基于酵母双杂交技术的PRR11相互作用蛋白筛选与初步分析
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摘要
目的:采用酵母双杂交技术,筛选PRR11的相互作用蛋白,为深入研究其分子行为机制奠定基础。方法:构建用于酵母双杂交筛选的PRR11诱饵质粒并检测其自激活作用,根据结果进一步构建突变体获得具有较低自激活作用的突变体。对人胎脑cDNA文库进行筛选,获得初始阳性克隆。通过LacZ报告基因检测、回转验证、阳性克隆测序及BLAST序列比对分析,排除假阳性克隆和重复克隆,确定PRR11的候选相互作用蛋白。结果:PRR11具有自激活作用,构建了一个较低自激活作用的PRR11突变体。通过酵母双杂交筛选,获得102个初始阳性克隆转化子。经LacZ报告基因检测,将阳性克隆锁定至39个。通过测序和序列比对分析,证实这39个克隆属于编码15种不同蛋白的基因。最终有RNF41等4个蛋白通过回转验证。结论:RNF41、SCG5、BCYRN1和PRR13是PRR11的潜在相互作用蛋白。
Objective:To screen PRR11 interacting proteins by the yeast two-hybrid technique,and to provide a basis for further studies of its molecular mechanism of action. Methods:PRR11 bait plasmids were made for the yeast two-hybrid system and tested for auto-activation. Mutants were constructed to obtain lower auto-activation. The human fetal brain cDNA library was screened to obtain initial positive clones. The lacZ reporter gene assay,retransformation,positive clone sequencing,and BLAST sequence alignment analysis were used to rule out false positive and repeated clones and identify candidate PRR11 interacting proteins. Results:PRR11 showed auto-activation and a PRR11 mutant with lower auto-activation was constructed. The yeast two-hybrid screening resulted in102 initial positive clones. The lacZ reporter gene assay further validated 39 positive clones. According to gene sequencing and sequence alignment analysis,the 39 clones were from genes encoding 15 different proteins. Finally,four candidate proteins including RNF41 were verified by retransformation. Conclusion:RNF41,SCG5,BCYRN1,and PRR13 are potential PRR11 interacting proteins.
Objective:To screen PRR11 interacting proteins by the yeast two-hybrid technique,and to provide a basis for further studies of its molecular mechanism of action. Methods:PRR11 bait plasmids were made for the yeast two-hybrid system and tested for auto-activation. Mutants were constructed to obtain lower auto-activation. The human fetal brain cDNA library was screened to obtain initial positive clones. The lacZ reporter gene assay,retransformation,positive clone sequencing,and BLAST sequence alignment analysis were used to rule out false positive and repeated clones and identify candidate PRR11 interacting proteins. Results:PRR11 showed auto-activation and a PRR11 mutant with lower auto-activation was constructed. The yeast two-hybrid screening resulted in102 initial positive clones. The lacZ reporter gene assay further validated 39 positive clones. According to gene sequencing and sequence alignment analysis,the 39 clones were from genes encoding 15 different proteins. Finally,four candidate proteins including RNF41 were verified by retransformation. Conclusion:RNF41,SCG5,BCYRN1,and PRR13 are potential PRR11 interacting proteins.
引文
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[15] Wauman J,De Ceuninck L,Vanderroost N,et al. RNF41(Nrdp1)controls type 1 cytokine receptor degradation and ectodomain shedding[J]. J Cell Sci,2011,124(Pt 6):921-932.
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[17] Waha A,Felsberg J,Hartmann W,et al. Frequent epigenetic inactivation of the chaperone SGNE1/7B2 in human gliomas[J]. Int J Cancer,2012,131(3):612-622.
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[19] Samson J,Cronin S,Dean K. BC200(BCYRN1)--the shortest,long,non-coding RNA associated with cancer[J]. Noncoding RNA Res,2018,22(3):131-143.
[2] Zhang C,Zhang Y,Li Y,et al. PRR11 regulates late-S to G2/M phase progression and induces premature chromatin condensation(PCC)[J]. Biochem Biophys Res Commun,2015,458(3):501-508.
[3] Wang Y,Zhang Y,Zhang C,et al. The gene pair PRR11 and SKA2shares a NF-Y-regulated bidirectional promoter and contributes to lung cancer development[J]. Biochim Biophys Acta,2015,1849(9):1133-1144.
[4] Zhang L,Lei Y,Zhang Y,et al. Silencing of PRR11 suppresses cell proliferation and induces autophagy in NSCLC cells[J]. Genes&Diseases,2017,5(2):158-166.
[5] Qiao W,Wang HY,Zhang XZ,et al. Proline-rich protein 11 silencing inhibits hepatocellular carcinoma growth and epithelial-mesenchymal transition throughβ-catenin signaling[J]. Gene,2019,681(10):7-14.
[6] Ying C,Zhanshan C,Wenzheng F,et al. The prognostic potential and oncogenic effects of PRR11 expression in hilar cholangiocarcinoma[J]. Oncotarget,2015,6(24):20419-20433.
[7] Song Z,Liu W,Xiao Y,et al. PRR11 is a prognostic marker and potential oncogene in patients with gastric cancer[J]. PLoS One,2015,10(8):e0128943.
[8] Valkonen J. Protein-protein interactions:the yeast two-hybrid system[J]. Methods in Molecular Biology,2008,451(451):421.
[9] Chien CT,Bartel PL,Sternglanz R,et al. The two-hybrid system:a method to identify and clone genes for proteins that interact with a protein of interest[J]. Proceedings of the National Academy of Sciences,1991,88(21):9578-9582.
[10] Ball LJ,Kuehne R,Schneider-Mergener J,et al. Recognition of proline-rich motifs by protein-protein-interaction domains[J]. Angew Chem Int Ed Engl,2005,44(19):2852-2869.
[11] Kay BK,Williamson MP,Sudol M. The importance of being proline:the interaction of proline-rich motifs in signaling proteins with their cognate domains[J]. Faseb,2000,14(2):231-241.
[12] Yen L,Cao Z,Wu X,et al. Loss of Nrdp1 Enhances ErbB2/ErbB3-dependent breast tumor cell growth[J]. Cancer Research,2006,66(23):11279-11286.
[13] Zhang Y,Zeng Y,Wang M,et al. Cardiac-specific overexpression of E3 ligase Nrdp1 increases ischemia and reperfusion-induced cardiac injury[J]. Basic Research in Cardiology,2011,106(3):371-383.
[14] Wang C,Chen T,Zhang J,et al. The E3 ubiquitin ligase Nrdp1‘preferentially’promotes TLR-mediated production of type I interferon[J]. Nat Immunol,2009,10(7):744-752.
[15] Wauman J,De Ceuninck L,Vanderroost N,et al. RNF41(Nrdp1)controls type 1 cytokine receptor degradation and ectodomain shedding[J]. J Cell Sci,2011,124(Pt 6):921-932.
[16] Shi H,Du J,Wang L,et al. Lower expression of Nrdp1 in human glioma contributes tumor progression by reducing apoptosis[J]. IUBMB Life,2014,66(10):704-710.
[17] Waha A,Felsberg J,Hartmann W,et al. Frequent epigenetic inactivation of the chaperone SGNE1/7B2 in human gliomas[J]. Int J Cancer,2012,131(3):612-622.
[18] Lih CJ,Wei W,Cohen SN. Txr1:a transcriptional regulator of thrombospondin-1 that modulates cellular sensitivity to taxanes[J].Genes Dev,2006,20(15):2082-2095.
[19] Samson J,Cronin S,Dean K. BC200(BCYRN1)--the shortest,long,non-coding RNA associated with cancer[J]. Noncoding RNA Res,2018,22(3):131-143.