Departamento de Bioquímica Instituto de Química, Universidade de São Paulo, São Paulo, São Paulo, Brazil.
Centro de Oncologia Molecular, Hospital Sírio Libanês, São Paulo, Brazil.
PLoS Negl Trop Dis. 2020 May 29;14(5):e0008262. doi: 10.1371/journal.pntd.0008262. eCollection 2020 May.
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.
锥虫游离体与细胞外基质（ECM）成分的黏附是其侵入哺乳动物宿主细胞的重要步骤。我们最近描述了游离体与 ECM 成分孵育时组蛋白 H2A 和 H4 的酪氨酸硝化水平显著增加。在这项工作中，我们使用抗硝基酪氨酸抗体进行染色质免疫沉淀（ChIP），然后进行质谱分析，以鉴定锥虫中的硝化 DNA 结合蛋白，并检测寄生虫与 ECM 孵育时硝化水平的变化。使用这种蛋白质组学方法，在 9 种最丰富的硝化 DNA 结合蛋白中检测到组蛋白 H1、H2B、H2A 和 H3。在 MS/MS 图谱中鉴定到组蛋白 H2B 中有一个硝化酪氨酸残基（Y29）。此外，我们观察到寄生虫与 ECM 孵育后组蛋白 H1、H2B、H2A 和 H4 的硝化水平显著增加。最后，我们使用 ChIP-Seq 来绘制硝化蛋白的 DNA 结合谱的全局变化。我们观察到寄生虫与 ECM 孵育后硝化蛋白与 DNA 的结合模式发生了显著变化。这项工作提供了锥虫中硝化 DNA 结合蛋白的首个全谱，并进一步证明了寄生虫与 ECM 孵育时组蛋白硝化谱的修饰。我们的数据还表明，寄生虫与 ECM 的相互作用会导致染色质结构发生改变，可能会影响核功能。
