【摘要】 路易小体(Lewy body, LB) ,位于神经细胞核周(perikaryon)的嗜酸性包含体(eosinophilic inclusion) ,含有广泛的蛋白质组分,其中一部分是组成型蛋白质(consistent organization) ,另外一部分则是选择型蛋白质(selective composition) .为了在体外获得LB中未知蛋白质的新线索,通过人工合成蛋白酶体抑制剂PSI(proteasomal inhibitor ,10μmol/L)作用PC 12细胞48 h,使其产生嗜酸性(staining for eosin)和抗α-synuclein阳性(immunostaining for α-synuclein)的PSI诱导性包含体(PSI-inducedinclusion) ,通过成功的分级分离(fractionation)纯化了完整、纯净的包含体,通过有效的双向电泳(two-dimensional electrophoresis ,2-DE)分离了包含体蛋白质,通过无偏差的基质辅助激光解析-离子化飞行时间质谱(matrix-assisted laser desorption/ionization time-of-flight massspectrometry,MALDITOF MS)鉴定了真核细胞翻译起始因子-3亚单位5(eukaryotic translation initiation factor 3 subunit 5 ,eIF-3ε)、真核细胞延伸因子-2 (eukaryotic elongation factor 2 , eEF-2)和线粒体延伸因子-Tu(mitochondrial elongation factor Tu, EF-Tumt)等真核细胞翻译因子(eukaryotic translation factors) .这一结果提示,当蛋白酶体受到抑制时真核细胞翻译因子被富集到PSI诱导性包含体中,并且可能影响其形成过程.更多还原

【Abstract】 Lewy body (LB), an eosinophilic inclusion localized in the neuronal perikaryon, consists of a wide range of proteins, including the consistent organization and the selective composition. Treatment of PC12 cells with synthetic proteasome inhibitor (PSI) at 10 μmol/L for 48 hours induced the formation of inclusions, which were detected by eosin staining and immunostaining for α-synuclein. To investigate the potential new components of PSI-induced inclusions in vitro, pure intact inclusions were successfully obtained by fractionation and subjected to two-dimensional electrophoresis (2-DE) then analyzed with unequivocal matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Eukaryotic translation initiation factor 3 subunit 5 (eIF-3ε), eukaryotic elongation factor 2 (eEF-2) and mitochondrial elongation factor Tu (EF-Tumt) were identified. The results suggest that 3 eukaryotic translation factors recruited in PSI-induced inclusions may influence formation of the intermediate organelles following the inhibition of proteasomes.更多还原