La protéogénomique comme approche pour la découverte de protéines chez une espèce non-modèle

International audienc

Documenting the unique biology of gammarids by means of high-throughput proteogenomics

International audienceNext-generation proteomics is able to offer in-depth insights into the physiology of organisms by identifying and quantifying thousands of proteins. However, this is only possible for model organisms for which the genome has been sequenced and carefully annotated, a requirement far from being a standard for amphipods. Here, we proposed a novel approach for quickly identifying novel proteins of the freshwater amphipod, Gammarus fossarum, an ecologically relevant species, by the alliance of genomic and proteomic, i.e. proteogenomics. For identifing protein-encoding genes, we characterized by deep RNA-seq the transcriptome of key physiological organs: the female and male reproductive system, the cephalon and the hepatopancreatic caecum. Then, we generated a six-reading frame translation protein database for interpreting protein data measured in parallel on the four tissues by high-throughput proteomics. A total of 1,033,282 MS/MS spectra was recorded and assigned with the RNA-seq derived database, resulting in 1,624 proteins identified. The function of these proteins was tentatively assigned by PSI-BLAST searches but functional annotation resulted quite scarce with the identification of numerous orphans. As demonstrated from the Daphnia pulex genome, lineage specific genes are more likely linked to the organism unique biology and have been shown to be amongst the most responsive to environmental challenges. Therefore, our dataset, the first identification of genes and proteins ever done at such large scale for the Gammarus genus, is of utmost importance for a comprehensive understanding of their key physiological functions and molecular mechanisms related to stress response. The perspectives of this work will address the use of this resource in ecotoxicology. We are currently focusing our attention on proteins involved in reproduction processes. Our study should result in interesting biomarkers for freshwater biomonitoring based on the G. fossarum sentinel

Protéogénomique chez Gammarus fossarum pour investiguer le système reproducteur des amphipodes

International audienceBecause of their ecological importance, amphipod crustacea are employed worldwide as test species in environmental risk assessment. Although proteomics allows new insights into the molecular mechanisms related to the stress response, such investigations are rare for these organisms because of the lack of comprehensive protein sequence databases. Here, we propose a proteogenomic approach for identifying specific proteins of the freshwater amphipod Gammarus fossarum, a keystone species in European freshwater ecosystems. After deep RNA sequencing, we created a comprehensive ORF database. We identified and annotated the most relevant proteins detected through a shotgun tandem mass spectrometry analysis carried out on the proteomes from three major tissues involved in the organism's reproductive function: the male and female reproductive systems, and the cephalon, where different neuroendocrine glands are present. The 1,873 mass-spectrometry-certified proteins represent the largest crustacean proteomic resource to date, with 218 proteins being lineage specific. Comparative proteomics between the male and female reproductive systems indicated key proteins with strong sexual dimorphism. Protein expression profiles during spermatogenesis at seven different stages highlighted the major gammarid proteins involved in the different facets of reproduction

Protéomique comparative chez Gammarus fossarum: focus sur la régulation endocrine

International audienceAmong all contaminants present in the aquatic ecosystems,endocrine-disrupting chemicals (EDCs) are currently a great concern because of their high potential induction of alterations in reproductive function and consequently population dynamics. Their mode of action and associated effects on hormonal regulation have been intensively studied for fish, resulting in robust, specific and reliable biomarkers for risk assessment. Meanwhile, similar tools are lacking for invertebrates, especially crustaceans, while they are major biocenose components, playing key role in ecosystemic processes. While disruption of endocrine regulation is documented in-situ, causality between these adverse effects and their cause can not be established due to the lack of knowledge on hormonal regulation for these models. A few biomarkers have been transposed from vertebrates. However, different evolution paths restraint this type of approach, involving that candidate biomarkers must be specifically identified and characterized. The recent trends in high-throughput proteomics allow large-scale characterization of the whole proteome of model organisms. We started to document the reproductive proteome of the freshwater amphipod, Gammarus fossarum, an ecologically and ecotoxicology relevant species. For this, we used the most advanced approaches in shotgun proteomics for an extensive comparison of the female and male reproductive tissues of G. fossarum. Tandem mass spectrometry analysis of a large pool of peptides arising from trypsin digestion of both tissues was done with a high resolution LTQ-Orbitrap XL hybrid mass spectrometer (ThermoFisher). This led to the identification of 37,167 peptides. A total of 1,336 proteins were identified with high confidence and quantified using the spectral count approach. The cross-comparison of the relative abundance of these proteins in both tissues has shown that 66 and 103 proteins were preferentially expressed in the female and the male reproductive tissues, respectively. The functions of these sexspecific proteins were tentatively assigned by PSI-BLAST searches but functional annotation resulted quite scare with the identification of numerous orfans. As gammarids reproductive biology is well described, involvement of each sex-specific protein in reproduction processes will be assessed by studying their modulation throughout the reproductive cycle. Our results should facilitate further bio biomarker development of EDCs exposure for crustaceans

A randomized clinical trial indicates that levamisole increases the time to relapse in children with steroid-sensitive idiopathic nephrotic syndrome

Item does not contain fulltex