Overexpression and Refolding of the Hydrophobic Ribosomal P0 Protein from Trypanosoma cruzi: A Component of the P1/P2/P0 Complex

Ayub, Maximiliano Juri; Levìn, Mariano J.; Aguilar, Carlos Fernando

doi:10.1006/prep.2001.1427

Cited by 9 publications

(2 citation statements)

References 33 publications

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“…Finally, proteins involved in the parasite cell defense mechanism (tryparedoxin peroxidase), autoimmune disorders after infection (ribosomal PO proteins), and control of cell-cycle progression (RACK proteins) were detected. The PO protein is part of the ribosomal stalk of the large ribosomal subunit, involved in the translocation step of protein synthesis . Its carboxi-terminal region is highly antigenic consisting of a major target for the antibody response in Chagas disease patients.…”

Section: Resultsmentioning

confidence: 99%

Proteomic Analysis of Trypanosoma cruzi Developmental Stages Using Isotope-Coded Affinity Tag Reagents

et al. 2004

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Comparative proteome analysis of developmental stages of the human pathogen Trypanosoma cruzi was carried out by isotope-coded affinity tag technology (ICAT) associated with liquid cromatography-mass spectrometry peptide sequencing (LC-MS/MS). Protein extracts of the protozoan trypomastigote and amastigote stages were labeled with heavy (D8) and light (D0) ICAT reagents and subjected to cation exchange and avidin affinity chromatographies followed by LC-MS/MS analysis. High confidence sequence information and expression levels for 41 T. cruzi polypeptides, including metabolic enzymes, paraflagellar rod components, tubulins, and heat-shock proteins were reported. Twenty-nine proteins displayed similar levels of expression in both forms of the parasite, nine proteins presented higher levels in trypomastigotes, whereas three were more expressed in amastigotes.

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Section: Resultsmentioning

confidence: 99%

Proteomic Analysis of Trypanosoma cruzi Developmental Stages Using Isotope-Coded Affinity Tag Reagents

et al. 2004

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“…The ribosomal P proteins were obtained and purified by means as His 6 -tag as described [44]. The purity and specificity of the recombinant proteins were analyzed by SDS-PAGE gels and Western-blot with a pool of chagasic and non-infected sera.…”

Section: Methodsmentioning

confidence: 99%

Cytokine Production but Lack of Proliferation in Peripheral Blood Mononuclear Cells from Chronic Chagas' Disease Cardiomyopathy Patients in Response to T. cruzi Ribosomal P Proteins

et al. 2014

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Background Trypanosoma cruzi ribosomal P proteins, P2β and P0, induce high levels of antibodies in patients with chronic Chagas' disease Cardiomyopathy (CCC). It is well known that these antibodies alter the beating rate of cardiomyocytes and provoke apoptosis by their interaction with β1-adrenergic and M2-muscarinic cardiac receptors. Based on these findings, we decided to study the cellular immune response to these proteins in CCC patients compared to non-infected individuals.Methodology/Principal findingsWe evaluated proliferation, presence of surface activation markers and cytokine production in peripheral blood mononuclear cells (PBMC) stimulated with P2β, the C-terminal portion of P0 (CP0) proteins and T. cruzi lysate from CCC patients predominantly infected with TcVI lineage. PBMC from CCC patients cultured with P2β or CP0 proteins, failed to proliferate and express CD25 and HLA-DR on T cell populations. However, multiplex cytokine assays showed that these antigens triggered higher secretion of IL-10, TNF-α and GM-CSF by PBMC as well as both CD4+ and CD8+ T cells subsets of CCC subjects. Upon T. cruzi lysate stimulation, PBMC from CCC patients not only proliferated but also became activated within the context of Th1 response. Interestingly, T. cruzi lysate was also able to induce the secretion of GM-CSF by CD4+ or CD8+ T cells.Conclusions/SignificanceOur results showed that although the lack of PBMC proliferation in CCC patients in response to ribosomal P proteins, the detection of IL-10, TNF-α and GM-CSF suggests that specific T cells could have both immunoregulatory and pro-inflammatory potential, which might modulate the immune response in Chagas' disease. Furthermore, it was possible to demonstrate for the first time that GM-CSF was produced by PBMC of CCC patients in response not only to recombinant ribosomal P proteins but also to parasite lysate, suggesting the value of this cytokine to evaluate T cells responses in T. cruzi infection.

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Chagas disease: a homology model for the three-dimensional structure of the Trypanosoma cruzi ribosomal P0 antigenic protein

Barroso

Aguilar

2014

Eur Biophys J

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Ribosomal P proteins form a "stalk" complex in the large subunit of the ribosomes. In Trypanosoma cruzi, the etiological agent of Chagas disease, the complex is formed by five P protein members: TcP0, TcP1α, TcP1β, TcP2α and TcP2β. The TcP0 protein has 34 kDa, and TcP1 and TcP2 proteins have 10 kDa. The structure of T. cruzi P0 and the stalk complex TcP0-TcP1α-TcP1β-TcP2α-TcP2β have not been solved to date. In this work, we constructed a three-dimensional molecular model for TcP0 using homology modeling as implemented in the MODELLER 9v12 software. The model was constructed using different templates: the X-ray structures of the protein P0 from Pirococcus horikoshii, a segment from the Danio renio Ca(+2)/K(+) channel and the C-terminal peptide (C13) from T. cruzi ribosomal P2 protein; the Cryo-EM structure of Triticum aestivum P0 protein and the NMR structure of Homo sapiens P1 ribosomal protein. TcP0 has a 200-residue-long N-terminal, which is an α/β globular stable domain, and a flexible C-terminal, 120-residue-long domain. The molecular surface electrostatic potential and hydrophobic surface were calculated. The surface properties are important for the C-terminal's antigenic properties. They are also responsible for P0-specific binding to RNA26S and the binding to the P1-P2 proteins. We explored and identified protein interactions that may be involved in conformational stability. The structure proposed in this work represents a first structural report for the TcP0 protein.

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Overexpression and Refolding of the Hydrophobic Ribosomal P0 Protein from Trypanosoma cruzi: A Component of the P1/P2/P0 Complex

Cited by 9 publications

References 33 publications

Proteomic Analysis of Trypanosoma cruzi Developmental Stages Using Isotope-Coded Affinity Tag Reagents

Proteomic Analysis of Trypanosoma cruzi Developmental Stages Using Isotope-Coded Affinity Tag Reagents

Cytokine Production but Lack of Proliferation in Peripheral Blood Mononuclear Cells from Chronic Chagas' Disease Cardiomyopathy Patients in Response to T. cruzi Ribosomal P Proteins

Chagas disease: a homology model for the three-dimensional structure of the Trypanosoma cruzi ribosomal P0 antigenic protein

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