Interaction Network of the 14-3-3 Protein in the Ancient Protozoan Parasite <i>Giardia duodenalis</i>

14-3-3s are phosphoserine/phosphotreonine binding proteins that play pivotal roles as regulators of multiple cellular processes in eukaryotes. The flagellated protozoan parasite Giardia duodenalis, the causing agent of giardiasis, is a valuable simplified eukaryotic model. A single 14-3-3 isoform (g14-3-3) is expressed in Giardia, and it is directly involved in the differentiation of the parasite into cyst. To define the overall functions of g14-3-3, the protein interactome has been investigated. A transgenic G. duodenalis strain was engineered to express a FLAG-tagged g14-3-3 under its own promoter. Affinity chromatography coupled with tandem mass spectrometry analysis have been used to purify and identify FLAG-g14-3-3-associated proteins from trophozoites and encysting parasites. A total of 314 putative g14-3-3 interaction partners were identified, including proteins involved in several pathways. Some interactions seemed to be peculiar of one specific stage, while others were shared among the different stages. Furthermore, the interaction of g14-3-3 with the giardial homologue of the CDC7 protein kinase (gCDC7) was characterized, leading to the identification of a multiprotein complex containing not only g14-3-3 and gCDC7 but also a newly identified and highly divergent homologue of DBF4, the putative regulatory subunit of gCDC7. The relevance of g14-3-3 interactions in G. duodenalis biology was discussed.14-3-3蛋白是一类磷酸丝氨酸/磷酸苏氨酸结合蛋白，在真核生物中作为多种细胞过程的调控因子发挥关键作用。具鞭毛的原生动物寄生虫十二指肠贾第虫（Giardia duodenalis）是贾第虫病的致病菌，同时也是极具价值的简化真核生物模型。该寄生虫仅表达一种14-3-3同工型（g14-3-3），且该蛋白直接参与寄生虫向包囊的分化过程。为明确g14-3-3的整体功能，研究人员对其蛋白质相互作用组（protein interactome）展开了探究。研究人员构建了转基因十二指肠贾第虫菌株，使其在自身启动子的调控下表达带有FLAG标签的g14-3-3。通过亲和色谱结合串联质谱（tandem mass spectrometry）分析，研究人员从滋养体和包囊形成期的寄生虫中纯化并鉴定了与FLAG-g14-3-3结合的蛋白。共计鉴定出314个潜在的g14-3-3互作伴侣蛋白，这些蛋白涉及多条信号通路。部分互作仅特异性存在于某一发育阶段，而另一些则在不同阶段间保守存在。此外，研究还对g14-3-3与CDC7蛋白激酶的贾第虫同源蛋白（gCDC7）的相互作用进行了表征，成功鉴定得到一个多蛋白复合物：该复合物不仅包含g14-3-3与gCDC7，还包含一个新鉴定的、高度趋异的DBF4同源蛋白——后者是gCDC7的潜在调控亚基。本文讨论了g14-3-3的互作蛋白在十二指肠贾第虫生物学中的相关意义。