Dataset Information

Profiling the Trypanosoma cruzi phosphoproteome.

ABSTRACT: Protein phosphorylation is a reversible post-translational modification essential for the regulation of several signal transduction pathways and biological processes in the living cell. Therefore, the identification of protein phosphorylation sites is crucial to understand cell signaling control at the molecular level. Based on mass spectrometry, recent studies have reported the large-scale mapping of phosphorylation sites in various eukaryotes and prokaryotes. However, little is known about the impact of phosphorylation in protozoan parasites. To in depth characterize the phosphoproteome of Trypanosoma cruzi, a parasite of the Kinetoplastida class, protein samples from cells at different phases of the metacyclogenesis--differentiation process of the parasites from non-infective epimastigotes to infective metacyclic trypomastigotes--were enriched for phosphopeptides using TiO(2) chromatography and analyzed on an LTQ-Orbitrap mass spectrometer. In total, 1,671 proteins were identified, including 753 phosphoproteins, containing a total of 2,572 phosphorylation sites. The distribution of phosphorylated residues was 2,162 (84.1%) on serine, 384 (14.9%) on threonine and 26 (1.0%) on tyrosine. Here, we also report several consensus phosphorylation sequence motifs and as some of these conserved groups have enriched biological functions, we can infer the regulation by protein kinases of this functions. To our knowledge, our phosphoproteome is the most comprehensive dataset identified until now for Kinetoplastida species. Here we also were able to extract biological information and infer groups of sites phosphorylated by the same protein kinase. To make our data accessible to the scientific community, we uploaded our study to the data repositories PHOSIDA, Proteome Commons and TriTrypDB enabling researchers to access information about the phosphorylation sites identified here.

SUBMITTER: Marchini FK

PROVIDER: S-EPMC3178638 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Profiling the Trypanosoma cruzi phosphoproteome.

Marchini Fabricio K FK de Godoy Lyris M F LM Rampazzo Rita C P RC Pavoni Daniela P DP Probst Christian M CM Gnad Florian F Mann Matthias M Krieger Marco A MA

PloS one 20110922 9

Protein phosphorylation is a reversible post-translational modification essential for the regulation of several signal transduction pathways and biological processes in the living cell. Therefore, the identification of protein phosphorylation sites is crucial to understand cell signaling control at the molecular level. Based on mass spectrometry, recent studies have reported the large-scale mapping of phosphorylation sites in various eukaryotes and prokaryotes. However, little is known about the ...[more]

PMID: 21966514

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Project description: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.

Project description:IntroductionTrypanosoma cruzi, the causative agent of Chagas disease, can infect almost any nucleated cell in the mammalian host. Although previous studies have described the transcriptomic changes that occur in host cells during parasite infection, the understanding of the role of post-transcriptional regulation in this process is limited. MicroRNAs, a class of short non-coding RNAs, are key players in regulating gene expression at the post-transcriptional level, and their involvement in the host-T. cruzi interplay is a growing area of research. However, to our knowledge, there are no comparative studies on the microRNA changes that occur in different cell types in response to T. cruzi infection.Methods and resultsHere we investigated microRNA changes in epithelial cells, cardiomyocytes and macrophages infected with T. cruzi for 24 hours, using small RNA sequencing followed by careful bioinformatics analysis. We show that, although microRNAs are highly cell type-specific, a signature of three microRNAs -miR-146a, miR-708 and miR-1246, emerges as consistently responsive to T. cruzi infection across representative human cell types. T. cruzi lacks canonical microRNA-induced silencing mechanisms and we confirm that it does not produce any small RNA that mimics known host microRNAs. We found that macrophages show a broad response to parasite infection, while microRNA changes in epithelial and cardiomyocytes are modest. Complementary data indicated that cardiomyocyte response may be greater at early time points of infection.ConclusionsOur findings emphasize the significance of considering microRNA changes at the cellular level and complement previous studies conducted at higher organizational levels, such as heart samples. While miR-146a has been previously implicated in T. cruzi infection, similarly to its involvement in many other immunological responses, miR-1246 and miR-708 are demonstrated here for the first time. Given their expression in multiple cell types, we anticipate our work as a starting point for future investigations into their role in the post-transcriptional regulation of T. cruzi infected cells and their potential as biomarkers for Chagas disease.

Dataset Information

Profiling the Trypanosoma cruzi phosphoproteome.

Publications

Profiling the Trypanosoma cruzi phosphoproteome.

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