背景:环纹斜纹虫可感染反刍动物的单核细胞/巨噬细胞和B淋巴细胞,并引起严重的淋巴增殖性疾病。同时,环状锥虫感染通过调节宿主细胞的信号通路导致细胞群的永久增殖。半胱氨酸蛋白酶(CPs)是一种蛋白质水解酶,通常在寄生虫的毒力中起关键作用,宿主入侵,营养和宿主免疫反应。然而,环状T.nulataCP(TaCP)的生物学功能尚不清楚。在这项研究中,进行酵母双杂交试验以筛选与TaCP相互作用的宿主蛋白,提供信息,以帮助我们理解T.annulata和宿主细胞之间的分子机制。
方法:将纯化的牛B细胞的cDNA插入pGADT7-SfiI载体(pGADT7-SfiI-BcDNA,猎物质粒),用于构建酵母双杂交cDNA文库。将TaCP克隆到pGBKT7载体(pGBKT7-TaCP)中,并在评估表达后被视为诱饵质粒,酵母菌株Y2HGold的自动激活和毒性试验。通过将诱饵和猎物质粒共转化到酵母菌株Y2HGold中进行酵母双杂交筛选。使用BLAST分析了阳性猎物的序列,基因本体论,UniProt和STRING。
结果:两种宿主蛋白,CRBN(Bostauruscereblon转录变体X2)和Ppp4C(Bosindicus蛋白磷酸酶4催化亚基)被鉴定为与TaCP相互作用。功能分析结果表明,这两种蛋白质参与了许多细胞过程,如泛素化调节,微管组织,DNA修复,细胞凋亡和剪接体snRNPs的成熟。
结论:本研究首次筛选牛B细胞与TaCP相互作用的宿主蛋白,和2种蛋白质,CRBN和Ppp4C,使用酵母双杂交技术进行鉴定。功能分析的结果表明,这两种蛋白质参与了许多细胞过程,如泛素化调节,微管组织,DNA修复,细胞凋亡和剪接体snRNPs的成熟。与CRBN和Ppp4C的相互作用表明TaCP可能参与调节信号通路和细胞增殖,这有助于理解T.annulata与宿主细胞之间的相互作用。
BACKGROUND: Theileria annulata can infect monocytes/macrophages and B lymphocytes and causes severe lymphoproliferative disease in ruminants. Meanwhile, infection by T. annulata leads to the permanent proliferation of cell population through regulating signaling pathways of host cells. Cysteine proteinases (CPs) are one kind of protein hydrolase and usually play critical roles in parasite virulence, host invasion, nutrition and host immune response. However, the biological function of T. annulata CP (TaCP) is still unclear. In this study, a yeast-two-hybrid assay was performed to screen host proteins interacting with TaCP, to provide information to help our understanding of the molecular mechanisms between T. annulata and host cells.
METHODS: The cDNA from purified bovine B cells was inserted into pGADT7-SfiI vector (pGADT7-SfiI-BcDNA, Prey plasmid) for constructing the yeast two-hybrid cDNA library. TaCP was cloned into the pGBKT7 vector (pGBKT7-TaCP) and was considered as bait plasmid after evaluating the expression, auto-activation and toxicity tests in the yeast strain Y2HGold. The yeast two-hybrid screening was carried out via co-transforming bait and prey plasmids into yeast strain Y2HGold. Sequences of positive preys were analyzed using BLAST, Gene Ontology, UniProt and STRING.
RESULTS: Two host proteins, CRBN (Bos taurus cereblon transcript variant X2) and
Ppp4C (Bos indicus protein phosphatase 4 catalytic subunit) were identified to interact with TaCP. The results of functional analysis showed that the two proteins were involved in many cellular processes, such as ubiquitylation regulation, microtubule organization, DNA repair, cell apoptosis and maturation of spliceosomal snRNPs.
CONCLUSIONS: This study is the first to screen the host proteins of bovine B cells interacting with TaCP, and 2 proteins, CRBN and
Ppp4C, were identified using yeast two-hybrid technique. The results of functional analysis suggest that the two proteins are involved in many cellular processes, such as ubiquitylation regulating, microtubule organization, DNA repair, cell apoptosis and maturation of spliceosomal snRNPs. The interaction with CRBN and
Ppp4C indicate that TaCP possibly is involved in regulating signaling pathways and cell proliferation, which is helpful for understanding the interaction between T. annulata and host cells.