CRBN基因突变引起轻型智力发育迟滞分子机制

Molecular mechanism of ARNSMR by nonsense mutant CRBN

[目的]为了研究CRBN基因与常染色体隐性遗传非综合征轻型智力发育迟滞(ARNSMR)的关系。[方法]从293T细胞中克隆了CRBN和CRBN突变体基因(CRBNm),并克隆至pc DNA-GFP、pc DNA-Flag质粒中,将重组质粒用脂质体转染到293T细胞中,Co-IP与GFP-CRBN、CRBNm融合蛋白相互作用的293T细胞蛋白,通过PAGE胶电泳、银染分析差异蛋白。同时我们还做了CRBN和CRBNm蛋白稳定性和泛素化修饰研究。[结果]发现CRBN基因无义突变后没有改变蛋白的细胞定位,但影响了相互作用蛋白,同时突变的CRBN蛋白自我泛素化修饰增强,与野生型相比更容易降解。[结论]CRBN基因的无义突变后蛋白泛素化修饰增强,细胞内相互作用蛋白发生改变,从而影响了CRBN基因的功能。

[ Objective ] To explore the relationship of CRBN gene with autosomal recessive nonsyndromic mental retardation （ARNSMR）. [ Methods] CRBN and mutant CRBN （CRBNm） genes were cloned from 293T cell line. After constructed to pcDAN - GFP and pcDNA - Flag vectors,transfected vectors of CRBN and CRBNm to 293T cell line with Lipo 2000. Co - IP proteins that can bind GFP - CRBN or CRBNm from 293T cell lysis solution; After electrophoresed by PAGE gel, analyzed diversity bands using silver staining method. At the same time, proteins stability and ubiquitin modification were detected. [ Results ] After the premature stop codons of CRBN, CRBNm gene localization does not change,but combined the special interaction proteins. Mutant CRBN can increase ubiquitin modification and is more readily degraded than WT CRBN. [ Conclusion] These changes may influence CRBN gene functions and contribute to ARNSMR.