Université PSL

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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.
Selection Dynamics in Transient Compartmentalization.
Laboratoire Biochimie - Blokhuis A, Lacoste D, Nghe P, Peliti L
Phys. Rev. Lett. - 158101 120(15): - doi: 10.1371/journal.pcbi.1004972 - 2018
Transient compartments have been recently shown to be able to maintain functional replicators in the context of prebiotic studies. Here, we show that a broad class of selection dynamics is able to achieve this goal. We identify two key parameters, the relative amplification of nonactive replicators (parasites) and the size of compartments. These parameters account for competition and diversity, and the results are relevant to similar multilevel selection problems, such as those found in virus-host ecology and trait group selection.
Coupled catabolism and anabolism in autocatalytic RNA sets.
Laboratoire Biochimie - Arsène S, Ameta S, Lehman N, Griffiths AD, Nghe P.
Nucleic Acids Res. - 46(18) 9660-9666 - doi: 10.1093/nar/gky598. - 2018
The ability to process molecules available in the environment into useable building blocks characterizes catabolism in contemporary cells and was probably critical for the initiation of life. Here we show that a catabolic process in collectively autocatalytic sets of RNAs allows diversified substrates to be assimilated. We modify fragments of the Azoarcus group I intron and find that the system is able to restore the original native fragments by a multi-step reaction pathway. This allows in turn the formation of catalysts by an anabolic process, eventually leading to the accumulation of ribozymes. These results demonstrate that rudimentary self-reproducing RNA systems based on recombination possess an inherent capacity to assimilate an expanded repertoire of chemical resources and suggest that coupled catabolism and anabolism could have arisen at a very early stage in primordial living systems.
Sign epistasis caused by hierarchy within signalling cascades
Laboratoire Biochimie - Nghe P, Kogenaru M, Tans SJ
Nat Commun - 1451 9660-9666 - 10.1038/s41467-018-03644-8. - 2018
The ability to process molecules available in the environment into useable building blocks characterizes catabolism in contemporary cells and was probably critical for the initiation of life. Here we show that a catabolic process in collectively autocatalytic sets of RNAs allows diversified substrates to be assimilated. We modify fragments of the Azoarcus group I intron and find that the system is able to restore the original native fragments by a multi-step reaction pathway. This allows in turn the formation of catalysts by an anabolic process, eventually leading to the accumulation of ribozymes. These results demonstrate that rudimentary self-reproducing RNA systems based on recombination possess an inherent capacity to assimilate an expanded repertoire of chemical resources and suggest that coupled catabolism and anabolism could have arisen at a very early stage in primordial living systems.
Selection Dynamics in Transient Compartmentalization
Laboratoire Biochimie - doi.org/10.1103/PhysRevLett.120.158101
Phys. Rev. Lett. - 120 158101 - doi.org/10.1103/PhysRevLett.120.158101 - 2018
Transient compartments have been recently shown to be able to maintain functional replicators in the context of prebiotic studies. Here, we show that a broad class of selection dynamics is able to achieve this goal. We identify two key parameters, the relative amplification of nonactive replicators (parasites) and the size of compartments. These parameters account for competition and diversity, and the results are relevant to similar multilevel selection problems, such as those found in virus-host ecology and trait group selection.
Spontaneous migration of cellular aggregates from giant keratocytes to running spheroids.
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Grégory Beaune, Carles Blanch-Mercader, Stéphane Douezan, Julien Dumond, David Gonzalez-Rodriguez, Damien Cuvelier, Thierry Ondarçuhu, Pierre Sens, Sylvie Dufour, Michael P Murrell, Françoise Brochard-Wyart
Proceedings of the National Academy of Sciences of the United States of America - 12926-12931 - 10.1073/pnas.1811348115 - 2018
Despite extensive knowledge on the mechanisms that drive single-cell migration, those governing the migration of cell clusters, as occurring during embryonic development and cancer metastasis, remain poorly understood. Here, we investigate the collective migration of cell on adhesive gels with variable rigidity, using 3D cellular aggregates as a model system. After initial adhesion to the substrate, aggregates spread by expanding outward a cell monolayer, whose dynamics is optimal in a narrow range of rigidities. Fast expansion gives rise to the accumulation of mechanical tension that leads to the rupture of cell-cell contacts and the nucleation of holes within the monolayer, which becomes unstable and undergoes dewetting like a liquid film. This leads to a symmetry breaking and causes the entire aggregate to move as a single entity. Varying the substrate rigidity modulates the extent of dewetting and induces different modes of aggregate motion: « giant keratocytes, » where the lamellipodium is a cell monolayer that expands at the front and retracts at the back; « penguins, » characterized by bipedal locomotion; and « running spheroids, » for nonspreading aggregates. We characterize these diverse modes of collective migration by quantifying the flows and forces that drive them, and we unveil the fundamental physical principles that govern these behaviors, which underscore the biological predisposition of living material to migrate, independent of length scale.
Innate Immune Signals Induce Anterograde Endosome Transport Promoting MHC Class I Cross-Presentation.
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Weimershaus M, Mauvais FX, Saveanu L, Adiko C, Babdor J, Abramova A, Montealegre S, Lawand M, Evnouchidou I, Huber KJ, Chadt A, Zwick M, Vargas P, Dussiot M, Lennon-Dumenil AM, Brocker T, Al-Hasani H, van Endert P.
Cell Reports - 24(13) 3568-3581 - doi: 10.1016/j.celrep.2018.08.041 - 2018
Both cross-presentation of antigens by dendritic cells, a key pathway triggering T cell immunity and immune tolerance, and survival of several pathogens residing in intracellular vacuoles are intimately linked to delayed maturation of vesicles containing internalized antigens and microbes. However, how early endosome or phagosome identity is maintained is incompletely understood. We show that Toll-like receptor 4 (TLR4) and Fc receptor ligation induces interaction of the GTPase Rab14 with the kinesin KIF16b mediating plus-end-directed microtubule transport of endosomes. As a result, Rab14 recruitment to phagosomes delays their maturation and killing of an internalized pathogen. Enhancing anterograde transport by overexpressing Rab14, promoting the GTP-bound Rab14 state, or inhibiting retrograde transport upregulates cross-presentation. Conversely, reducing Rab14 expression, destabilizing Rab14 endosomes, and inhibiting anterograde microtubule transport by Kif16b knockdown compromise cross-presentation. Therefore, regulation of early endosome trafficking by innate immune signals is a critical parameter in cross-presentation by dendritic cells.

391 publications.