CAJ | 학술논문

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-induced inclusion),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,MALDI-TOF 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.
로역소체(Lewy body,LB),위우신경세포핵주(perikaryon)적기산성포함체(eosinophilic inclusion),함유엄범적단백질조분,기중일부분시조성형단백질(consistent organization),령외일부분칙시선택형단백질(selective composition).위료재체외획득LB중미지단백질적신선색,통과인공합성단백매체억제제PSI(proteasomal inhibitor,10μmol/L)작용PC 12세포48 h,사기산생기산성(staining for eosin)화항α-synuclein양성(immunostaining for α-synuclein)적PSI유도성포함체(PSI-induced inclusion),통과성공적분급분리(fractionation)순화료완정、순정적포함체,통과유효적쌍향전영(two-dimensional electrophoresis,2-DE)분리료포함체단백질,통과무편차적기질보조격광해석-리자화비행시간질보(matrix-assisted laser desorption/ionization time-of-flight massspectrometry,MALDI-TOF MS)감정료진핵세포번역기시인자-3아단위5(eukaryotic translation initiation factor 3 subunit 5,eIF-3ε)、진핵세포연신인자-2(eukaryotic elongation factor 2,eEF-2)화선립체연신인자-Tu(mitochondrial elongation factor Tu,EF-Tumt)등진핵세포번역인자(eukaryotic translation factors).저일결과제시,당단백매체수도억제시진핵세포번역인자피부집도PSI유도성포함체중,병차가능영향기형성과정.
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 matrixassisted 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.