Proteomic analysis of increased Parkin expression and its interactants provides evidence for a role in modulation of mitochondrial function
Proteomic analysis of increased Parkin expression and its interactants provides evidence for a role in modulation of mitochondrial function
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DOI：
10.1002/pmic.200900126
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发表时间：
2009-09-01
期刊：
影响因子：
3.4
通讯作者：
Robinson, Philip A.
中科院分区：
文献类型：
--
作者：
Davison, Eleanor J.;Pennington, Kyla;Robinson, Philip A.
帕金森氏病是一种泛素蛋白连接酶（E3），其突变可引起少年发病-常染色体隐性帕金森氏病，并导致酶活性降低。相反，增加的水平对线粒体功能障碍和神经变性有保护作用，其机制在很大程度上是未知的。在本研究中，利用2-DE和MS蛋白质组学技术，在HEK293细胞中诱导Parkin表达系统中，研究了Parkin水平升高对全细胞裂解物中蛋白表达的影响，并利用串联亲和纯化和MS分离了Parkin的潜在相互作用，使用2-DE分析，9个蛋白的表达显著差异（+/- 2倍变化，p < 0.05）。ms发现这些蛋白分别为ACAT2、HNRNPK、HSPD1、PGK1、PRDX6、VCL、VIM、TPI1和IMPDH2。其中前7个基因表达减少。Western blot分析证实了其中一种蛋白（HNRNPK）的减少，其水平依赖于26S蛋白酶体活性。串联亲和纯化/质谱法发现了14种潜在的Parkin相互作用物；Ckb、dbt、hspd1、hsp9、lrpprc、ndufs2、prdx6、slc25a5、tpi1、uch1、uqcrc1、vcl、ywhaz、ywhae。其中9个直接参与线粒体能量代谢和糖酵解；在2-DE研究中也发现了4个（HSP60、PRDX6、TPI1和VCL）。这项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。
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. M S 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.