Cloning and functional analysis of PKZ (PKR-like) from grass carp (Ctenopharyngodon idellus).
Cloning and functional analysis of PKZ (PKR-like) from grass carp (Ctenopharyngodon idellus).
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DOI：
10.1016/j.fsi.2011.10.012
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发表时间：
2011-12
影响因子：
4.7
通讯作者：
Peng-Jie Yang;Chu-Xin Wu;Wen Li;Lihua Fan;Gang Lin;Chengyu Hu
中科院分区：
文献类型：
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作者：
Peng-Jie Yang;Chu-Xin Wu;Wen Li;Lihua Fan;Gang Lin;Chengyu Hu
从草鱼（Ctenopharyngodon idellus）中克隆并鉴定了一个新的硬骨鱼PKZ（PKR-like）全长cDNA（GU 299765）。草鱼PKZ（CiPKZ）cDNA全长2185 bp，最大开放阅读框（ORF）编码513个氨基酸。CiPKZ具有eIF 2 α激酶家族保守的C-末端催化结构域。N端有两个结合结构域Zα，分别命名为Zα1（1- 67 aa）和Zα2（81- 152 aa）。BLAST同源性搜索显示，CiPKZ与其他鱼类PKZ和PKR具有高度同源性。与其他鱼类PKZ和PKR一样，CiPKZ是普遍存在的组织表达基因，其具有非常低的组成型表达水平，但响应于Poly I：C或热应激（34 °C）而上调。为了研究CiPKZ的潜在功能，我们通过大肠杆菌Rosetta原核表达获得CiPKZ多肽，并通过Ni-NTA His-Bind Resin亲和层析纯化。CiPKZ多肽用于体外磷酸化eIF 2 α的测试。结果表明，CiPKZ能被Z-DNA激活，而不能被Poly I：C激活，并能使eIF 2 α磷酸化。同时构建了pcDNA3.1/PKZ-wet、pcDNA3.1/PKZ-wet-K198 R、pcDNA3.1/PKZ-wet-C、pcDNA3.1/PKZ-wet-C-K198 R四种重组质粒。将pcDNA3.1/PKZ重组质粒和PGL-3启动子质粒瞬时共转染小鼠骨髓瘤细胞（Sp2/0）和人脐静脉内皮细胞（HUVEC）。结果显示，CiPKZ可显著降低这些细胞中的荧光素酶水平。Zα和K198氨基酸残基可能在其功能中起关键作用。
The new teleost fish PKZ (PKR-like) full-length cDNA (GU299765) had been cloned and identified from grass carp (Ctenopharyngodon idellus). The cDNA of grass carp PKZ (CiPKZ) has 2185 bp in length with a largest open reading frame (ORF) encoding 513aa. CiPKZ possesses a conserved C-terminal catalytic domain of eIF2α kinase family. Within its N-terminal there are two binding domain (Zα) named Zα1 (1–67aa) and Zα2 (81–152aa). BLAST homologous search reveals that CiPKZ has a high-level homology with other fish PKZs and PKRs. Like other fish PKZs and PKRs, CiPKZ is a ubiquitous tissue expression gene that had a very low level of constitutive expression but up-regulated in response to Poly I:C or hot stress (34 °C). For the purpose of searching for the potential function of CiPKZ, we obtained CiPKZ polypeptide via Escherichia coli Rosetta prokaryotic expression and purified with Ni-NTA His-Bind Resin affinity chromatography. CiPKZ polypeptide was used for the test of phosphorylating eIF2αin vitro. The results demonstrated that CiPKZ could be activated by Z-DNA but not by Poly I:C, and with subsequent could phosphorylate eIF2α. Meanwhile, four pcDNA3.1/PKZ recombinant plasmids, including pcDNA3.1/PKZ-wet, pcDNA3.1/PKZ-wet-K198R, pcDNA3.1/PKZ-wet-C, pcDNA3.1/PKZ-wet-C-K198R had been constructed, respectively. Mouse Myeloma cells (Sp2/0) and Human Umbilical Vein Endothelial Cells (HUVEC) were transiently cotransfected with pcDNA3.1/PKZ recombinant plasmid and PGL-3-promoter plasmid. The results revealed that CiPKZ could greatly decrease luciferase level in these cells. Zα and the K198 amino acid residue may play a key role in its function.