利用酵母双杂交系统从人胚胎肝脏cDNA文库中筛选与JNKK2相互作用的蛋白
|
摘要
目的:利用酵母双杂交系统,以149位赖氨酸突变成甲硫氨酸的c-Jun N端激酶激酶2(JNKK2)失活突变体(JNKK2KM)为诱饵,从人胎肝文库中筛选能与JNKK2作用的蛋白。方法:构建诱饵质粒p GBKT7-JNKK2KM,将其转化到AH109感受态酵母菌中进行扩增,而后检测融合蛋白表达、自激活活性以及对宿主的毒性;将诱饵酵母菌与含有人胎肝c DNA文库质粒的Y187交配进行酵母双杂交筛选,经缺陷型培养基筛选排除假阳性克隆;取阳性菌落抽提质粒进行酶切鉴定,对鉴定后的阳性克隆进行测序及生物信息学分析,明确筛选出的蛋白信息,通过免疫共沉淀实验验证筛选出的蛋白与JNKK2的相互作用。结果:构建了诱饵质粒,检测到融合蛋白的表达,且无自激活作用,未发现对宿主存在毒性;初步筛选到120个阳性克隆,经多轮重复验证得到阳性克隆11个,经生物信息学分析最后确定3个可以与JNKK2相互作用的新蛋白质分子为鸟嘌呤核苷酸结合蛋白β多肽2样蛋白1(GNB2L1)、开放读框60(ORF60)与泛素样修饰物激活酶3(UBA3),免疫共沉淀实验表明它们均能与JNKK2发生相互作用。结论:筛选到3个新的JNKK2相互作用蛋白,为深入探究JNKK2在肝脏中的作用奠定了基础。
Objective: To investigate the biological function of c-Jun N-terminal kinase kinase 2(JNKK2) in liver, we used the inactivated JNKK2(K149 M) as a bait to screen interactive protein from human fetal liver c DNA library by yeast two-hybrid system. Methods: We constructed the bait plasmid p GBKT7-JNKK2 KM, and transformed it into AH109 yeast strains, followed by BD-JNKK2 KM fusion protein expression and transcriptional activation and toxicity test. A yeast two-hybrid screening was performed by mating AH109 with Y187 containing human fetal liver c DNA library plasmids. After elimination of false positive clones, the interaction between JNKK2 and proteins obtained from positive colonies were further confirmed by yeast two-hybrid screen again. Plasmids were extracted from positive colonies and sequenced, and analyzed by BLAST online. Finally, the specific interaction between positive proteins and JNKK2 were confirmed by co-immunoprecipitation assay. Results: The bait plasmid p GBKT7-JNKK2 KM was successfully constructed and transformed into yeast strain AH109, and this recombinant plasmid showed no self-activation and toxicity in host yeast. After repeating yeast two-hybrid screen, 11 positive colonies were selected and sequenced. Finally, three proteins(ORF60, GNB2 L1, UBA3) interacting with JNKK2 which confirmed by co-immunoprecipitation assay. Conclusion: Three proteins interacted with JNKK2 were successfully screened. Our work might be facilitated to elucidate the complicated mechanisms of JNKK2 in liver.
Objective: To investigate the biological function of c-Jun N-terminal kinase kinase 2(JNKK2) in liver, we used the inactivated JNKK2(K149 M) as a bait to screen interactive protein from human fetal liver c DNA library by yeast two-hybrid system. Methods: We constructed the bait plasmid p GBKT7-JNKK2 KM, and transformed it into AH109 yeast strains, followed by BD-JNKK2 KM fusion protein expression and transcriptional activation and toxicity test. A yeast two-hybrid screening was performed by mating AH109 with Y187 containing human fetal liver c DNA library plasmids. After elimination of false positive clones, the interaction between JNKK2 and proteins obtained from positive colonies were further confirmed by yeast two-hybrid screen again. Plasmids were extracted from positive colonies and sequenced, and analyzed by BLAST online. Finally, the specific interaction between positive proteins and JNKK2 were confirmed by co-immunoprecipitation assay. Results: The bait plasmid p GBKT7-JNKK2 KM was successfully constructed and transformed into yeast strain AH109, and this recombinant plasmid showed no self-activation and toxicity in host yeast. After repeating yeast two-hybrid screen, 11 positive colonies were selected and sequenced. Finally, three proteins(ORF60, GNB2 L1, UBA3) interacting with JNKK2 which confirmed by co-immunoprecipitation assay. Conclusion: Three proteins interacted with JNKK2 were successfully screened. Our work might be facilitated to elucidate the complicated mechanisms of JNKK2 in liver.
引文
[1]Haeusgen W,Herdegen T,Waetzig V.The bottleneck of JNK signaling:molecular and functional characteristics of MKK4 and MKK7[J].Eur J Cell Biol,2011,90(6-7):536-544.
[2]Davies C,Tournier C.Exploring the function of the JNK(c-Jun N-terminal kinase)signalling pathway in physiological and pathological processes to design novel therapeutic strategies[J].Biochem Soc Trans,2012,40(1):85-89.
[3]Guo Y,Wang W,Wang J,et al.Receptor for activated C kinase 1 promotes hepatocellular carcinoma growth by enhancing mitogen-activated protein kinase kinase 7 activity[J].Hepatology,2013,57(1):140-151.
[4]Wang J,Tang R,Lv M,et al.Defective anchoring of JNK1 in the cytoplasm by MKK7 in Jurkat cells is associated with resistance to Fas-mediated apoptosis[J].Mol Biol Cell,2011,22(1):117-127.
[5]Zhu T,Wang J,Pei Y,et al.Neddylation controls basal MKK7 kinase activity in breast cancer cells[J].Oncogene,2016,35(20):2624-2633.
[6]Tornatore L,Sandomenico A,Raimondo D,et al.Cancer-selective targeting of the NF-kappa B survival pathway with GADD45beta/MKK7 inhibitors[J].Cancer Cell,2014,26(4):495-508.
[7]Moosavi B,Mousavi B,Yang W C,et al.Yeastbased assays for detecting protein-protein/drug interactions and their inhibitors[J].Eur J Cell Biol,2017,96(6):529-541.
[8]Rajagopala S V.Mapping the protein-protein interactome networks using yeast two-hybrid screens[J].Adv Exp Med Biol,2015,883:187-214.
[9]Chien C T,Bartel P L,Sternglanz R,et al.The twohybrid system:a method to identify and clone genes for proteins that interact with a protein of interest[J].Proc Natl Acad Sci USA,1991;88(21):9578-9582.
[10]Saada A,Vogel R O,Hoefs SJ,et al.Mutations in NDUFAF3(C3ORF60),encoding an NDUFAF4(C6ORF66)-interacting complex I assembly protein,cause fatal neonatal mitochondrial disease[J].Am J Hum Genet,2009,84(6):718-727.
[11]Duff D,Long A.Roles for RACK1 in cancer cell migration and invasion[J].Cell Signal,2017,35:250-255.
[12]Li J J,Xie D.RACK1,a versatile hub in cancer[J].Oncogene,2015,34(15):1890-1898.
[13]Gandin V,Senft D,Topisirovic I,et al.RACK1 function in cell motility and protein synthesis[J].Genes Cancer,2013,4(9-10):369-377.
[14]Islas-Flores T,Rahman A,Ullah H,et al.The receptor for activated C kinase in plant signaling:tale of a promiscuous little molecule[J].Frontiers Plant Sci.2015,6:1090.
[15]Berns H,Humar R,Hengerer B,et al.RACK1 is upregulated in angiogenesis and human carcinomas[J].FASEB J,2000,14(15):2549-2558.
[16]Wang Z,Zhang B,Jiang L,et al.RACK1,an excellent predictor for poor clinical outcome in oral squamous carcinoma,similar to Ki67[J].Eur J Cancer.,2009,45(3):490-496.
[17]Al-Reefy S,Osman H,Jiang W,et al.Evidence for a pro-apoptotic function of RACK1 in human breast cancer[J].Oncogene,2010,29(41):5651.
[18]Nagashio R,Sato Y,Matsumoto T,et al.Expression of RACK1 is a novel biomarker in pulmonary adenocarcinomas[J].Lung Cancer,2010,69(1):54-59.
[19]Ruan Y,Sun L,Hao Y,et al.Ribosomal RACK1 promotes chemoresistance and growth in human hepatocellular carcinoma[J].J Clin Investig,2012,122(7):2554-2566.
[20]Gao Q,Yu G Y,Shi J Y,et al.Neddylation pathway is up-regulated in human intrahepatic cholangiocarcinoma and serves as a potential therapeutic target[J].Oncotarget,2014,5(17):7820-7832.
[21]Hua W,Li C,Yang Z,et al.Suppression of glioblastoma by targeting the overactivated protein neddylation pathway[J].Neuro Oncol,2015,17(10):1333-1343.
[22]Li L,Wang M,Yu G,et al.Overactivated neddylation pathway as a therapeutic target in lung cancer[J].J Natl Cancer Inst,2014,106(6):dju083.
[23]Soucy T A,Smith P G,Milhollen M A,et al.An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer[J].Nature,2009,458(7239):732-736.
[2]Davies C,Tournier C.Exploring the function of the JNK(c-Jun N-terminal kinase)signalling pathway in physiological and pathological processes to design novel therapeutic strategies[J].Biochem Soc Trans,2012,40(1):85-89.
[3]Guo Y,Wang W,Wang J,et al.Receptor for activated C kinase 1 promotes hepatocellular carcinoma growth by enhancing mitogen-activated protein kinase kinase 7 activity[J].Hepatology,2013,57(1):140-151.
[4]Wang J,Tang R,Lv M,et al.Defective anchoring of JNK1 in the cytoplasm by MKK7 in Jurkat cells is associated with resistance to Fas-mediated apoptosis[J].Mol Biol Cell,2011,22(1):117-127.
[5]Zhu T,Wang J,Pei Y,et al.Neddylation controls basal MKK7 kinase activity in breast cancer cells[J].Oncogene,2016,35(20):2624-2633.
[6]Tornatore L,Sandomenico A,Raimondo D,et al.Cancer-selective targeting of the NF-kappa B survival pathway with GADD45beta/MKK7 inhibitors[J].Cancer Cell,2014,26(4):495-508.
[7]Moosavi B,Mousavi B,Yang W C,et al.Yeastbased assays for detecting protein-protein/drug interactions and their inhibitors[J].Eur J Cell Biol,2017,96(6):529-541.
[8]Rajagopala S V.Mapping the protein-protein interactome networks using yeast two-hybrid screens[J].Adv Exp Med Biol,2015,883:187-214.
[9]Chien C T,Bartel P L,Sternglanz R,et al.The twohybrid system:a method to identify and clone genes for proteins that interact with a protein of interest[J].Proc Natl Acad Sci USA,1991;88(21):9578-9582.
[10]Saada A,Vogel R O,Hoefs SJ,et al.Mutations in NDUFAF3(C3ORF60),encoding an NDUFAF4(C6ORF66)-interacting complex I assembly protein,cause fatal neonatal mitochondrial disease[J].Am J Hum Genet,2009,84(6):718-727.
[11]Duff D,Long A.Roles for RACK1 in cancer cell migration and invasion[J].Cell Signal,2017,35:250-255.
[12]Li J J,Xie D.RACK1,a versatile hub in cancer[J].Oncogene,2015,34(15):1890-1898.
[13]Gandin V,Senft D,Topisirovic I,et al.RACK1 function in cell motility and protein synthesis[J].Genes Cancer,2013,4(9-10):369-377.
[14]Islas-Flores T,Rahman A,Ullah H,et al.The receptor for activated C kinase in plant signaling:tale of a promiscuous little molecule[J].Frontiers Plant Sci.2015,6:1090.
[15]Berns H,Humar R,Hengerer B,et al.RACK1 is upregulated in angiogenesis and human carcinomas[J].FASEB J,2000,14(15):2549-2558.
[16]Wang Z,Zhang B,Jiang L,et al.RACK1,an excellent predictor for poor clinical outcome in oral squamous carcinoma,similar to Ki67[J].Eur J Cancer.,2009,45(3):490-496.
[17]Al-Reefy S,Osman H,Jiang W,et al.Evidence for a pro-apoptotic function of RACK1 in human breast cancer[J].Oncogene,2010,29(41):5651.
[18]Nagashio R,Sato Y,Matsumoto T,et al.Expression of RACK1 is a novel biomarker in pulmonary adenocarcinomas[J].Lung Cancer,2010,69(1):54-59.
[19]Ruan Y,Sun L,Hao Y,et al.Ribosomal RACK1 promotes chemoresistance and growth in human hepatocellular carcinoma[J].J Clin Investig,2012,122(7):2554-2566.
[20]Gao Q,Yu G Y,Shi J Y,et al.Neddylation pathway is up-regulated in human intrahepatic cholangiocarcinoma and serves as a potential therapeutic target[J].Oncotarget,2014,5(17):7820-7832.
[21]Hua W,Li C,Yang Z,et al.Suppression of glioblastoma by targeting the overactivated protein neddylation pathway[J].Neuro Oncol,2015,17(10):1333-1343.
[22]Li L,Wang M,Yu G,et al.Overactivated neddylation pathway as a therapeutic target in lung cancer[J].J Natl Cancer Inst,2014,106(6):dju083.
[23]Soucy T A,Smith P G,Milhollen M A,et al.An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer[J].Nature,2009,458(7239):732-736.