Tsigankov Polina, Gherardini Pier Federico, Helmer-Citterich Manuela, Späth Gerald F, Myler Peter J, Zilberstein Dan
From the ‡Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel;
§Center for Molecular Bioinformatics, Department of Biology, University of Rome Tor Vergata, Rome, Italy;
Mol Cell Proteomics. 2014 Jul;13(7):1787-99. doi: 10.1074/mcp.M114.037705. Epub 2014 Apr 16.
Leishmania are obligatory intracellular parasitic protozoa that cause a wide range of diseases in humans, cycling between extracellular promastigotes in the mid-gut of sand flies and intracellular amastigotes in the phagolysosomes of mammalian macrophages. Although many of the molecular mechanisms of development inside macrophages remain a mystery, the development of a host-free system that simulates phagolysosome conditions (37 °C and pH 5.5) has provided new insights into these processes. The time course of promastigote-to-amastigote differentiation can be divided into four morphologically distinct phases: I, signal perception (0-5 h after exposure); II, movement cessation and aggregation (5-10 h); III, amastigote morphogenesis (10-24 h); and IV, maturation (24-120 h). Transcriptomic and proteomic analyses have indicated that differentiation is a coordinated process that results in adaptation to life inside phagolysosomes. Recent phosphoproteomic analysis revealed extensive differences in phosphorylation between promastigotes and amastigotes and identified stage-specific phosphorylation motifs. We hypothesized that the differentiation signal activates a phosphorylation pathway that initiates Leishmania transformation, and here we used isobaric tags for relative and absolute quantitation to interrogate the dynamics of changes in the phosphorylation profile during Leishmania donovani promastigote-to-amastigote differentiation. Analysis of 163 phosphopeptides (from 106 proteins) revealed six distinct kinetic profiles; with increases in phosphorylation predominated during phases I and III, whereas phases II and IV were characterized by greater dephosphorylation. Several proteins (including a protein kinase) were phosphorylated in phase I after exposure to the complete differentiation signal (i.e. signal-specific; 37 °C and pH 5.5), but not after either of the physical parameters separately. Several other protein kinases (including regulatory subunits) and phosphatases also showed changes in phosphorylation during differentiation. This work constitutes the first genome-scale interrogation of phosphorylation dynamics in a parasitic protozoa, revealing the outline of a signaling pathway during Leishmania differentiation. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (identifier PXD000671). Data can be viewed using ProteinPilot™ software.
利什曼原虫是专性细胞内寄生原生动物，可在人类中引发多种疾病，在白蛉中肠的细胞外前鞭毛体与哺乳动物巨噬细胞吞噬溶酶体中的细胞内无鞭毛体之间循环。尽管巨噬细胞内发育的许多分子机制仍是未解之谜，但模拟吞噬溶酶体条件（37℃和pH 5.5）的无宿主系统的开发为这些过程提供了新的见解。前鞭毛体向无鞭毛体分化的时间进程可分为四个形态上不同的阶段：I，信号感知（暴露后0 - 5小时）；II，运动停止和聚集（5 - 10小时）；III，无鞭毛体形态发生（10 - 24小时）；IV，成熟（24 - 120小时）。转录组学和蛋白质组学分析表明，分化是一个协调的过程，导致适应吞噬溶酶体内的生活。最近的磷酸化蛋白质组学分析揭示了前鞭毛体和无鞭毛体之间磷酸化的广泛差异，并鉴定了阶段特异性磷酸化基序。我们假设分化信号激活了一条磷酸化途径，启动了利什曼原虫的转化，在此我们使用等压标签进行相对和绝对定量，以探究杜氏利什曼原虫前鞭毛体向无鞭毛体分化过程中磷酸化谱变化的动力学。对163个磷酸肽（来自106种蛋白质）的分析揭示了六种不同的动力学谱；磷酸化增加在阶段I和III占主导，而阶段II和IV的特征是去磷酸化程度更高。几种蛋白质（包括一种蛋白激酶）在暴露于完整分化信号（即信号特异性；37℃和pH 5.5）后的阶段I被磷酸化，但在单独的任何一个物理参数作用后未被磷酸化。其他几种蛋白激酶（包括调节亚基）和磷酸酶在分化过程中也显示出磷酸化的变化。这项工作构成了对寄生原生动物磷酸化动力学的首次全基因组规模探究，揭示了利什曼原虫分化过程中一条信号通路的轮廓。质谱蛋白质组学数据已存入蛋白质组交换联盟（标识符PXD000671）。数据可使用ProteinPilot™软件查看。
