brain tissue when compared with control, Pin1 is hyperphosphorylated at serine 16 and found in the most insoluble hyperphosphorylated tau fraction of AD brain tissue. Furthermore, in all tau pathology conditions, acetylation of Pin1 may also contribute to the differences observed. In conclusion, Pin1 displays several posttranslational modifications, which are specific in tauopathies and may be useful as biomarker.
On-bead tryptic proteolysis: An attractive procedure for LC-MS/MS analysis of the Drosophila caspase 8 protein complex during immune response against bacteria
Laboratoire Spectrométrie de masse biologique et protéomique - Fukuyama H & Ndiaye S, Hoffmann J, Rossier J, Liuu S, Vinh J, Verdier Y.
J Proteomics - 75(15) :4610-9 - DOI:10.1016/j.jprot.2012.03.003 - 2012
This study aims to characterize the immune response against bacteria in Drosophila melanogaster. Obtaining a description of the in vivo state of protein complexes requires their isolation as a snapshot of physiological conditions before their identification. Affinity purification with streptavidin-biotin system is widely used to address this issue. However, because of the extraordinary stability of the interaction between streptavidin and biotin, the release of biotin-labeled bait remains a challenge. We transfected Drosophila cells with a DNA construct encoding a biotin-tagged Dredd protein (ortholog of caspase 8). After affinity purification, different strategies were evaluated, and proteins analyzed by LC-MS/MS mass spectrometry. The on-bead digestion allowed the identification of more proteins associated to the Dredd complex than different protocols using competitive or acid elution. A functional assay showed that a large part of the proteins specifically identified in the Dredd sample are functionally involved in the activation of the Imd pathway. These proteins are immune response proteins (BG4, Q9VP57), stress response proteins (HSP7C, Q9VXQ5), structural proteins (TBB1, CP190), a protein biosynthesis protein (Q9W1B9) and an antioxidant system protein (SODC). Our results clearly show that on-bead digestion of proteins is an attractive procedure for the study of protein complexes by mass spectrometry. This article is part of a Special Issue entitled: Translational Proteomics.

28 publications.