蛋白酶体活性测定与组分分析方法的建立

Methods for proteasome activity measurement and component analysis

背景:26S蛋白酶体对维持细胞周期、增殖、生存等细胞生物学行为的正常具有极其重要的作用,但目前对细胞体内蛋白酶体活性的测定及组分分析缺少明确的方法。目的:建立蛋白酶体活性与组分的分析方法,用以检测肿瘤组织、细胞蛋白酶体的活性状态与组分表达情况。方法:应用3种特异性的荧光多肽底物Suc-LLVY-AMC、Z-ARR-AMC和Z-LLE-AMC建立对蛋白酶体的糜蛋白酶样、胰蛋白酶样和肽基谷氨酰肽水解酶样蛋白酶活性的测定方法。并基于蛋白酶体特异亲和性探针AdaK(Bio)Ahx3L3VS建立对蛋白酶体各亚基(包括β1/β1i,β2/β2i,β5/β5i)组分分析的方法。结果与结论:利用特异性的荧光多肽底物检测蛋白酶活性及蛋白酶体特异亲和性探针检测蛋白酶体亚基组分分析的方法发现在K562红白血病细胞,β2、β5亚基表达较高,而蛋白酶体抑制剂PS341及PSI对K562细胞的蛋白酶体活性与组分的抑制作用主要是靶定在β5和β5i亚基,抑制其糜蛋白酶样活性。实验建立了对以上3种蛋白酶体活性测定及组分分析的方法,为蛋白酶体抑制剂的体外活性鉴定提供了实验方法。

BACKGROUND：26S proteasome plays an important role in maintaining normal cell cycle,proliferation,and survival.However,there is not a precise method can test in vivo proteasome activity and composition.OBJECTIVE：To establish methods for analyzing proteasome activity and composition in cancer tissues.METHODS：Three specific fluorogenic peptide substrates：Suc-LLVY-AMC,Z-ARR-AMC and Z-LLE-AMC were used to monitor chymotrypsin-like,trypsin-like and peptidly-glutamyl peptide-hydrolyzing-like（PGPH-like） proteolytic activity,and a proteasome-specific affinity probe AdaK（Bio）Ahx3L3VS was employed to assay the β1/β1i,β2/β2i,and β5/β5i subunits of proteasome.RESULTS AND CONCLUSION：We investigated the catalytic activity and the expression of the component of proteasome,and the effects of proteasome inhibitor PS341 and PSI on the activity and composition of proteasome in erythroleukemia K562 cells.Our results showed that the expression of the β2 and β5 subunits of proteasome was high,while PS341 and PSI inhibited β5/β5i subunits,and the chymotrypsin-like activity of proteasome in K562 cells.Methods for analyzing proteasome activity and composition was established and supplied experimental methods for identifying in vitro activity of proteasome inhibitors.