Elucidation of the fumctional network among the genes for familial Parkinson disease

1)我们已经阐明了PINK1和Omi/HtrA2、PARK6和PARK13基因产物分别在五路易小体中共定位。PINK1和Qmi/HtrA2分别是线粒体定位蛋白激酶和丝氨酸蛋白酶，提示线粒体蛋白可能参与了这些park相关基因的路易糖形成和/或隔离，可能有助于散发性帕金森病多巴胺能神经症的产生。2)帕金森氏症，一种家族性帕金森氏病（PD）称为常染色体隐性遗传性少年帕金森氏症(AR-JP)/PARK2的基因。帕金已被证明可以保护细胞免受内质网（ER）和氧化应激源的影响，这可能是由于它的泛素连接酶活性，其目标是蛋白酶体降解的蛋白质。虽然我们发现parkin在内质网应激下上调，但随后的报道表明，它并不代表一种普遍的未折叠蛋白反应（UPR）。我们报道了不同细胞系对内质网应激的不同调节，证明了parkin的上调是一种细胞类型特异性的内质网应激反应。2-巯基乙醇（2-ME）和tunicamycin增加了SH-SY5Y (H)细胞、神经-2a细胞、Goto-P3细胞中parkin的表达，而SH-SY5Y (J)细胞和IMR32细胞中parkin的表达没有增加。与这些研究同时，我们还观察到内质网应激对parkin协同调节基因(PACRG)/ parkin邻近基因（Glup）的上调。荧光素酶检测未能显示200bp的parkin /Glup启动子在内质网应激下的转录激活。这些结果表明内质网应激诱导parkin是一种细胞类型特异性反应。3)我们发现在细胞中PINK1与Hsp90和Cdc37/p50形成复合物。在热休克蛋白90抑制剂格尔达霉素或新生物素处理的细胞中，PINK1蛋白的稳定性大大降低。格尔达霉素处理通过蛋白酶体依赖途径导致泛素化增加和PINK1的快速降解。此外，我们发现PINK1的致病突变体L347P降低了与Hsp90和Cdc37/p50的相互作用，与野生型PINK1相比，其蛋白稳定性降低。这些结果强烈提示Hsp90和Cdc37是调节PINK1稳定性的重要因子，参与帕金森病的发生。少

1) We have elucidated that PINK1 and Omi/HtrA2, PARK6 and PARK13 gene products, respectively, are co-localized in fee Lewy bodies. PINK1 and Qmi/HtrA2 are mitochondria-localizing protein kinase and serine protease, respectively, suggesting that mitochondrial proteins may be involved in the Lewy hody formation and/or sequestration of these PARK-related genes may contribute to the dopamin ergic neurode generation in sporadic Parkinson's disease.2) Parkin, the gene responsible for a familial form of Parkinson's disease (PD) termed autosomal recessive juvenile parkinsonism (AR-JP)/PARK2. Parkin has been shown to protect cells from endoplasmic reticulum (ER) and oxidative stressors presumably due to its ubiquitin ligase activity that targets proteins for proteasomal degradation. Although we showed that parkin is upregulated in response to ER stress, subsequent reports suggest that it does not represent a universal unfolded protein response (UPR). We report different regulation of parkin in … More response to ER stress in different cell lines, demonstrating upregulation of parkin as a cell type-specific response to ER stress. 2-mercaptoethanol (2-ME) and tunicamycin increased the expression of parkin in SH-SY5Y (H) cells, Neuro-2a cells, Goto-P3 cells, but not in SH-SY5Y (J) cells and IMR32 cells. In parallel with these studies, we also observed similar upregulation of the parkin coregulated gene (PACRG)/gene adjacent to parkin(Glup) by ER stress. Luciferase assays failed to show the transcriptional activation of 200bp parkin /Glup promoter in response to ER stress. These results indicate that induction of parkin by ER stress represents a cell type-specific response.3) We found that PINK1 forms complexes with Hsp90 and Cdc37/p50 in the cells. The protein stability of PINK1 was greatly reduced in the cells treated with either geldanamycin or novobiocin, inhibitors of Hsp90. Geldanamycin treatment led to the increased ubiquitination and the rapid degradation of PINK1 through the proteasome-dependent pathway. Furthermore, we found that L347P, a pathogenic mutant of PINK1, decreased the interaction with both Hsp90 and Cdc37/p50, and exhibited reduced protein stability compared with wild-type PINK1. These results strongly suggest that Hsp90 and Cdc37 are important factors regulating the stability of PINK1, which is involved in Parkinson s disease. Less