The arginine methyltransferase PRMT5 and PRMT1 distinctly regulate the degradation of anti-apoptotic protein CFLARL in human lung cancer cells
The arginine methyltransferase PRMT5 and PRMT1 distinctly regulate the degradation of anti-apoptotic protein CFLARL in human lung cancer cells
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精氨酸甲基转移酶PRMT5和PRMT1明显调节人肺癌细胞中抗凋亡蛋白CFLAR(L)的降解
DOI：
10.1186/s13046-019-1064-8
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发表时间：
2019-02-08
影响因子：
11.3
通讯作者：
Liu, Xiangguo
中科院分区：
文献类型：
--
作者：
Li, Mingyue;An, Wentao;Liu, Xiangguo
背景CFLAR（L）又称c-FLIPL，是一种重要的抗凋亡蛋白，能抑制哺乳动物细胞中caspase 8的活化。以往的研究表明CFLAR（L）的精氨酸122可以被单甲基化。然而，CFLAR（L）精氨酸甲基转移酶的确切作用仍不清楚。PRMT 5和PRMT 1是PRMT家族的重要成员，催化甲基转移到底物蛋白的精氨酸上。PRMT 5可单甲基化或对称二甲基化精氨酸残基，而PRMT 1可单甲基化或不对称二甲基化精氨酸残基。MethodsLung cancer cells were cultured after standard protocol and the cell lysings were prepared to detect the given proteins by Western Blot analysis，and the protein interaction was assessed by co-immunoprecipitation（Co-IP）or GST pull-down assay.采用蛋白酶体抑制剂处理结合HA-Ub转染和WB法检测CFLAR（L）泛素化水平。结果PRMT 5基因通过下调CFLAR（L）的泛素化水平，上调CFLAR（L）的蛋白水平，从而抑制CFLAR（L）的泛素化。此外，PRMT 1通过增加CFLAR（L）的泛素化和降解来下调CFLAR（L）的蛋白水平。PRMT 5的过表达可以抑制CFLAR（L）和ITCH之间的相互作用，其已被鉴定为CFLAR（L）的E3泛素连接酶，而过表达的PRMT 1增强CFLAR（L）和ITCH之间的相互作用。此外，我们验证了PRMT 5或PRMT 1的死亡突变对CFLAR（L）的影响与野生型突变相同，这表明CFLAR和PRMT 1/5之间的物理相互作用调节CFLAR（L）降解，而不是其酶活性。结论PRMT 5和PRMT 1通过调节E3连接酶ITCH的结合，对NSCLC细胞中CFLAR（L）的降解有不同的影响。本研究确定了一种细胞死亡机制，该机制由PRMT 1/5微调，调节人NSCLC细胞中CFLAR（L）降解。
BackgroundCFLAR(L), also known as c-FLIPL, is a critical anti-apoptotic protein that inhibits activation of caspase 8 in mammalian cells. Previous studies have shown that arginine 122 of CFLAR(L) can be mono-methylated. However, the precise role of arginine methyltransferase of CFLAR(L) remains unknown. PRMT5 and PRMT1, which are important members of the PRMT family, catalyze the transfer of methyl groups to the arginine of substrate proteins. PRMT5 can monomethylate or symmetrically dimethylate arginine residues, while PRMT1 can monomethylate or asymmetrically dimethylate arginine residues.MethodsLung cancer cells were cultured following the standard protocol and the cell lysates were prepared to detect the given proteins by Western Blot analysis, and the protein interaction was assayed by co-immunoprecipitation (Co-IP) or GST pull-down assay. CFLAR(L) ubiquitination level was evaluated by proteasomal inhibitor treatment combined with HA-Ub transfection and WB assay. PRMT1 and PRMT5 genes were knocked down by siRNA technique.ResultsWe show that PRMT5 up-regulated the protein levels of CFLAR(L) by decreasing the ubiquitination and increasing its protein level. Additionally, PRMT1 down-regulated the protein level of CFLAR(L) by increasing the ubiquitination and degradation. The overexpression of PRMT5 can inhibit the interaction between CFLAR(L) and ITCH, which has been identified as an E3 ubiquitin ligase of CFLAR(L), while overexpressed PRMT1 enhances the interaction between CFLAR(L) and ITCH. Furthermore, we verified that dead mutations of PRMT5 or PRMT1 have the same effects on CFLAR(L) as the wild-type ones have, suggesting it is the physical interaction between CFLAR and PRMT1/5 that regulates CFLAR(L) degradation other than its enzymatic activity. Finally, we showed that PRMT5 and PRMT1 could suppress or facilitate apoptosis induced by doxorubicin or pemetrexed by affecting CFLAR(L) in NSCLC cells.ConclusionsPRMT5 and PRMT1 mediate the distinct effects on CFLAR(L) degradation by regulating the binding of E3 ligase ITCH in NSCLC cells. This study identifies a cell death mechanism that is fine-tuned by PRMT1/5 that modulate CFLAR(L) degradation in human NSCLC cells.