Abstract
Adhesion of T. cruzi trypomastigotes to components of the extracellular matrix (ECM) is an important step in mammalian host cell invasion. We have recently described a significant increase in the tyrosine nitration levels of histones H2A and H4 when trypomastigotes are incubated with components of the ECM. In this work, we used chromatin immunoprecipitation (ChIP) with an anti-nitrotyrosine antibody followed by mass spectrometry to identify nitrated DNA binding proteins in T. cruzi and to detect alterations in nitration levels induced upon parasite incubation with the ECM. Histone H1, H2B, H2A and H3 were detected among the 9 most abundant nitrated DNA binding proteins using this proteomic approach. One nitrated tyrosine residue (Y29) was identified in Histone H2B in the MS/MS spectrum. In addition, we observed a significant increase in the nitration levels of histones H1, H2B, H2A and H4 upon parasite incubation with ECM. Finally, we used ChIP-Seq to map global changes in the DNA binding profile of nitrated proteins. We observed a significant change in the binding pattern of nitrated proteins to DNA after parasite incubation with ECM. This work provides the first global profile of nitrated DNA binding proteins in T. cruzi and additional evidence for modification in the nitration profile of histones upon parasite incubation with ECM. Our data also indicate that the parasite interaction with the ECM induces alterations in chromatin structure, possibly affecting nuclear functions.
Highlights
Trypanosoma cruzi, the causative agent of Chagas’ disease, completes its life cycle alternating between invertebrate and vertebrate hosts
We have recently reported that nitric oxide (NO) signaling is down-regulated when T. cruzi trypomastigotes are incubated with extracellular matrix (ECM)
Antibody characterization and identification of DNA binding nitrated proteins The immunoprecipitated proteins from the enriched nuclear fraction of T. cruzi trypomastigotes using the anti-nitrotyrosine antibody were analyzed by bottom up proteomic approach
Summary
Trypanosoma cruzi, the causative agent of Chagas’ disease, completes its life cycle alternating between invertebrate (triatomine insects) and vertebrate (mammals, including humans) hosts. Adhesion of T. cruzi trypomastigotes to components of the extracellular matrix (ECM) is an important step in mammalian host cell invasion [3,4]. We have recently reported that nitric oxide (NO) signaling is down-regulated when T. cruzi trypomastigotes are incubated with ECM. Inhibition of NOS activity, with the expected decrease in NO production, as well as decrease in cGMP concentration, was observed when trypomastigotes are incubated with ECM. We observed that parasite adhesion to ECM significantly modulates NOmediated protein post-translational modifications (PTMs). In spite of a global down regulation of protein S-nitrosylation and nitration, a significant increase in the tyrosine nitration levels of histones H2A and H4 was observed upon incubation of the parasites with the ECM [4], pointing out to the specificity of the modification
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