环生Theileriaarnulata是一种通过tick传播的尖丛寄生虫,在寄生真核生物中具有独特的转化宿主细胞的能力,在牛身上诱发致命的癌症样疾病。了解宿主细胞和恶性Theileria物种之间的机制相互作用,从而驱动这种转化,需要鉴定负责任的寄生虫效应蛋白。在这项研究中,我们使用了基于TurboID的邻近度标签,它毫不偏倚地鉴定了宿主细胞区室中分泌的寄生虫蛋白。通过将TurboID融合到核出口或本地化信号中,我们在受感染的巨噬细胞的细胞核或细胞质中的连接酶附近的生物素化蛋白质,其次是质谱分析。我们的方法以高置信度揭示了宿主细胞区室中的9个核和4个胞质候选寄生虫蛋白,其中8个在非转化的东方毛虫中没有直系同源物。引人注目的是,所有这八种蛋白质都被预测为高度内在无序的蛋白质。我们发现了一个新的串联排列蛋白家族,核内在无序蛋白质(NIDP)1-4,具有保守蛋白质结构域预测的多种功能。特别是,NIDP2表现出双相宿主细胞周期依赖性定位,与裂殖体表面的EB1/CD2AP/CLASP1寄生虫膜复合物和肿瘤抑制基质抗原2(STAG2)相互作用,一个内聚复杂亚基,在宿主核中。除STAG2外,还鉴定了许多与多种癌症有关的NIDP2相关宿主核蛋白,阐明了环状锥虫输出蛋白家族NIDP在宿主细胞转化和癌症相关途径中的潜在作用。IMPORTANCETurboID邻近标记用于鉴定环状Theileria的分泌蛋白,一种负责致命疾病的牙尖丛寄生虫,牛的增殖性疾病代表了北非的重大社会经济负担,中亚,和印度。我们的调查为独特的宿主-寄生虫相互作用提供了重要的见解,揭示了分泌的寄生虫蛋白,其特征是内在无序的蛋白质结构。值得注意的是,这些蛋白质在非转化的Theileria物种中明显不存在,强烈暗示它们在宿主细胞内的转化过程中的核心作用。我们的研究发现了一个新的串联排列的蛋白质家族,核本质上无序的蛋白质2成为与已建立的肿瘤基因相互作用的中心参与者。重要的是,这项工作代表了对Theileria中出口蛋白质的首次无偏见筛查,并为了解免疫细胞恶性转化背后的分子复杂性提供了必要的见解。
Theileria annulata is a tick-transmitted apicomplexan parasite that gained the unique ability among parasitic eukaryotes to transform its host cell, inducing a fatal cancer-like disease in cattle. Understanding the mechanistic interplay between the host cell and malignant Theileria species that drives this transformation requires the identification of responsible parasite effector proteins. In this study, we used TurboID-based proximity labeling, which unbiasedly identified secreted parasite proteins within host cell compartments. By fusing TurboID to nuclear export or localization signals, we biotinylated proteins in the vicinity of the ligase enzyme in the nucleus or cytoplasm of infected macrophages, followed by mass spectrometry analysis. Our approach revealed with high confidence nine nuclear and four cytosolic candidate parasite proteins within the host cell compartments, eight of which had no orthologs in non-transforming T. orientalis. Strikingly, all eight of these proteins are predicted to be highly intrinsically disordered proteins. We discovered a novel tandem arrayed protein family, nuclear intrinsically disordered proteins (NIDP) 1-4, featuring diverse functions predicted by conserved protein domains. Particularly, NIDP2 exhibited a biphasic host cell-cycle-dependent localization, interacting with the EB1/CD2AP/CLASP1 parasite membrane complex at the schizont surface and the tumor suppressor stromal antigen 2 (STAG2), a cohesion complex subunit, in the host nucleus. In addition to STAG2, numerous NIDP2-associated host nuclear proteins implicated in various cancers were identified, shedding light on the potential role of the T. annulata exported protein family NIDP in host cell transformation and cancer-related pathways.IMPORTANCETurboID proximity labeling was used to identify secreted proteins of Theileria annulata, an apicomplexan parasite responsible for a fatal, proliferative disorder in cattle that represents a significant socio-economic burden in North Africa, central Asia, and India. Our investigation has provided important insights into the unique host-parasite interaction, revealing secreted parasite proteins characterized by intrinsically disordered protein structures. Remarkably, these proteins are conspicuously absent in non-transforming Theileria species, strongly suggesting their central role in the transformative processes within host cells. Our study identified a novel tandem arrayed protein family, with nuclear intrinsically disordered protein 2 emerging as a central player interacting with established tumor genes. Significantly, this work represents the first unbiased screening for exported proteins in Theileria and contributes essential insights into the molecular intricacies behind the malignant transformation of immune cells.