Davison Eleanor J, Pennington Kyla, Hung Chao-Chun, Peng Jianhe, Rafiq Rumana, Ostareck-Lederer Antje, Ostareck Dirk H, Ardley Helen C, Banks Rosamonde E, Robinson Philip A
Section of Ophthalmology and Neuroscience, Leeds Institute for Molecular Medicine, Wellcome Trust Brenner Building, St. James's University Hospital, Leeds, UK.
Proteomics. 2009 Sep;9(18):4284-97. doi: 10.1002/pmic.200900126.
Parkin is an ubiquitin-protein ligase (E3), mutations of which cause juvenile onset - autosomal recessive Parkinson's disease, and result in reduced enzymic activity. In contrast, increased levels are protective against mitochondrial dysfunction and neurodegeneration, the mechanism of which is largely unknown. In this study, 2-DE and MS proteomic techniques were utilised to investigate the effects of increased Parkin levels on protein expression in whole cell lysates using in an inducible Parkin expression system in HEK293 cells, and also to isolate potential interactants of Parkin using tandem affinity purification and MS. Nine proteins were significantly differentially expressed (+/-2-fold change; p<0.05) using 2-DE analysis. MS revealed the identity of these proteins to be ACAT2, HNRNPK, HSPD1, PGK1, PRDX6, VCL, VIM, TPI1, and IMPDH2. The first seven of these were reduced in expression. Western blot analysis confirmed the reduction in one of these proteins (HNRNPK), and that its levels were dependent on 26S proteasomal activity. Tandem affinity purification/MS revealed 14 potential interactants of Parkin; CKB, DBT, HSPD1, HSPA9, LRPPRC, NDUFS2, PRDX6, SLC25A5, TPI1, UCHL1, UQCRC1, VCL, YWHAZ, YWHAE. Nine of these are directly involved in mitochondrial energy metabolism and glycolysis; four were also identified in the 2-DE study (HSP60, PRDX6, TPI1, and VCL). This study provides further evidence for a role for Parkin in regulating mitochondrial activity within cells.
帕金蛋白是一种泛素蛋白连接酶（E3），其突变会导致青少年型常染色体隐性帕金森病，并致使酶活性降低。相比之下，其水平升高对线粒体功能障碍和神经退行性变具有保护作用，但其机制在很大程度上尚不清楚。在本研究中，利用二维凝胶电泳（2-DE）和质谱（MS）蛋白质组学技术，在人胚肾293细胞的可诱导帕金蛋白表达系统中，研究帕金蛋白水平升高对全细胞裂解物中蛋白质表达的影响，并通过串联亲和纯化和质谱技术分离帕金蛋白的潜在相互作用蛋白。采用2-DE分析，有9种蛋白质的表达存在显著差异（变化倍数为±2倍；p<0.05）。质谱鉴定出这些蛋白质为乙酰辅酶A乙酰基转移酶2（ACAT2）、不均一核糖核蛋白K（HNRNPK）、热休克蛋白60（HSPD1）、磷酸甘油酸激酶1（PGK1）、过氧化物还原酶6（PRDX6）、纽蛋白（VCL）、波形蛋白（VIM）、磷酸丙糖异构酶1（TPI1）和肌苷酸脱氢酶2（IMPDH2）。其中前7种蛋白质的表达降低。蛋白质印迹分析证实了其中一种蛋白质（HNRNPK）的表达降低，且其水平依赖于26S蛋白酶体活性。串联亲和纯化/质谱分析揭示了帕金蛋白的14种潜在相互作用蛋白；肌酸激酶B（CKB）、二氢硫辛酰胺转乙酰基酶（DBT）、热休克蛋白60（HSPD1）、热休克蛋白A9（HSPA9）、富含亮氨酸的五肽重复序列蛋白C（LRPPRC）、NADH脱氢酶[泛醌]铁硫蛋白2（NDUFS2）、过氧化物还原酶6（PRDX6）、溶质载体家族25成员5（SLC25A5）、磷酸丙糖异构酶1（TPI1）、泛素羧基末端水解酶L1（UCHL1）、泛醌-细胞色素c还原酶核心蛋白1（UQCRC1）、纽蛋白（VCL）、14-3-3ζ蛋白（YWHAZ）、14-3-3ε蛋白（YWHAE）。其中9种直接参与线粒体能量代谢和糖酵解；在二维凝胶电泳研究中也鉴定出了4种（热休克蛋白60、过氧化物还原酶6、磷酸丙糖异构酶1和纽蛋白）。本研究为帕金蛋白在调节细胞内线粒体活性中的作用提供了进一步的证据。
