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首页> 外文期刊>Molecular & cellular proteomics: MCP >Quantitative Proteomic Analysis of Replicative and Nonreplicative Forms Reveals Important Insights into Chromatin Biology of Trypanosoma cruzi
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Quantitative Proteomic Analysis of Replicative and Nonreplicative Forms Reveals Important Insights into Chromatin Biology of Trypanosoma cruzi

机译：复制和非重种形式的定量蛋白质组学分析揭示了睾丸瘤Cruzi染色质生物学的重要见解

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摘要

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.
机译：染色质相关蛋白质是细胞中许多重要过程的关键调节因子。触发器组Cruzi，一种原因导致Chagas疾病的原生动物鞭毛，复制和非重种形式之间的交替伴随着全局转录水平的转变和染色质架构的变化。在这里，我们使用三种不同的方案研究了T.Cruzi染色质蛋白质组，并通过高分辨率质谱比较了复制（癫痫术）和非重种（胰蛋白酶术）形式。在染色质和非溴磷脂酶中鉴定出超过2000个蛋白质并定量蛋白质。除了组蛋白和其他已知的核蛋白外，睾丸形式染色质还含有代谢（主要来自碳水化合物途径）4，细胞骨架和许多具有未知功能的其他蛋白质。引人注目的是，两种寄生虫形式有很大差异，差异是它们的染色质相关因素组成和量。虽然核心含量与寿命形式相同（如MNASE消化所见），但剩余的蛋白质以非更换形式较少检测到，表明它们具有裸露的染色质。与DNA增殖相关的蛋白质，如PCNA。 RPA和DNA拓扑异构酶专门在复制阶段的染色质中发现。另一方面，非手术阶段具有富集组蛋白H2B变体。此外，几乎20％的复制阶段染色质相关蛋白质以非连续形式表达，但位于非溴酸素空间。我们鉴定了不同类别的蛋白质，包括磷酸酶和具有慢性结合蛋白的蛋白质，其可以在分化期间染色质和非溴脲空间之间穿梭。选择七种蛋白质，包括具有未知功能的蛋白质进行进一步验证。我们使用蛋白质印迹测定和染色质免疫沉淀（芯片）确认了它们在染色质谱和差异表达中的位置。我们的结果表明，锥虫的复制态涉及染色质相关蛋白质含量的增加。我们详细讨论了该染色质染色质染色质生物蛋白酶中该染色质的定性和定量含义。因为锥虫是早期分支的生物，所以这种数据可以提高我们对其他细胞类型的染色质相关过程的理解。

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