Identification and function analysis of the three dsRBMs in the N terminal dsRBD of grass carp (Ctenopharyngodon idella) PKR
Identification and function analysis of the three dsRBMs in the N terminal dsRBD of grass carp (Ctenopharyngodon idella) PKR
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草鱼PKR N端dsRBD中3个dsRBM的鉴定及功能分析
DOI：
10.1016/j.fsi.2016.01.011
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发表时间：
2016
影响因子：
4.7
通讯作者：
Chengyu Hu
中科院分区：
文献类型：
--
作者：
Yousheng Hu;Lihua Fan;Chuxin Wu;Binhua Wang;Zhicheng Sun;Chengyu Hu
蛋白激酶R(PKR)在病毒入侵等多种应激条件下，可抑制蛋白质翻译，诱导细胞凋亡。PKR是一种具有dsRBD和Kd结构域的dsRNA结合蛋白。DsRBD主要由两个(哺乳动物PKR)或三个(某些鱼类PKR)dsRNA结合基序(DsRBM)组成。多序列比对和系统发育分析表明，鱼类PKR的3个dsRBM具有相似的结构，但显示为发散起源。本研究对本实验室克隆的草鱼(Ctenophyngodon Idellus)PKR(CiPKR)的三个dsRBM进行了鉴定和分析。CiPKR的dsRBM有两个或三个参与dsRNA结合的保守区。在3个dsRBM中，dsRBM1是某些鱼类PKR所特有的，而dsRBM2和dsRBM3分别与哺乳动物PKR的dsRBM1和dsRBM2密切相关。二聚化分析表明，dsRBM1和dsRBM2不仅形成均二聚体，还形成均二聚体，而dsRBM3只形成均二聚体。同时，dsRBM1-2、dsRBM2-3和dsRBM1-2-3也能发生同源二聚和同源多聚体。Poly I：C下拉实验表明，在体外，dsRBM与Poly I：C的结合需要两到三个dsRBM的协同作用，这意味着来自CiPKR的一个dsRBM不能有效地与dsRNA结合。为了进一步研究dsRBM对CiPKR功能的影响，我们构建了pcDNA3.1/CiPKR-wt和一系列突变体重组质粒，包括pcDNA3.1/ciPKR-ΔdsRBM2-3、pcDNA3.1/ciPKR-ΔdsRBM1、pcDNA3.1/ciPKR-ΔdsRBM1-2、pcDNA3.1/ciPKR-ΔdsRBM3、pcDNA3.1/ciPKR-ΔdsRBM1。将重组载体分别与pGL3启动子共转染CIK细胞。与对照组相比，荧光素酶翻译抑制率分别为78.7%、15%、0、0.5%、61.8%、67.3%。结果表明，只含有1个dsRBM的CiPKR突变体对蛋白质翻译的抑制作用很弱，而含有2个或3个dsRBM的CiPKR突变体对蛋白质翻译的抑制作用很强。细胞存活率依次为34.2%、98.2%、112%、108%、50.3%、47.5%。细胞计数结果表明，pcDNA3.1/CiPKR-ΔdsRBM2-3、pcDNA3.1/CiPKR-ΔdsRBM1、pcDNA3.1/CiPKR-ΔdsRBM1-2、pcDNA3.1/CiPKR-ΔdsRBM3、pcDNA3.1/CiPKR-ΔdsRBM1均能抑制CIK细胞的蛋白质翻译，促进细胞数量的减少。综上所述，我们的观察结果表明，N端的两个dsRBM对CiPKR的功能是必不可少的，额外的dsRBM1的存在增强了它的功能。
The protein kinase R (PKR) can inhibit protein translation and lead to apoptosis under the circumstances of virus invasion and multiple other stress conditions. PKR is a dsRNA binding protein with a dsRBD and a kinase domain (KD). dsRBD is mostly composed of two (in mammal PKR) or three (in some fish PKR) dsRNA binding motifs (dsRBMs). Multiple sequences alignment and Phylogenetic analysis indicate that the three dsRBMs of fish PKR share analogous structure but show to be divergence origination. In this study, we have identified and analyzed the three dsRBMs from grass carp (Ctenopharyngodon idellus) PKR (CiPKR), which was cloned previously in our laboratory. dsRBMs of CiPKR have two or three conserved regions involved in dsRNA binding. Among the three dsRBMs, dsRBM1 was peculiar to some fish PKRs, while dsRBM2 and dsRBM3 were closely related to the dsRBM1 and dsRBM2 of mammal PKRs respectively. Dimerization assay indicated that dsRBM1 and dsRBM2 formed not only homo-dimer but also homo-multimer; whereas dsRBM3 formed merely homo-dimer. Meanwhile, dsRBM1-2, dsRBM2-3 and dsRBM1-2-3 could homo-dimerize and homo-multimerize also. Poly I:C pull-down assay showed that the binding of dsRBM to Poly I:C needed two or three dsRBMs to cooperate in vitro, meaning one dsRBM from CiPKR could not bind to dsRNA efficiently. To further investigate the effect of dsRBM on the function of CiPKR, we constructed pcDNA3.1/CiPKR-wt and a series of CiPKR mutants recombined plasmids including pcDNA3.1/CiPKR-ΔdsRBM2-3, pcDNA3.1/CiPKR-ΔdsRBM1,3, pcDNA3.1/CiPKR-ΔdsRBM1-2, pcDNA3.1/CiPKR-ΔdsRBM3, pcDNA3.1/CiPKR-ΔdsRBM1. The recombined plasmids respectively were co-transfected with plasmid PGL3 promoter into CIK cells. In comparison with the control group, the luciferase translation inhibitions were 78.7%, 15%, 0, 0.5%, 61.8%, 67.3% respectively. The results indicated that the protein translation inhibition caused by CiPKR mutants with only one dsRBM were very weak, while those with two or three dsRBMs inhibited the protein translation powerfully. Cell viability were 34.2%, 98.2%, 112%, 108%, 50.3%, 47.5% respectively after transfected with pcDNA3.1/CiPKR-wt, pcDNA3.1/CiPKR-ΔdsRBM2-3, pcDNA3.1/CiPKR-ΔdsRBM1,3, pcDNA3.1/CiPKR-ΔdsRBM1-2, pcDNA3.1/CiPKR-ΔdsRBM3, pcDNA3.1/CiPKR-ΔdsRBM1 in order into CIK cells for 48 h. The results from cell counting also indicated that transfection of CiPKR-wt and the mutants CiPKR-ΔdsRBM3, CiPKR-ΔdsRBM1 could inhibit the protein translation and facilitated the decrease of CIK cells number. In conclusion, our observations suggested that two dsRBMs ranking in tandem at N terminal were essential for the function of CiPKR, and the presence of the extra dsRBM1 enhanced its function.