蛋白酶体抑制剂诱导帕金森病PINK1蛋白聚集物的形成与特征

Proteasomal inhibitor induces PINK1 aggresome formation and aggregating features

目的研究在蛋白酶体抑制剂作用下,帕金森病 PINK1蛋白聚集物的形成及其特征。方法应用 PCR 从胎脑 cDNA 文库扩增全长 PINK1基因,亚克隆至增强型绿色荧光蛋白载体(pEGFP-N1),经测序证实载体构建正确。PINK1-pEGFP-N1表达载体转染非洲绿猴肾细胞株(COS-7细胞),应用 MG-132抑制蛋白酶体活性,Western blot 检测 PINK1蛋白表达量,荧光染色观察 PINK1蛋白是否形成蛋白聚集物,免疫荧光观察构成 Lewy 小体的两种主要成分:泛素和α-突触核蛋白是否存在于蛋白聚集物中。结果应用 MG-132诱导后 PINK1蛋白表达量明显增加,PINK1蛋白形成了蛋白聚集物,泛素和α-突触核蛋白与 PINK1蛋白聚集物共定位。结论在蛋白酶体抑制剂作用下,PINK1蛋白可形成蛋白聚集物,泛素和α-突触核蛋白是其组成成分。

Objective To study the PINK1 aggresome formation and it＇ s features in response to proteasomal inhibition, Methods Full-length PINK1 cDNA were amplified by polymerase chain reaction （PCR） from fetus brain cDNA library and subcloned into the EcoR I and BamH I sites of the vector pEGFP- N1. The integrity of the constructs was confirmed by sequencing. COS-7 cells were transiently transfected with PINK1-pEGFP-N1 using Lipofectamine 2000. Cells were treated by MG-132 in order to test the effect of proteasome inhibition on aggregation formation, The protein level of wild-type PINK1 with or without MG-132 treatment was confirmed by Western blot analysis. The formation of PINK1 aggregates was tested by fluorescence and the presence of ubiquitin, and α-synuclein in PINK1 aggregates was examined by immunofluorescence and confocal microscopy. Results The expression level of PINK1 was significant increased into the form of aggregate in cells treated with MG-132; immunostaining for endogenous ubiquitin and α-synuclein revealed a co-localization of both proteins in PINKl-positive aggregates. Conclusions In the presence of MG-132, overexpressed PINK1 forms into aggregates, whose components are ubiquitin and α-synuclein.