轴突型腓骨肌萎缩症2L型致病基因HSPB8突变导致细胞内聚集物形成的机理研究

Study on aggregate formation mechanism of HSPB8 gene mutation resulting in CMT2L

目的探讨轴突型腓骨肌萎缩症2L型(axonal Charcot-Marie-Tooth disease type 2L,CMT2L)致病基因小分子热休克蛋白HSPB8(smallheatshockproteinHSPB8,HSPB8)的K141N突变导致细胞内聚集物形成的可能机理。方法建立pEGFPN1-HSPB8、pEGFPN1-K141NHSPB8瞬时表达细胞模型,并进行EGFP-K141NHSPB8与小分子热休克蛋白HSPB1(smallheatshockproteinHSPB1,HSPB1)、神经丝轻链(neurofilamentlightchain,NEFL)的免疫荧光共定位分析,观察EGFP-K141NHSPB8在不同内源性表达细胞系的聚集物形成情况,采用t检验和单因素方差分析的统计学方法分析聚集物形成的可能机理。结果EGFP-K141NHSPB8形成以核周分布为主的聚集物,EGFP-K141NHSPB8与HSPB1、NEFL均存在免疫荧光共定位。EGFP-K141NHSPB8在不同内源性表达细胞系的聚集物形成百分率的差异有统计学意义。结论突变型HSPB8(K141N)形成以核周分布为主的胞内聚集物,聚集物中K141NHSPB8与HSPB1、NEFL均存在共定位。聚集物形成的可能机理包括K141NHSPB8多肽链构象发生改变后不能维持稳态而出现自身异常聚集;与家族内其他成员特别是HSPB1结合成异常的异源多聚体,在胞内形成不可溶性大分子后产生聚集。

Objective To study the possible mechanism of the intracellular aggregate formation of small heat shock protein HSPB8 （HSPB8） （K141N） mutation resulting in axonal Charcot-Marie-Tooth disease type 2L （CMT2L）. Methods The cell models which transiently expressed pEGFPN1 - HSPB8 and pEGFPNI-^K141N HSPB8 were established. The immunotluorescent co-location study of EGFP-^K141N HSPB8 and HSPB1, EGFP-^K141N HSPB8 and neurofilament light chain （NEFL） was carried out in the SHSYSY cell models. The aggregate formation of EGFP-^N141N HSPB8 in cell models was investigated and the possible mechanism of cellular aggregate formation was analyzed by t test and analysis of variance between groups （ANOVA）. Results EGFP-^K141N HSPB8 formed large aggregate which predominantly located around the nucleus in cell models. EGFP-^K141N HSPB8 co-localized perfectly with HSPB1 and NEFL in the SHSY5Y cell models. The aggregate formation was different in different cell types, there were fewer aggregates formed in an sHSPs deficient milieu than in HEK293T cells. Condusion ^K141N HSPB8 formed aggregates predominantly locate around the nucleus in cells. ^K141N HSPB8 co-localizes perfectly with HSPB1 and NEFL. The aggregate formation may be due to ^K141N HSPB8 conformational change leading to self aggregation and its abnormal interaction with other sHSPs such as HSPB1.