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Quantitative Proteomic Analysis of Replicative and Nonreplicative Forms Reveals Important Insights into Chromatin Biology of Trypanosoma cruzi

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Autor(es):
Leandro de Jesus, Teresa Cristina ; Calderano, Simone Guedes ; de Luna Vitorino, Francisca Nathalia ; Llanos, Ricardo Pariona ; Lopes, Mariana de Camargo ; de Araujo, Christiane Bezerra ; Thiemann, Otavio Henrique ; Reis, Marcelo da Silva ; Elias, Maria Carolina ; Chagas da Cunha, Julia Pinheiro
Número total de Autores: 10
Tipo de documento: Artigo Científico
Fonte: MOLECULAR & CELLULAR PROTEOMICS; v. 16, n. 1, p. 23-38, JAN 2017.
Citações Web of Science: 5
Resumo

Chromatin associated proteins are key regulators of many important processes in the cell. Trypanosome cruzi, a protozoa flagellate that causes Chagas disease, alternates between replicative and nonreplicative forms accompanied by a shift on global transcription levels and by changes in its chromatin architecture. Here, we investigated the T. cruzi chromatin proteome using three different protocols and compared it between replicative (epimastigote) and nonreplicative (trypomastigote) forms by high-resolution mass spectrometry. More than 2000 proteins were identified and quantified both in chromatin and nonchromatin extracts. Besides histones and other known nuclear proteins, trypanosomes chromatin also contains metabolic (mainly from carbohydrate pathwa)4, cytoskeleton and many other proteins with unknown functions. Strikingly, the two parasite forms differ greatly regarding their chromatin-associated factors composition and amount. Although the nucleosome content is the same for both life forms (as seen by MNase digestion), the remaining proteins were much less detected in nonreplicative forms, suggesting that they have a naked chromatin. Proteins associated to DNA proliferation, such as PCNA. RPA, and DNA topoisomerases were exclusively found in the chromatin of replicative stages. On the other hand, the nonreplicative stages have an enrichment of a histone H2B variant. Furthermore, almost 20% of replicative stages chromatin-associated proteins are expressed in nonreplicative forms, but located at nonchromatin space. We identified different classes of proteins including phosphatases and a Ran-binding protein, that may shuttle between chromatin and nonchromatin space during differentiation. Seven proteins, including those with unknown functions, were selected for further validation. We confirmed their location in chromatin and their differential expression, using Western blotting assays and chromatin immunoprecipitation (ChIP). Our results indicate that the replicative state in trypanosomes involves an increase of chromatin associated proteins content. We discuss in details, the qualitative and quantitative implication of this chromatin set in trypanosome chromatin biology. Because trypanosomes are early-branching organisms, this data can boost our understanding of chromatin-associated processes in other cell types. (AU)

Processo FAPESP: 11/22619-7 - Alterações nucleares e na cromatina ao longo do ciclo celular e senescência de células de mamíferos
Beneficiário:Julia Pinheiro Chagas da Cunha
Linha de fomento: Auxílio à Pesquisa - Apoio a Jovens Pesquisadores
Processo FAPESP: 13/07467-1 - CeTICS - Centro de Toxinas, Imuno-Resposta e Sinalização Celular
Beneficiário:Hugo Aguirre Armelin
Linha de fomento: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs