摘要
在部分朊病毒病(priondiseases)中,高度磷酸化的微管相关蛋白tau与朊蛋白(prionprotein,PrP)发生共定位,tau蛋白可能在朊病毒病的病理机制中有重要作用.本室已经证明二者可以发生分子间相互作用,本文进一步分析了tau蛋白与prion的体外相互作用及作用位点.利用RTPCR方法从人源细胞系SHSY5YcDNA中扩增出微管相关蛋白tau全长cDNA序列,克隆至质粒pGEX2T载体,在大肠杆菌中诱导表达融合蛋白GSTtau.利用GSTpulldown及免疫共沉淀方法检测全长tau蛋白与PrP23231的分子间相互作用.进一步表达tau蛋白的各种缺失突变体,确定tau蛋白与PrP蛋白的相互作用位点.结果表明,所表达的全长tau蛋白及各种缺失突变体均为可溶性蛋白,Western印迹结果显示,各种蛋白均能很好的被tau蛋白单抗识别.GSTpulldown和免疫共沉淀实验均显示,原核表达的全长tau蛋白可与全长的PrP蛋白在体外发生相互作用,并确定相互作用位点位于tau蛋白的N端序列及中段的重复区.上述结果为研究tau蛋白与PrP的相互作用在朊病毒病的发病机制中的意义提供了一定的理论基础.
Co-deposition of hyperphosphorylated tau （p-tau） and prion amyloid was detected in the brain tissues of patients in certain prion diseases. Therefore tan protein may play roles in pathological mechanism in prion diseases. Our previous study has proved the molecular interaction between prion protein （PrP） and tan protein. In this study, we further analyzed the in vitro interaction between PrP and tau protein and identified the binding region on tau with PrP. The full-length cDNA sequence encoding human microtubule-associated protein tan was amplified with RT-PCR using total RNA from the cell line SH-SY5Y as the template, and inserted into a prokaryotic expressing vector pGEX-2T. GST-tau fusion protein was expressed in E. coli. The possible molecular interaction between full-length tan protein and PrP23-231 was tested by GST pull-down and immunoprecipitation assay. To map the binding domain within tau protein with PrP, various truncated tau segments were individually expressed. Protein analyses revealed that all GST-tau fusion proteins were expressed in soluble form and were specifically recognized by tau specific monoclonal antibody with Western blotting. Remarkable molecular interactions between tan and PrP were observed both in GST pull-down and immunoprecipitation assays. Subsequently, the binding regions for PrP within tau peptide were mapped in its N-terminus and the microtubule-binding domain in the middle portion. The results provide useful evidences for further studies of potential role of interaction between tau protein and PrP in pathogenicity of prion disease.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2006年第5期409-414,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金重点项目(No.30130070)
国家自然科学基金项目(No.30571672)
国家高技术研究发展计划(863计划)项目(No.2001AA215391)
欧盟项目(No.QLRT200001441)
国家科技攻关计划项目(No.2003BA712A0402)资助.~~
关键词
微管相关蛋白tau
朊蛋白
分子间相互作用
microtubule-associated protein tau
prion protein
molecular interaction
