Structural insights into protein–uranyl interaction: towards an in silico detection method

Documenting the modes of interaction of uranyl (UO 2 2+) with large biomolecules, and particularly with proteins, is instrumental for the interpretation of its behavior in vitro and in vivo. The gathering of three-dimensional information concerning uranyl-first shell atoms from two structural databases, the Cambridge Structural Databank and the Protein Data Bank (PDB) allowed a screening of corresponding topologies in proteins of known structure. In the computer-aided procedure, all potentially bound residues from the template structure were granted full flexibility using a rotamer library. The Amber force-field was used to loosen constraints and score each predicted site. Our algorithm was validated as a first stage through the recognition of existing experimental data in the PDB. The coherent localization of missing atoms in the density map of an ambiguous uranium/uranyl-protein complex exemplified the efficiency of our approach, which is currently suggesting the experimental investigation of uranyl-protein binding site

Caractérisation haut-débit de la dynamique du protéome pour la découverte des protéines clefs chez les espèces sentinelles : une surprenante diversité des vitellogénines chez le crustacé Gammarus fossarum

International audienceIn environmental science,omics-based approaches arewidely used for the identification of gene products related to stress response. However,when dealingwith non-model species, functional prediction of genes is challenging. Indeed, functional predictions are often obtained by sequence similarity searches and functional data from phylogenetically distant organisms, which can lead to inaccurate predictions due to quite different evolutionary scenarios. In oviparous females, vitellogenin production is vital for embryonic development, ensuring population viability. Its abnormal presence in fish male organisms is commonly employed as a biomarker of exposure to xenoestrogens, named endocrine disruptors. Here, in the freshwater amphipod Gammarus fossarum, we identified vitellogenin proteins by means of a proteometemporal dynamics analysis during oogenesis and embryogenesis. This exhaustive approach allows several functional molecular hypotheses in the oogenesis process to be drawn. Moreover, we revealed an unsuspected diversity of molecular players involved in yolk formation as eight proteins originating from different families of the large lipid transfer protein superfamily were identified as “true vitellogenins”. Biological significance: In non-model species, next generation sequencing technologies development enables quickly deciphering gene and protein sequences but accuracy of associated functional prediction remains to be established. Here, in the crustacean Gammarus fossarum, a key sentinel species in freshwater biomonitoring, we identified key molecular players involved in the female reproduction by studying the proteome dynamics of ovaries and embryos. An unsuspected diversity of vitellogenin proteins was evidenced. These proteins being vital for offspring development, their high diversity may be advantageous for the organism's reproduction. Phylogenetic analysis showed that some forms are true vitellogenin orthologs while others are included in the apolipoprotein family, a paralogous group from the vitellogenin family. Among crustaceans, Gammarus fossarum is the first documented case where diverse protein families are involved in the yolk formation process

Espèces non-modèles, des espèces en voies de disparition en protéogénomique

International audiencePreviously, large-scale proteomics was possible only for organisms whose genomes were sequenced, meaning the most common model organisms. The use of next-generation sequencers is now changing the deal. With "proteogenomics", the use of experimental proteomics data to refine genome annotations, a higher integration of omics data is gaining ground. By extension, combining genomic and proteomic data is becoming routine in many research projects. "Proteogenomic"-flavored approaches are currently expanding, enabling the molecular studies of non-model organisms at an unprecedented depth. Today draft genomes can be obtained using next-generation sequencers in a rather straightforward way and at a reasonable cost for any organism. Unfinished genome sequences can be used to interpret tandem mass spectrometry proteomics data without the need for time-consuming genome annotation, and the use of RNA-seq to establish nucleotide sequences that are directly translated into protein sequences appears promising. There are, however, certain drawbacks that deserve further attention for RNA-seq to become more efficient. Here, we discuss the opportunities of working with non-model organisms, the proteomic methods that have been used until now, and the dramatic improvements proffered by proteogenomics. These put the distinction between model and non-model organisms in great danger, at least in terms of proteomics! BIOLOGICAL SIGNIFICANCE: Model organisms have been crucial for in-depth analysis of cellular and molecular processes of life. Focusing the efforts of thousands of researchers on the Escherichia coli bacterium, Saccharomyces cerevisiae yeast, Arabidopsis thaliana plant, Danio rerio fish and other models for which genetic manipulation was possible was certainly worthwhile in terms of fundamental and invaluable biological insights. Until recently, proteomics of non-model organisms was limited to tedious, homology-based techniques, but today draft genomes or RNA-seq data can be straightforwardly obtained using next-generation sequencers, allowing the establishment of a draft protein database for any organism. Thus, proteogenomics opens new perspectives for molecular studies of non-model organisms, although they are still difficult experimental organisms. This article is part of a Special Issue entitled: Proteomics of non-model organisms

Caractérisation par la protéogénomique du core-protéome des tissus reproductifs femelles chez les crustacés amphipodes

International audienceAs a result of the poor genome sequence coverage of crustacean amphipods, characterization of their evolutionary biology relies mostly on phenotypic traits. Here, we analyzed the proteome of ovaries from five amphipods, all from the Senticaudata suborder, with the objective to obtain insights into the core-proteome of female reproductive systems. These amphipods were from either the Gammarida infraorder: Gammarus fossarum, Gammarus pulex, Gammarus roeseli, or the Talitrida infraorder: Parhyale hawaiensis and Hyalella azteca. Ovaries from animals sampled at the end of their reproductive cycle were dissected. Their whole protein contents were extracted and their proteomes were recorded by high-throughput nanoLC–MS/MS with a high-resolution mass spectrometer. We interpreted tandem mass spectrometry data with the protein sequence resource from G. fossarum and P. hawaiensis, both recently established by RNA sequencing. The large molecular biodiversity within amphipods was assessed by the ratio of MS/MS spectra assigned for each sample, which tends to diverge rapidly along the taxonomic level considered. The core-proteomewas defined as the proteins conserved along all samples, thus detectable by the homology-based proteomic assignment procedure. This specific subproteome may be further enriched in the future with the analysis of new species and update of the protein sequence resource

Etude protéomique de la fonction mâle chez Gammarus fossarum en réponse de pertubateurs endocriniens

International audienceWhile the decrease in human sperm count in response to pollutants is a worldwide concern, little attention is being devoted to its causes and occurrence in the biodiversity of the animal kingdom. Arthropoda is the most species-rich phyla, inhabiting all aquatic and terrestrial ecosystems. During evolution, key molecular players of the arthropod endocrine system have diverged from the vertebrate counterparts. Consequently, arthropods may have different sensitivities toward endocrine disrupting chemicals (EDCs). Here alteration of sperm quality in a crustacean, Gammarus fossarum, a popular organism in freshwater risk assessment, was investigated after laboratory exposure to various concentrations of three different xenobiotics: cadmium, methoxyfenozide, and pyriproxyfen. The integrity of the reproductive process was assessed by means of sperm-quality markers. For each substance, semiquantitative/relative proteomics based on spectral counting procedure was carried out on male gonads to observe the biological impact. The changes in a total of 871 proteins were monitored in response to toxic pressure. A drastic effect was observed on spermatozoon production, with a dose-response relationship. While exposure to EDCs leads to strong modulations of male-specific proteins in testis, no induction of female-specific proteins was noted. Also, a significant portion of orphans proved to be sensitive to toxic stress

Proteomics meets blue biotechnology: A wealth of novelties and opportunities.

[eng] Blue biotechnology, in which aquatic environments provide the inspiration for various products such as food additives, aquaculture, biosensors, green chemistry, bioenergy, and pharmaceuticals, holds enormous promise. Large-scale efforts to sequence aquatic genomes and metagenomes, as well as campaigns to isolate new organisms and culture-based screenings, are helping to push the boundaries of known organisms. Mass spectrometry-based proteomics can complement 16S gene sequencing in the effort to discover new organisms of potential relevance to blue biotechnology by facilitating the rapid screening of microbial isolates and by providing in depth profiles of the proteomes and metaproteomes of marine organisms, both model cultivable isolates and, more recently, exotic non-cultivable species and communities. Proteomics has already contributed to blue biotechnology by identifying aquatic proteins with potential applications to food fermentation, the textile industry, and biomedical drug development. In this review, we discuss historical developments in blue biotechnology, the current limitations to the known marine biosphere, and the ways in which mass spectrometry can expand that knowledge. We further speculate about directions that research in blue biotechnology will take given current and near-future technological advancements in mass spectrometry

Dataset de protéomique shotgun aquis chez Gammarus pulex sur le terrain

International audienceThis data article associated with the manuscript "Comparative proteomics in the wild: accounting for intrapopulation variability improves describing proteome response in a Gammarus pulex field population exposed to cadmium" refers to the shotgun proteomics analysis performed on 40 Gammarus pulex animals sampled from the wild. Proteins were extracted, digested with trypsin, and the resulting peptides were identified by tandem mass spectrometry. Here, we present the list of proteins from males and the list of proteins from females that are differentially detected between the Brameloup and the Pollon populations. Data are available via ProteomeXchange with identifiers PXD013656 and PXD013712, respectively

When proteogenomics helps ecotoxicology

International audienceAs pioneers in biosurveillance, Kolkwitz and Marsson proposed, in 1909, a saprobic classification system for freshwater ecosystems based on the presence/absence of indicator species with differing organic pollution tolerances. Although chemical analysis is a useful tool to address this question, it is still impossible to quantify all chemical compounds and their associated degradation products, to predict their bioavailabilities, and their toxic effects on biota. Hence, biological indicators such as biochemical responses (molecular changes) have been proposed to link the presence of chemical compounds and their effects. These indicators have to be designed as an early warning signal for ecosystem degradation, but also as specific of key physiological functions failures or specific of a contaminant in relation to its mode of action. By studying proteomes of “sentinel species”, the challenge of identification of biological indicators as key proteins involved in the molecular mechanism in response to xenobiotic can be reached. But most of these “sentinel species” are non-model species, with an important lack of genomic characterization, and consequently, proteome exploration is still a big challenge. The rapid advance of mass spectrometry technology and next generation sequencing of transcriptome made possible the quick exploration of proteomes of non-model species and deciphering protein signatures in response to toxicants. Here we describe a pipeline, from discovery of biomarkers to their validation. This pipeline includes mRNA sequencing, assembling of reads, and six-reading frame translation for the design of a protein database specific of Gammarus fossarum, a freshwater crustacean. This pipeline was applied to the research of reproduction biomarkers. This study consisted in the analysis of the proteome of Gammarus fossarum exposed to three toxic compounds as compared to non-treated animals. Differentially expressed proteins were identified as potential biomarkers of reproductive impairments, and were quantified by targeted proteomics in multiplexed assays by selected reaction monitoring (SRM)

Transcriptomes de novo transcriptomes de 14 gammaridae pour analyse proteogenomique de 7 groupes taxonomiques

International audienceGammarids are amphipods found worldwide distributed in fresh and marine waters. They play an important role in aquatic ecosystems and are well established sentinel species in ecotoxicology. In this study, we sequenced the transcriptomes of a male individual and a female individual for seven different taxonomic groups belonging to the two genera Gammarus and Echinogammarus: Gammarus fossarum A, G. fossarum B, G. fossarum C, Gammarus wautieri, Gammarus pulex, Echinogammarus berilloni, and Echinogammarus marinus. These taxa were chosen to explore the molecular diversity of transcribed genes of genotyped individuals from these groups. Transcriptomes were de novo assembled and annotated. High-quality assembly was confirmed by BUSCO comparison against the Arthropod dataset. The 14 RNA-Seq-derived protein sequence databases proposed here will be a significant resource for proteogenomics studies of these ecotoxicologically relevant non-model organisms. These transcriptomes represent reliable reference sequences for whole-transcriptome and proteome studies on other gammarids, for primer design to clone specific genes or monitor their specific expression, and for analyses of molecular differences between gammarid species