RESUMEN
In living systems, protein assemblies have essential functions, serving as structural supports, transport highways for molecular cargo, and containers of genetic material. The construction of protein assemblies, which involves control over space and time, remains a significant challenge in biotechnology. Here, we show that anionic boron clusters, 3,3'-commo-bis[closo-1,2-dicarba-3-cobaltadodecaborane] (COSAN-), and halogenated closo-dodecarboranes (B12X122-, X = Η, Cl or I), described as super-chaotropic nano-ions, induce the formation of 2D assemblies of model proteins, myoglobin, carbonic anhydrase, and trypsin inhibitor. We found that the nano-ion concentration reversibly controls the size of the protein assemblies. Furthermore, the secondary structures of the proteins are only slightly affected by assembly formation. For myoglobin, the formation of these assemblies even prevents temperature denaturation, highlighting a preservation effect of nano-ions. Our study reveals that inorganic boron-based nano-ions act as a reversible molecular glue for proteins, providing a potential starting point for the further development of controlled protein assemblies.
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Carbamazepine is one of the most abundant pharmaceutical active compounds detected in aquatic systems. Based on laboratory exposures, carbamazepine has been proven to adversely affect aquatic organisms. However, the underlying molecular events remain poorly understood. This study aims to investigate the molecular mechanisms potentially associated with toxicological effects of carbamazepine on the mussel Mytilus galloprovincialis exposed for 3 days at realistic concentrations encountered in coastal environments (80 ng/L and 8 µg/L). An integrated metabolomics and proteogenomics approach, including data fusion strategy, was applied to gain more insight in molecular events and cellular processes triggered by carbamazepine exposure. Consistent metabolic and protein signatures revealed a metabolic rewiring and cellular stress at both concentrations (e.g. intensification of protein synthesis, transport and catabolism processes, disruption of lipid and amino acid metabolisms). These highlighted molecular signatures point to the induction of autophagy, closely related with carbamazepine mechanism of action, as well as a destabilization of the lysosomal membranes and an enzymatic overactivity of the peroxisomes. Induction of programmed cell death was highlighted by the modulation of apoptotic cognate proteins. The proposed integrative omics data analysis was shown to be highly relevant to identify the modulations of the two molecular levels, i.e. metabolites and proteins. Multi-omics approach is able to explain the resulting complex biological system, and document stronger toxicological pieces of evidence on pharmaceutical active compounds at environmental concentrations in sentinel organisms.
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Mytilus , Proteogenómica , Contaminantes Químicos del Agua , Animales , Carbamazepina/toxicidad , Masculino , Metabolómica , Mytilus/genética , Contaminantes Químicos del Agua/toxicidadRESUMEN
The transfer of methyl-Hg (MeHg) from food is central for its effects in aquatic animals, but we still lack knowledge concerning its impact on invertebrate primary consumers. In aquatic environments, cell walls of plants are particularly recalcitrant to degradation and as such remain available as a food source for long periods. Here, the impact at the proteomic level of dietary MeHg in Gammarus fossarum was established and linked to subcellular distribution of Hg. Individuals of G. fossarum were fed with MeHg in cell wall or intracellular compartments of Elodea nuttallii. Hg concentrations in subcellular fractions were 2 to 6 times higher in animals fed with cell wall than intracellular compartments. At the higher concentrations tested, the proportion of Hg in metal-sensitive fraction increased from 30.0 ± 6.1 to 41.0 ± 5.7% for individuals fed with intracellular compartment, while biologically detoxified metal fraction increased from 30.0 ± 6.1 to 50.0 ± 2.8% when fed with cell wall compartment. Data suggested that several thresholds of proteomic response are triggered by increased bioaccumulation in each subcellular fraction in correlation with Hg exclusively bound to the metal-sensitive fraction, while the increase of biologically detoxified metal likely had a cost for fitness. Proteomics analysis supported that the different binding sites and speciation in shoots subsequently resulted in different fate and cellular toxicity pathways to consumers. Our data confirmed that Hg bound in cell walls of plants can be assimilated by G. fossarum, which is consistent with its feeding strategy, hence pointing cell walls as a significant source for Hg transfers and toxicity in primary consumers. The high accumulation of Hg in macrophytes makes them a risk for food web transfer in shallow ecosystems. The present results allowed gaining new insights into the effects and uptake mechanisms of MeHg in aquatic primary consumers.
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Anfípodos , Mercurio , Compuestos de Metilmercurio , Contaminantes Químicos del Agua , Animales , Ecosistema , Cadena Alimentaria , Humanos , Proteoma , Proteómica , Contaminantes Químicos del Agua/análisisRESUMEN
Omics approaches are continuously providing new clues on the mechanisms of action of contaminants in species of environmental relevance, contributing to the emergence of molecular ecotoxicology. Co-expression network approaches represent a suitable methodological framework for studying the rich content of omics datasets. This study aimed to find evidence of key pathways and proteins related to the testicular toxicity in the sentinel crustacean species Gammarus fossarum exposed to endocrine disruptors using a weighted protein co-expression network analysis. From a shotgun proteomics dataset of male gonads of G. fossarum organisms exposed to cadmium (Cd), pyriproxyfen (Pyr) and methoxyfenozide (Met) in laboratory conditions, four distinct modules were identified as significantly correlated to contaminants' exposure. Protein set enrichment analysis identified modules involved in cytoskeleton organization and oxidative stress response associated with the Cd exposure. The module associated with Pyr exposure was associated with endoplasmic reticulum stress (ER) response, and the module correlated with Met exposure was characterized by a significant proportion of amphipod-restricted proteins whose functions are still not characterized. Our results show that co-expression networks are efficient and adapted tools to identify new potential mode of actions from environmental sentinel species, such as G. fossarum, using a proteogenomic approach, even without an annotated genome.
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Anfípodos/fisiología , Cadmio/toxicidad , Piridinas/toxicidad , Contaminantes Químicos del Agua/toxicidad , Anfípodos/metabolismo , Animales , Ecotoxicología/métodos , Disruptores Endocrinos/toxicidad , Genoma , Hidrazinas , Hormonas Juveniles , Masculino , Proteómica/métodos , Especies Centinela/genética , Testículo/efectos de los fármacosRESUMEN
Bcl-xL, a member of the Bcl-2 family, is a pro-survival protein involved in apoptosis regulation. We have previously reported the ability of Bcl-xL to form various types of fibers, from native to amyloid conformations. Here, we have mimicked the effect of apoptosis-induced caspase activity on Bcl-xL by limited proteolysis using trypsin. We show that cleaved Bcl-xL (ΔN-Bcl-xL) forms fibers that exhibit the features of amyloid structures (BclxLcf37). Moreover, three monoclonal antibodies (mAbs), produced by mouse immunization and directed against ΔN-Bcl-xL or Bcl-xL fibers, were selected and characterized. Our results show that these mAbs specifically target ΔN-Bcl-xL in amyloid fibers in vitro. Upon metal-stress-induced apoptosis, these mAbs are able to detect the presence of Bcl-xL in amyloid aggregates in neuroblastoma SH-SY5Y cell lines. In conclusion, these specific mAbs directed against amyloidogenic conformations of Bcl-xL constitute promising tools for studying, in vitro and in cellulo, the contribution of Bcl-xL in apoptosis. These mAbs may further help in developing new diagnostics and therapies, considering Bcl-xL as a strategic target for treating brain lesions relevant to stroke and neurodegenerative diseases.
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Amiloide/inmunología , Anticuerpos Monoclonales/inmunología , Neuroblastoma/metabolismo , Proteína bcl-X/inmunología , Amiloide/química , Animales , Apoptosis , Línea Celular Tumoral , Humanos , Metales Pesados/toxicidad , Ratones , Neuroblastoma/etiología , Oxidantes/toxicidad , Conformación Proteica , Proteína bcl-X/químicaRESUMEN
A highly multiplexed liquid chromatography mass spectrometry-multiple reaction monitoring (MRM)-based assay has been developed for evaluating 107 candidate immune biomarkers in both hemocytes and plasma of the zebra mussel Dreissena polymorpha. The Scout-MRM strategy was employed for the first time, shortening the implementation of a targeted MRM bottom-up proteomics assay using selected immune protein-related peptides identified by shotgun discovery proteogenomics. This strategy relies on spiking scout peptides during the discovery phase and using them to build and deploy the MRM targeted proteomics method. It proved to be highly relevant, since about 90% of the targeted peptides and proteins were monitored and rapidly measured in both hemocyte and plasma samples. The sample preparation protocol was optimized by evaluating the digestion efficiency of tryptic peptides over time. The accuracy and precision of 50 stable isotope-labeled peptides were evaluated for use as internal standards. Finally, the specificity of the transitions was thoroughly assessed to ensure the reliable measurement of protein biomarkers. Several analytical and biological validation criteria were evaluated across hemocytes and plasma samples exposed ex vivo to biological contaminants, resulting in the validation of two Scout-MRM assays for the relative quantitation of 85 and 89 proteins in hemocytes and plasma, respectively. Graphical abstract.
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Dreissena/metabolismo , Proteómica/métodos , Animales , Biomarcadores/metabolismo , Cromatografía Liquida/métodos , Dreissena/inmunología , Espectrometría de Masas/métodosRESUMEN
Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a pandemic and is continuing to spread rapidly around the globe. No effective vaccine is currently available to prevent COVID-19, and intense efforts are being invested worldwide into vaccine development. In this context, all technology platforms must overcome several challenges resulting from the use of an incompletely characterized new virus. These include finding the right conditions for virus amplification for the development of vaccines based on inactivated or attenuated whole viral particles. Here, we describe a shotgun tandem mass spectrometry workflow, the data produced can be used to guide optimization of the conditions for viral amplification. In parallel, we analysed the changes occurring in the host cell proteome following SARS-CoV-2 infection to glean information on the biological processes modulated by the virus that could be further explored as potential drug targets to deal with the pandemic.
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Antígenos Virales/biosíntesis , Betacoronavirus/inmunología , Proteómica/métodos , Vacunas Virales/inmunología , Virión/inmunología , Animales , Antígenos Virales/inmunología , Chlorocebus aethiops , SARS-CoV-2 , Espectrometría de Masas en Tándem , Células VeroRESUMEN
Ecotoxicoproteomics employs mass spectrometry-based approaches centered on proteins of sentinel organisms to assess for instance, chemical toxicity in fresh water. In this study, we combined proteogenomics experiments and a novel targeted proteomics approach free from retention time scheduling called Scout-MRM. This methodology will enable the measurement of simultaneously changes in the relative abundance of multiple proteins involved in key physiological processes and potentially impacted by contaminants in the freshwater sentinel Gammarus fossarum. The development and validation of the assay were performed to target 157 protein biomarkers of this non-model organism. We carefully chose and validated the transitions to monitor using conventional parameters (linearity, repeatability, LOD, LOQ). Finally, the potential of the methodology is illustrated by measuring 277-peptide-plex assay (831 transitions) in sentinel animals exposed in natura to different agricultural sites potentially exposed to pesticide contamination. Multivariate data analyses highlighted the modulation of several key proteins involved in feeding and molting. This multiplex-targeted proteomics assay paves the way for the discovery and the use of a large panel of novel protein biomarkers in emergent ecotoxicological models for environmental monitoring in the future. BIOLOGICAL SIGNIFICANCE: The study contributed to the development of Scout-MRM for the high-throughput quantitation of a large panel of proteins in the Gammarus fossarum freshwater sentinel. Increasing the number of markers in ecotoxicoproteomics is of most interest to assess the impact of pollutants in freshwater organisms. The development and validation of the assay enabled the monitoring of a large panel of reporter peptides of exposed gammarids. To illustrate the applicability of the methodology, animals from different agricultural sites were analysed. The application of the assay highlighted the modulation of some biomarker proteins involved in key physiological pathways, such as molting, feeding and general stress response. Increasing multiplexing capabilities and field test will provide the development of diagnostic protein biomarkers for emergent ecotoxicological models in future environmental biomonitoring programs.
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Anfípodos , Animales , Biomarcadores , Ecotoxicología , Monitoreo del Ambiente , ProteómicaRESUMEN
Biological responses of zebra mussel Dreissena polymorpha are investigated to assess the impact of contaminants on aquatic organisms and ecosystems. In addition to concentrate chemical contaminants in their tissues, zebra mussels accumulate several microorganisms such as viruses, protozoa and bacteria. In order to understand the molecular mechanisms involved in the defence against microorganisms this study aims at identifying immune proteins from D. polymorpha hemolymph involved in defence against protozoa and viruses. For this purpose, hemolymph were exposed ex vivo to Cryptosporidium parvum and RNA poly I:C. Differential proteomics on both hemocytes and plasma revealed immune proteins modulated under exposures. Different patterns of response were observed after C. parvum and RNA poly I:C exposures. The number of modulated proteins per hemolymphatic compartments suggest that C. parvum is managed in cells while RNA poly I:C is managed in plasma after 4 h exposure. BLAST annotation and GO terms enrichment analysis revealed further characteristics of immune mechanisms. Results showed that many proteins involved in the recognition and destruction of microorganisms were modulated in both exposure conditions, while proteins related to phagocytosis and apoptosis were exclusively modulated by C. parvum. This differential proteomic analysis highlights in zebra mussels modulated proteins involved in the response to microorganisms, which reflect a broad range of immune mechanisms such as recognition, internalization and destruction of microorganisms. This study paves the way for the identification of new markers of immune processes that can be used to assess the impact of both chemical and biological contaminations on the health status of aquatic organisms.
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Dreissena/inmunología , Hemocitos/metabolismo , Hemolinfa/inmunología , Interacciones Microbiota-Huesped/inmunología , Animales , Apoptosis/inmunología , Cryptosporidium parvum/inmunología , Dreissena/parasitología , Dreissena/virología , Hemocitos/inmunología , Hemolinfa/citología , Hemolinfa/metabolismo , Inmunidad Innata , Fagocitosis/inmunología , Poli I-C/inmunología , ProteómicaRESUMEN
Proteogenomics is gaining momentum as, today, genomics, transcriptomics, and proteomics can be readily performed on any new species. This approach allows key alterations to molecular pathways to be identified when comparing conditions. For animals and plants, RNA-seq-informed proteomics is the most popular means of interpreting tandem mass spectrometry spectra acquired for species for which the genome has not yet been sequenced. It relies on high-performance de novo RNA-seq assembly and optimized translation strategies. Here, several pre-treatments for Illumina RNA-seq reads before assembly are explored to translate the resulting contigs into useful polypeptide sequences. Experimental transcriptomics and proteomics datasets acquired for individual Gammarus fossarum freshwater crustaceans are used, the most relevant procedure is defined by the ratio of MS/MS spectra assigned to peptide sequences. Removing reads with a mean quality score of less than 17-which represents a single probable nucleotide error on 150-bp reads-prior to assembly, increases the proteomics outcome. The best translation using Transdecoder is achieved with a minimal open reading frame length of 50 amino acids and systematic selection of ORFs longer than 900 nucleotides. Using these parameters, transcriptome assembly and translation informed by proteomics pave the way to further improvements in proteogenomics.
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Proteogenómica/métodos , Proteómica , RNA-Seq , Transcriptoma/genética , Secuencia de Aminoácidos/genética , Animales , Biología Computacional , Bases de Datos de Proteínas/normas , Genoma/genética , Genómica/tendencias , Análisis de Secuencia de ARNRESUMEN
This 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.
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Gammarids 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.
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Anfípodos/genética , Proteogenómica , Transcriptoma , Animales , Bases de Datos de Proteínas , Femenino , Francia , Agua Dulce , Masculino , RNA-SeqRESUMEN
High-throughput proteomics can be performed on animal sentinels for discovering key molecular biomarkers signing the physiological response and adaptation of organisms. Ecotoxicoproteomics is today amenable by means of proteogenomics to small arthropods such as Gammarids which are well known sentinels of aquatic environments. Here, we analysed two regional Gammarus pulex populations to characterize the potential proteome divergence induced in one site by natural bioavailable mono-metallic contamination (cadmium) compared to a non-contaminated site. Two RNAseq-derived protein sequence databases were established previously on male and female individuals sampled from the reference site. Here, individual proteomes were acquired on 10 male and 10 female paired organisms sampled from each site. Proteins involved in protein lipidation, carbohydrate metabolism, proteolysis, innate immunity, oxidative stress response and lipid transport were found more abundant in animals exposed to cadmium, while hemocyanins were found in lower abundance. The intrapopulation proteome variability of long-term exposed G. pulex was inflated relatively to the non-contaminated population. These results show that, while remaining a challenge for such organisms with not yet sequenced genomes, taking into account intrapopulation variability is important to better define the molecular players induced by toxic stress in a comparative field proteomics approach.
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Anfípodos/metabolismo , Cadmio/toxicidad , Proteoma/metabolismo , Proteómica , Anfípodos/efectos de los fármacos , Animales , Análisis por Conglomerados , Femenino , Masculino , Análisis de Componente Principal , Contaminantes Químicos del Agua/toxicidadRESUMEN
Next generation sequencing and mass spectrometry technologies have recently expanded the availability of whole transcriptomes and proteomes beyond classical model organisms in molecular biology, even in absence of an annotated genome. However, the fragmented nature of transcriptomic and proteomic data reduces the ability to interpret the data, notably in non-model organisms. Network-based approaches may help extracting important biological information from -omics datasets. The reproductive cycle of the freshwater crustacean Gammarus fossarum.provides an excellent case study to test the relevance of a network analysis in non-model organisms. Here, we illustrated how the use of a co-expression network analysis (based on Weighted Gene Co-expression Network Analysis algorithm, WGCNA) allowed identifying protein modules whose expression profiles described germ cell maturation and embryonic development in the freshwater crustacean Gammarus fossarum. Proteome datasets included testes, ovaries or embryos samples at different maturation or developmental stages, respectively. We identified an embryonic module correlated with mid-developmental stages corresponding to the organogenesis and it was characterized by enrichment in proteins involved in RNA editing and splicing. An ovarian module was enriched in vitellogenin-like proteins and clottable proteins, confirming the diversity of proteins belonging to the large lipid transfer family involved in oocytes maturations in this freshwater amphipod. Moreover, our results found evidence of a fine-tuned regulation between energy production by glycolysis and actin-myosin-dependent events in G. fossarum spermatogenesis. This study illustrates the importance of applying systems biology approaches to emergent animal models to improve the understanding of the molecular mechanisms regulating important physiological events with ecological relevance.
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Anfípodos/embriología , Anfípodos/fisiología , Mapas de Interacción de Proteínas , Anfípodos/genética , Animales , Femenino , Regulación del Desarrollo de la Expresión Génica , Gónadas/fisiología , Masculino , Proteómica/métodos , Empalme del ARN , Reproducción , Especies Centinela/embriología , Especies Centinela/genética , Especies Centinela/fisiologíaRESUMEN
The immune system of bivalves is of great interest since it reflects the health status of these organisms during stressful conditions. While immune molecular responses are well documented for marine bivalves, few information is available for continental bivalves such as the zebra mussel, Dreissena polymorpha. A proteogenomic approach was conducted on both hemocytes and plasma to identified immune proteins of this non-model species. Combining transcriptomic sequences with mass spectrometry data acquired on proteins is a relevant strategy since 3020 proteins were identified, representing the largest protein inventory for this sentinel organism. Functional annotation and gene ontology (GO) analysis performed on the identified proteins described the main molecular players of hemocytes and plasma in immunity. GO analysis highlights the complementary immune functions of these two compartments in the management of micro-organisms. Functional annotation revealed new mechanisms in the immune defence of the zebra mussel. Proteins rarely observed in the hemolymph of bivalves were pinpointed such as natterin-like and thaumatin-like proteins. Furthermore, the high abundance of complement-related proteins observed in plasma suggested a strong implication of the complement system in the immune defence of D. polymorpha. This work brings a better understanding of the molecular mechanisms involved in zebra mussel immunity. SIGNIFICANCE: Although the molecular mechanisms of marine bivalves are widely investigated, little information is known for continental bivalves. Moreover, few proteomic studies described the complementarity of both hemolymphatic compartments (cellular and plasmatic) in the immune defence of invertebrates. The recent proteogenomics concept made it possible to discover proteins in non-model organisms. Here, we propose a proteogenomic strategy with the zebra mussel, a key sentinel species for biomonitoring of freshwater, to identify and describe the molecular actors involved in the immune system in both hemocytes and plasma compartments. More widely, this study provided new insight into bivalve immunity.
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Bivalvos/inmunología , Hemocitos/inmunología , Hemolinfa/inmunología , Proteogenómica , Animales , Agua DulceRESUMEN
Anthropogenic pollutants are found worldwide. Their fate and effects on human and ecosystem health must be appropriately monitored. Today, ecotoxicology is focused on the development of new methods to assess the impact of pollutant toxicity on living organisms and ecosystems. In situ biomonitoring often uses sentinel animals for which, ideally, molecular biomarkers have been defined thanks to which environmental quality can be assessed. In this context, high-throughput proteomics methods offer an attractive approach to study the early molecular responses of organisms to environmental stressors. This approach can be used to identify toxicity pathways, to quantify more precisely novel biomarkers, and to draw the possible adverse outcome pathways. In this review, we discuss the major advances in ecotoxicoproteomics made over the last decade and present the current state of knowledge, emphasizing the technological and conceptual advancements that allowed major breakthroughs in this field, which aims to "make our planet great again". SIGNIFICANCE: Ecotoxicoproteomics is a protein-centric methodology that is useful for ecotoxicology and could have future applications as part of chemical risk assessment and environmental monitoring. Ecotoxicology employing non-model sentinel organisms with highly divergent phylogenetic backgrounds aims to preserve the functioning of ecosystems and the overall range of biological species supporting them. The classical proteomics workflow involves protein identification, functional annotation, and extrapolation of toxicity across species. Thus, it is essential to develop multi-omics approaches in order to unravel molecular information and construct the most suitable databases for protein identification and pathway analysis in non-model species. Current instrumentation and available software allow relevant combined transcriptomic/proteomic studies to be performed for almost any species. This review summarizes these approaches and illustrates how they can be implemented in ecotoxicology for routine biomonitoring.
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Ecosistema , Ecotoxicología , Monitoreo del Ambiente/métodos , Proteómica , Animales , Ecotoxicología/métodos , Ecotoxicología/tendencias , Humanos , Proteómica/métodos , Proteómica/tendenciasRESUMEN
As a proof of principle, a selected reaction monitoring (SRM) mass spectrometry-based methodology was applied to the simultaneous quantification of dozens of protein biomarkers in caged amphipods (Gammarus fossarum). We evaluated the suitability of the methodology to assess complex field contaminations through its application in the framework of a regional river monitoring network. Thanks to the high throughput acquisition of biomarker levels in G. fossarum exposed in four reference and 13 contaminated sites, we analyzed the individual responses of 38 peptides reporting for 25 proteins of interest in 170 organisms. Responses obtained in contaminated sites included inductions of vitellogenin-like proteins in male organisms, inductions of Na+K+/ATPases, and strong inhibitions of molt-related proteins such as chitinase and JHE-carboxylesterase. Proteins from detoxification and immunity processes were also found modulated in abundance. Summarizing, the results presented here show that the SRM strategy developed for multibiomarker measurement paves a very promising way to define multiple indicators of the health status of sentinel organisms for environmental hazard assessment.
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Anfípodos , Monitoreo del Ambiente , Proteómica , Animales , Bioensayo , Masculino , RíosRESUMEN
A highly multiplexed liquid chromatography mass spectrometry-selected reaction monitoring (SRM)-based assay for determination of 40 potential protein biomarkers from Gammarus fossarum, an ecotoxicological relevant species, was described. The assay relies on 71 stable isotope-labeled reported peptide standards for the quantitation of proteins of interest in relation to essential physiological functions such as reproductive cycle, defense mechanism, and enzymes involved in homeostasis process and in energy. A direct linear relationship between the spiked peptide concentration and the area under the peak was clearly demonstrated in biological extracts. Precision and accuracy were determined to be between 1.1 and 21% and between 79 and 120%, respectively, depending on the selected protein in a few samples after optimization of digestion conditions. The validity of the assay was documented for several biomarkers linked with reproduction and the molting process was performed with the assessment of protein levels throughout contrasted physiological process (sex, reproductive status). This assay is easy to use, robust, sensitive, and has high-throughput capabilities. The proposed strategy may be extended to any non-model organisms relevant in environmental science. Graphical abstract á .
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Anfípodos/química , Proteínas de Artrópodos/análisis , Espectrometría de Masas en Tándem/métodos , Anfípodos/fisiología , Animales , Proteínas de Artrópodos/metabolismo , Cromatografía Liquida/métodos , Monitoreo del Ambiente , Femenino , Masculino , Muda , ReproducciónRESUMEN
We showed that bee venom phospholipase A2 can be used as a membrane-binding vector to anchor to the surface of cells a soluble protein fused to its C-terminus. ZZ, a two-domain derivative of staphylococcal protein A capable of binding constant regions of antibodies was fused to the C-terminus of the phospholipase or to a mutant devoid of enzymatic activity. The fusion proteins bound to the surface of cells and could themselves bind IgGs. Their fate depended on the cell type to which they bound. On the A431 carcinoma cell line the proteins remained exposed on the cell surface. In contrast, on human dendritic cells the proteins were internalized into early endosomes.
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Venenos de Abeja/enzimología , Membrana Celular/metabolismo , Fosfolipasas A2/química , Animales , Línea Celular Tumoral , Células Dendríticas/metabolismo , Humanos , Fosfolipasas A2/aislamiento & purificación , Fosfolipasas A2/metabolismo , Proteínas Recombinantes de Fusión/químicaRESUMEN
As 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-proteome was 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.