RESUMEN
Although the subcellular dynamics of RNA and proteins are key determinants of cell homeostasis, their characterization is still challenging. Here we present an integrative framework to simultaneously interrogate the dynamics of the transcriptome and proteome at subcellular resolution by combining two methods: localization of RNA (LoRNA) and a streamlined density-based localization of proteins by isotope tagging (dLOPIT) to map RNA and protein to organelles (nucleus, endoplasmic reticulum and mitochondria) and membraneless compartments (cytosol, nucleolus and cytosolic granules). Interrogating all RNA subcellular locations at once enables system-wide quantification of the proportional distribution of RNA. We obtain a cell-wide overview of localization dynamics for 31,839 transcripts and 5,314 proteins during the unfolded protein response, revealing that endoplasmic reticulum-localized transcripts are more efficiently recruited to cytosolic granules than cytosolic RNAs, and that the translation initiation factor eIF3d is key to sustaining cytoskeletal function. Overall, we provide the most comprehensive overview so far of RNA and protein subcellular localization dynamics.
Asunto(s)
Retículo Endoplásmico , ARN , ARN/genética , ARN/metabolismo , Fracciones Subcelulares/metabolismo , Retículo Endoplásmico/metabolismo , Proteoma/análisisRESUMEN
The ability of the cellular immune system to discriminate self from foreign antigens depends on the appropriate calibration of the T cell receptor (TCR) signalling threshold. The lymphocyte homeostatic cytokine interleukin 7 (IL-7) is known to affect TCR thresholding, but the molecular mechanism is not fully elucidated. A better understanding of this process is highly relevant in the context of autoimmune disease therapy and cancer immunotherapy. We sought to characterise the early signalling events attributable to IL-7 priming; in particular, the altered phosphorylation of signal transduction proteins and their molecular localisation to the TCR. By integrating high-resolution proximity- phospho-proteomic and imaging approaches using primary T cells, rather than engineered cell lines or an in vitro expanded T cell population, we uncovered transduction events previously not linked to IL-7. We show that IL-7 leads to dephosphorylation of cytohesin interacting protein (CYTIP) at a hitherto undescribed phosphorylation site (pThr280) and alters the co-localisation of cytohesin-1 with the TCR and LFA-1 integrin. These results show that IL-7, acting via CYTIP and cytohesin-1, may impact TCR activation thresholds by enhancing the co-clustering of TCR and LFA-1 integrin.
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Factores de Intercambio de Guanina Nucleótido/metabolismo , Interleucina-7/farmacología , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/efectos de los fármacos , Linfocitos T/inmunología , Factores de Transcripción/metabolismo , Citoesqueleto de Actina/metabolismo , Donantes de Sangre , Células Cultivadas , Humanos , Activación de Linfocitos/efectos de los fármacos , Fosforilación/efectos de los fármacos , Proteínas Recombinantes/farmacología , Treonina/metabolismoRESUMEN
Following central nervous system (CNS) demyelination, adult oligodendrocyte progenitor cells (OPCs) can differentiate into new myelin-forming oligodendrocytes in a regenerative process called remyelination. Although remyelination is very efficient in young adults, its efficiency declines progressively with ageing. Here we performed proteomic analysis of OPCs freshly isolated from the brains of neonate, young and aged female rats. Approximately 50% of the proteins are expressed at different levels in OPCs from neonates compared with their adult counterparts. The amount of myelin-associated proteins, and proteins associated with oxidative phosphorylation, inflammatory responses and actin cytoskeletal organization increased with age, whereas cholesterol-biosynthesis, transcription factors and cell cycle proteins decreased. Our experiments provide the first ageing OPC proteome, revealing the distinct features of OPCs at different ages. These studies provide new insights into why remyelination efficiency declines with ageing and potential roles for aged OPCs in other neurodegenerative diseases.
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Envejecimiento/metabolismo , Células Precursoras de Oligodendrocitos/metabolismo , Proteoma/metabolismo , Animales , Animales Recién Nacidos , Biomarcadores/metabolismo , Separación Celular , Colesterol/metabolismo , Vaina de Mielina/metabolismo , Enfermedades Neurodegenerativas/patología , Células Precursoras de Oligodendrocitos/citología , Complejo de la Endopetidasa Proteasomal/metabolismo , Pliegue de Proteína , Proteómica , Proteostasis , Ratas Sprague-Dawley , Reproducibilidad de los ResultadosRESUMEN
Chagas disease is a tropical neglected disease endemic in Latin America caused by the protozoan Trypanosoma cruzi. The parasite has four major life stages: epimastigote, metacyclic trypomastigote, bloodstream trypomastigote, and amastigote. The differentiation from infective trypomastigotes into replicative amastigotes, called amastigogenesis, takes place in vivo inside mammalian host cells after a period of incubation in an acidic phagolysosome. This differentiation process can be mimicked in vitro by incubating tissue-culture-derived trypomastigotes in acidic DMEM. Here we used this well-established differentiation protocol to perform a comprehensive quantitative proteomic and phosphoproteomic analysis of T. cruzi amastigogenesis. Samples from fully differentiated forms and two biologically relevant intermediate time points were Lys-C/trypsin digested, iTRAQ-labeled, and multiplexed. Subsequently, phosphopeptides were enriched using a TiO2 matrix. Non-phosphorylated peptides were fractionated via hydrophilic interaction liquid chromatography prior to LC-MS/MS analysis. LC-MS/MS and bioinformatics procedures were used for protein and phosphopeptide quantitation, identification, and phosphorylation site assignment. We were able to identify regulated proteins and pathways involved in coordinating amastigogenesis. We also observed that a significant proportion of the regulated proteins were membrane proteins. Modulated phosphorylation events coordinated by protein kinases and phosphatases that are part of the signaling cascade induced by incubation in acidic medium were also evinced. To our knowledge, this work is the most comprehensive quantitative proteomics study of T. cruzi amastigogenesis, and these data will serve as a trustworthy basis for future studies, and possibly for new potential drug targets.
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Estadios del Ciclo de Vida/genética , Péptidos/química , Fosfoproteínas/genética , Proteínas Protozoarias/genética , Trypanosoma cruzi/genética , Medios de Cultivo/farmacología , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Concentración de Iones de Hidrógeno , Estadios del Ciclo de Vida/efectos de los fármacos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Anotación de Secuencia Molecular , Mapeo Peptídico , Péptidos/genética , Péptidos/metabolismo , Fosfoproteínas/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteómica/métodos , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/crecimiento & desarrollo , Trypanosoma cruzi/metabolismoRESUMEN
Chagas' disease is a neglected infectious illness, caused by the protozoan Trypanosoma cruzi. It remains a challenging health issue in Latin America, where it is endemic, and so far there is no immunoprophylatic vaccine or satisfactory chemotherapic treatment for its chronic stage. The present work addressed the analysis of the plasma membrane (PM) subproteome from T. cruzi human-hosted life stages, trypomastigote and axenic amastigote, by two complementary PM protein enrichment techniques followed by identification using an LC-MS/MS approach. The results revealed an extensive repertoire of proteins in the PM subproteomes, including enzymes that might be suitable candidates for drug intervention. The comparison of the cell surface proteome among the life forms revealed some potentially stage-specific enzymes, although the majority was shared by both stages. Bioinformatic analysis showed that the vast majority of the identified proteins are membrane-derived and/or possess predicted transmembrane domains. They are mainly involved in host cell infection, protein adhesion, cell signaling, and the modulation of mammalian host immune response. Several virulence factors and proteins potentially capable of acting at a number of metabolic pathways of the host and also to regulate cell differentiation of the parasite itself were also found.
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Estadios del Ciclo de Vida/genética , Proteínas de la Membrana/genética , Proteómica/métodos , Trypanosoma cruzi/genética , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/prevención & control , Cromatografía de Afinidad , Cromatografía Liquida/métodos , Biología Computacional , Descubrimiento de Drogas/métodos , Humanos , Espectrometría de Masas en Tándem/métodos , Trypanosoma cruzi/metabolismoRESUMEN
Trypanosoma cruzi is a protozoan that causes Chagas' disease, a neglected infectious illness that affects millions of people, mostly in Latin America. Here, the cell surface subproteome of the T. cruzi epimastigote life form was characterized. In order to prepare samples enriched in epimastigote plasma membrane protein, two distinct methodologies were optimized and evaluated. The first methodology was based on cell surface trypsinization (Shave) of intact living cells while the second approach used biotinylation of cell surface proteins followed by streptavidin affinity chromatography isolation of the labeled proteins. Both T. cruzi subproteomes were analyzed by LC-MS/MS. The results showed that the methodologies offered comprehensive and complementary information about the parasite's plasma membrane subproteome.
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Enfermedad de Chagas/diagnóstico , Proteínas de la Membrana/aislamiento & purificación , Proteoma/análisis , Trypanosoma cruzi/metabolismo , Biotinilación , Membrana Celular/metabolismo , Enfermedad de Chagas/patología , Humanos , Proteínas de la Membrana/metabolismo , Espectrometría de Masas en Tándem , Trypanosoma cruzi/patogenicidad , Tripsina/químicaRESUMEN
BACKGROUND: Cystic Fibrosis (CF) is a genetic disorder affecting around 1 in every 3000 newborns. In the most common mutation, F508del, the defective anion channel, CFTR, is prevented from reaching the plasma membrane (PM) by the quality check control of the cell. Little is known about how CFTR pharmacological rescue impacts the cell proteome. METHODS: We used high-resolution mass spectrometry, differential ultracentrifugation, machine learning and bioinformatics to investigate both changes in the expression and localization of the human bronchial epithelium CF model (F508del-CFTR CFBE41o-) proteome following treatment with VX-809 (Lumacaftor), a drug able to improve the trafficking of CFTR. RESULTS: The data suggested no stark changes in protein expression, yet subtle localization changes of proteins of the mitochondria and peroxisomes were detected. We then used high-content confocal microscopy to further investigate the morphological and compositional changes of peroxisomes and mitochondria under these conditions, as well as in patient-derived primary cells. We profiled several thousand proteins and we determined the subcellular localization data for around 5000 of them using the LOPIT-DC spatial proteomics protocol. CONCLUSIONS: We observed that treatment with VX-809 induces extensive structural and functional remodelling of mitochondria and peroxisomes that resemble the phenotype of healthy cells. Our data suggest additional rescue mechanisms of VX-809 beyond the correction of aberrant folding of F508del-CFTR and subsequent trafficking to the PM.
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Fibrosis Quística , Aminopiridinas , Benzodioxoles , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Epitelio/metabolismo , Humanos , Recién Nacido , Mitocondrias/metabolismo , Proteoma/metabolismoRESUMEN
Drosophila nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that represent a target for insecticides. Peptide neurotoxins are known to block nAChRs by binding to their target subunits, however, a better understanding of this mechanism is needed for effective insecticide design. To facilitate the analysis of nAChRs we used a CRISPR/Cas9 strategy to generate null alleles for all ten nAChR subunit genes in a common genetic background. We studied interactions of nAChR subunits with peptide neurotoxins by larval injections and styrene maleic acid lipid particles (SMALPs) pull-down assays. For the null alleles, we determined the effects of α-Bungarotoxin (α-Btx) and ω-Hexatoxin-Hv1a (Hv1a) administration, identifying potential receptor subunits implicated in the binding of these toxins. We employed pull-down assays to confirm α-Btx interactions with the Drosophila α5 (Dα5), Dα6, Dα7 subunits. Finally, we report the localisation of fluorescent tagged endogenous Dα6 during Drosophila CNS development. Taken together, this study elucidates native Drosophila nAChR subunit interactions with insecticidal peptide toxins and provides a resource for the in vivo analysis of insect nAChRs.
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Insecticidas , Receptores Nicotínicos , Animales , Bungarotoxinas/metabolismo , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Insecticidas/toxicidad , Neurotoxinas , Péptidos/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismoRESUMEN
Dipetalogaster maxima is a blood-sucking Hemiptera that inhabits sylvatic areas in Mexico. It usually takes its blood meal from lizards, but following human population growth, it invaded suburban areas, feeding also on humans and domestic animals. Hematophagous insect salivary glands produce potent pharmacologic compounds that counteract host hemostasis, including anticlotting, antiplatelet, and vasodilatory molecules. To obtain further insight into the salivary biochemical and pharmacologic complexity of this insect, a cDNA library from its salivary glands was randomly sequenced. Salivary proteins were also submitted to one- and two-dimensional gel electrophoresis (1DE and 2DE) followed by mass spectrometry analysis. We present the analysis of a set of 2728 cDNA sequences, 1375 of which coded for proteins of a putative secretory nature. The saliva 2DE proteome displayed approximately 150 spots. The mass spectrometry analysis revealed mainly lipocalins, pallidipins, antigen 5-like proteins, and apyrases. The redundancy of sequence identification of saliva-secreted proteins suggests that proteins are present in multiple isoforms or derive from gene duplications.
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Proteínas de Insectos/análisis , Proteoma/análisis , Triatominae/metabolismo , Secuencia de Aminoácidos , Animales , Análisis por Conglomerados , Electroforesis en Gel de Poliacrilamida , Perfilación de la Expresión Génica , Biblioteca de Genes , Proteínas de Insectos/clasificación , Proteínas de Insectos/metabolismo , Espectrometría de Masas , Datos de Secuencia Molecular , Mapeo Peptídico , Proteoma/metabolismo , ARN Mensajero/química , ARN Mensajero/aislamiento & purificación , Glándulas Salivales/química , Glándulas Salivales/metabolismo , Alineación de SecuenciaRESUMEN
RNA-protein interactions play a pivotal role in cell homeostasis and disease, but current approaches to study them require a considerable amount of starting material, favor the recovery of only a subset of RNA species or are complex and time-consuming. We recently developed orthogonal organic phase separation (OOPS): a quick, efficient and reproducible method to purify cross-linked RNA-protein adducts in an unbiased way. OOPS avoids molecular tagging or the capture of polyadenylated RNA. Instead, it is based on sampling the interface of a standard TRIzol extraction to enrich RNA-binding proteins (RBPs) and their cognate bound RNA. OOPS specificity is achieved by digesting the enriched interfaces with RNases or proteases to release the RBPs or protein-bound RNA, respectively. Here we present a step-by-step protocol to purify protein-RNA adducts, free protein and free RNA from the same sample. We further describe how OOPS can be applied in human cell lines, Arabidopsis thaliana, Schizosaccharomyces pombe and Escherichia coli and how it can be used to study RBP dynamics.
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Fraccionamiento Químico/métodos , Proteoma/aislamiento & purificación , Proteínas de Unión al ARN/aislamiento & purificación , ARN/aislamiento & purificación , Transcriptoma , Línea Celular , Humanos , Proteoma/metabolismo , ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Flujo de TrabajoRESUMEN
Protein-RNA interactions regulate all aspects of RNA metabolism and are crucial to the function of catalytic ribonucleoproteins. Until recently, the available technologies to capture RNA-bound proteins have been biased toward poly(A) RNA-binding proteins (RBPs) or involve molecular labeling, limiting their application. With the advent of organic-aqueous phase separation-based methods, we now have technologies that efficiently enrich the complete suite of RBPs and enable quantification of RBP dynamics. These flexible approaches to study RBPs and their bound RNA open up new research avenues for systems-level interrogation of protein-RNA interactions.
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Proteoma/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Proteoma/química , Proteómica/métodos , ARN/química , ARN Mensajero/química , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/aislamiento & purificaciónRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Existing high-throughput methods to identify RNA-binding proteins (RBPs) are based on capture of polyadenylated RNAs and cannot recover proteins that interact with nonadenylated RNAs, including long noncoding RNA, pre-mRNAs and bacterial RNAs. We present orthogonal organic phase separation (OOPS), which does not require molecular tagging or capture of polyadenylated RNA, and apply it to recover cross-linked protein-RNA and free protein, or protein-bound RNA and free RNA, in an unbiased way. We validated OOPS in HEK293, U2OS and MCF10A human cell lines, and show that 96% of proteins recovered were bound to RNA. We show that all long RNAs can be cross-linked to proteins, and recovered 1,838 RBPs, including 926 putative novel RBPs. OOPS is approximately 100-fold more efficient than existing methods and can enable analyses of dynamic RNA-protein interactions. We also characterize dynamic changes in RNA-protein interactions in mammalian cells following nocodazole arrest, and present a bacterial RNA-interactome for Escherichia coli. OOPS is compatible with downstream proteomics and RNA sequencing, and can be applied in any organism.
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ARN Mensajero/química , Proteínas de Unión al ARN/aislamiento & purificación , ARN/aislamiento & purificación , Línea Celular Tumoral , Análisis por Conglomerados , Reactivos de Enlaces Cruzados/química , Escherichia coli , Glicoproteínas/química , Células HEK293 , Humanos , Nocodazol/química , Unión Proteica , Proteoma , Proteómica , ARN/química , ARN Bacteriano/química , ARN Largo no Codificante/química , Proteínas de Unión al ARN/química , Análisis de Secuencia de ARN , Timidina/química , TranscriptomaRESUMEN
Chagas disease is caused by the protozoan Trypanosoma cruzi, affecting around 8 million people worldwide. After host cell invasion, the infective trypomastigote form remains 2-4 hours inside acidic phagolysosomes to differentiate into replicative amastigote form. In vitro acidic-pH-induced axenic amastigogenesis was used here to study this step of the parasite life cycle. After three hours of trypomastigote incubation in amastigogenesis promoting acidic medium (pH 5.0) or control physiological pH (7.4) medium samples were subjected to three rounds of centrifugation followed by ultrafiltration of the supernatants. The resulting exoproteome samples were trypsin digested and analysed by nano flow liquid chromatography coupled to tandem mass spectrometry. Computational protein identification searches yielded 271 and 483 protein groups in the exoproteome at pH 7.4 and pH 5.0, respectively, with 180 common proteins between both conditions. The total amount and diversity of proteins released by parasites almost doubled upon acidic incubation compared to control. Overall, 76.5% of proteins were predicted to be secreted by classical or non-classical pathways and 35.1% of these proteins have predicted transmembrane domains. Classical secretory pathway analysis showed an increased number of mucins and mucin-associated surface proteins after acidic incubation. However, the number of released trans-sialidases and surface GP63 peptidases was higher at pH 7.4. Trans-sialidases and mucins are anchored to the membrane and exhibit an enzyme-substrate relationship. In general, mucins are glycoproteins with immunomodulatory functions in Chagas disease, present mainly in the epimastigote and trypomastigote surfaces and could be enzymatically cleaved and released in the phagolysosome during amastigogenesis. Moreover, evidence for flagella discard during amastigogenesis are addressed. This study provides the first comparative analysis of the exoproteome during amastigogenesis, and the presented data evidence the dynamism of its profile in response to acidic pH-induced differentiation.
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Enfermedad de Chagas/parasitología , Proteómica/métodos , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/patogenicidad , Enfermedad de Chagas/metabolismo , Cromatografía Liquida , Células HeLa , Interacciones Huésped-Parásitos , Humanos , Concentración de Iones de Hidrógeno , Estadios del Ciclo de Vida , Espectrometría de Masas en Tándem , Trypanosoma cruzi/metabolismoRESUMEN
The canonical molecular machinery required for global mRNA translation and its control has been well defined, with distinct sets of proteins involved in the processes of translation initiation, elongation and termination. Additionally, noncanonical, trans-acting regulatory RNA-binding proteins (RBPs) are necessary to provide mRNA-specific translation, and these interact with 5' and 3' untranslated regions and coding regions of mRNA to regulate ribosome recruitment and transit. Recently it has also been demonstrated that trans-acting ribosomal proteins direct the translation of specific mRNAs. Importantly, it has been shown that subsets of RBPs often work in concert, forming distinct regulatory complexes upon different cellular perturbation, creating an RBP combinatorial code, which through the translation of specific subsets of mRNAs, dictate cell fate. With the development of new methodologies, a plethora of novel RNA binding proteins have recently been identified, although the function of many of these proteins within mRNA translation is unknown. In this review we will discuss these methodologies and their shortcomings when applied to the study of translation, which need to be addressed to enable a better understanding of trans-acting translational regulatory proteins. Moreover, we discuss the protein domains that are responsible for RNA binding as well as the RNA motifs to which they bind, and the role of trans-acting ribosomal proteins in directing the translation of specific mRNAs. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Translation > Translation Regulation Translation > Translation Mechanisms.
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Proteínas de Unión al ARN/metabolismo , Animales , Humanos , Biosíntesis de Proteínas , Dominios Proteicos , Proteínas de Unión al ARN/químicaRESUMEN
Plasmodium mature sexual cycle occurs in the vector mosquitoes and ensures the transmission to a new host. Gametogenesis takes place within minutes in the vector midgut. Gametocytes have to complete a deep nuclear reorganization, quick differentiation, and in the case of male gametocytes, intracytoplasmic flagellum assembly that results in free-swimming microgametes required for macrogamete fertilization. In efforts to improve our knowledge of molecular mechanisms involved in gamete morphogenesis, we carried out a nanoLC/MSMS based quantitative proteomic analysis throughout the xanthurenic acid-induced gametogenesis of the rodent parasite P. berghei. Time-course analyses were performed 7 and 15min after gametogenesis induction. From 2617 iTRAQ-labelled peptides, 49 proteins were found differentially abundant. Proteins related to RNA translation, DNA, and protein biosynthesis were most prominent and strongly regulated. The energetic metabolic pathway, glycolysis, environmental stress response, RNA/protein biosynthesis, mitosis and axoneme formation, both related to tubulin-associated cytoskeleton dynamic, were predominant regulated cell processes at protein level during the differentiation. Our results also include 26 phosphoproteins in gametocytes/gametes. This first iTRAQ-based proteomic time course analysis of Plasmodium gamete development sheds light on the biological protein orchestration within gametogenesis. SIGNIFICANCE: Malaria is one of the most serious and widespread parasitic diseases that affected humans in medicine history. The increasing emergence of resistance of parasites from Plasmodium genus to the available antimalarial drugs and the absence of efficient vaccines require an urgent need of development of new therapeutic strategies to fight against that disease. The sexual reproduction is a key step of Plasmodium life cycle and constitutes an attractive target for the development of new therapeutic approaches since it would control malaria based on an inhibition of the parasite transmission to Anopheles, and then to humans. Male and female gamete formation (gametogenesis) is thus a biological event that is determinant for the perpetuation of the parasite in which drastic morphological and metabolic changes occur in a short time interval, resulting in the production of 8 male gametes from a male gametocyte, and fertilization of the female gamete. Development of such transmission-blocking strategies required in deep understanding of the molecular and cellular events associated to gametogenesis. Despite several studies, our understanding on gametogenesis is still incomplete and requires further investigations. This work is the first large-scale quantitative proteomic insight into the P. berghei gamete morphogenesis providing valuable time course data. Plasmodium gametogenesis clearly requires regulation of expression and phosphorylation of proteins belonging to different metabolic pathways and functions, in order to produce mature male and female gametes.
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Gametogénesis/fisiología , Células Germinativas/metabolismo , Estadios del Ciclo de Vida/fisiología , Plasmodium berghei/metabolismo , Proteómica , Proteínas Protozoarias/metabolismo , Animales , Femenino , Ratones , Mosquitos Vectores/parasitologíaRESUMEN
The protozoan parasite Trypanosoma cruzi causes Chagas disease, one of the major neglected infectious diseases. It has the potential to infect any nucleated mammalian cell. The secreted/excreted protein repertoire released by T. cruzi trypomastigotes is crucial in host-pathogen interactions. In this study, mammalian tissue culture-derived trypomastigotes (Y strain) were used to characterize the exoproteome of the infective bloodstream life form. Proteins released into the serum-free culture medium after 3 h of incubation were harvested and digested with trypsin. NanoLC-MS/MS analysis resulted in the identification of 540 proteins, the largest set of released proteins identified to date in Trypanosoma spp. Bioinformatic analysis predicted most identified proteins as secreted, predominantly by non-classical pathways, and involved in host-cell infection. Some proteins possess predicted GPI-anchor signals, these being mostly trans-sialidases, mucin associated surface proteins and surface glycoproteins. Moreover, we enriched phosphopeptides and glycopeptides from tryptic digests. The majority of identified glycoproteins are trans-sialidases and surface glycoproteins involved in host-parasite interaction. Conversely, most identified phosphoproteins have no Gene Ontology classification. The existence of various proteins related to similar functions in the exoproteome likely reflects this parasite's enhanced mechanisms for adhesion, invasion, and internalization of different host-cell types, and escape from immune defenses.
RESUMEN
Giardiasis, currently considered a neglected disease, is caused by the intestinal protozoan parasite Giardia duodenalis and is widely spread in human as well as domestic and wild animals. The lack of appropriate medications and the spread of resistant parasite strains urgently call for the development of novel therapeutic strategies. Host microbiota or certain probiotic strains have the capacity to provide some protection against giardiasis. By combining biological and biochemical approaches, we have been able to decipher a molecular mechanism used by the probiotic strain Lactobacillus johnsonii La1 to prevent Giardia growth in vitro. We provide evidence that the supernatant of this strain contains active principle(s) not directly toxic to Giardia but able to convert non-toxic components of bile into components highly toxic to Giardia. By using bile acid profiling, these components were identified as deconjugated bile-salts. A bacterial bile-salt-hydrolase of commercial origin was able to mimic the properties of the supernatant. Mass spectrometric analysis of the bacterial supernatant identified two of the three bile-salt-hydrolases encoded in the genome of this probiotic strain. These observations document a possible mechanism by which L. johnsonii La1, by secreting, or releasing BSH-like activity(ies) in the vicinity of replicating Giardia in an environment where bile is present and abundant, can fight this parasite. This discovery has both fundamental and applied outcomes to fight giardiasis, based on local delivery of deconjugated bile salts, enzyme deconjugation of bile components, or natural or recombinant probiotic strains that secrete or release such deconjugating activities in a compartment where both bile salts and Giardia are present.
RESUMEN
The present study was carried out to evaluate the ability of Trichoderma harzianum (ALL 42-isolated from Brazilian Cerrado soil) to promote common bean growth and to modulate its metabolism and defense response in the presence or absence of the phytopathogenic fungi Rhizoctonia solani and Fusarium solani using a proteomic approach. T. harzianum was able to promote common bean plants growth as shown by the increase in root/foliar areas and by size in comparison to plants grown in its absence. The interaction was shown to modulate the expression of defense-related genes (Glu1, pod3 and lox1) in roots of P. vulgaris. Proteomic maps constructed using roots and leaves of plants challenged or unchallenged by T. harzianum and phytopathogenic fungi showed differences. Reference gels presented differences in spot distribution (absence/presence) and relative volumes of common spots (up or down-regulation). Differential spots were identified by peptide fingerprinting MALDI-TOF mass spectrometry. A total of 48 identified spots (19 for leaves and 29 for roots) were grouped into protein functional classes. For leaves, 33%, 22% and 11% of the identified proteins were categorized as pertaining to the groups: metabolism, defense response and oxidative stress response, respectively. For roots, 17.2%, 24.1% and 10.3% of the identified proteins were categorized as pertaining to the groups: metabolism, defense response and oxidative stress response, respectively.