IDENTIFICATION AND CHARACTERIZATION OF A NOVEL UBIQUITIN LIGASE SIAH THAT EXHIBITS COMPLEMENTARY ROLES WITH PARKIN

帕金森病是一种常见的神经退行性疾病，其特征在于多巴胺能神经元的损失和路易体的出现，路易体是高度富含泛素的细胞质内含物。Synphilin-1、α-synuclein和Parkin是Lewy小体的主要成分，参与了帕金森病的发病机制。Synphilin-1是一种α-突触核蛋白结合蛋白，被帕金泛素化。最近，在synphilin-1基因突变的报告在散发性帕金森病患者。虽然synphilin-1定位接近突触囊泡，其功能仍然未知。为了研究与synphilin-1相互作用的蛋白质，本研究进行了酵母双杂交筛选，并确定了一个新的相互作用蛋白，Siah-1泛素连接酶。Synphilin-1和Siah-1蛋白在中枢神经系统中内源性表达，并在大鼠脑匀浆中相互免疫共沉淀。共聚焦显微镜分析表明，这两种蛋白质在细胞中的共定位。发现Siah-1通过其底物结合结构域与synphilin-1的N-末端相互作用，并通过其RING指结构域特异性地泛素化synphilin-1。Siah-1通过泛素-蛋白酶体途径比Parkin更有效地促进synphilin-1降解。发现Siah-1不促进野生型或突变型α-突触核蛋白的泛素化和降解。Synphilin-1抑制高K^+诱导的PC 12细胞多巴胺释放。发现Siah-1消除了synphilin-1对多巴胺释放的抑制作用。这些发现表明Siah-1可能在synphilin-1功能的调节中发挥作用。

Parkinson's disease is a common neurodegenerative disorder characterized by loss of dopaminergic neurons and appearance of Lewy bodies, cytoplasmic inclusions that are highly enriched with ubiquitin. Synphilin-1, α-synuclein and Parkin represent the major components of Lewy bodies, and are involved in pathogenesis of Parkinson's disease. Synphilin-1 is anα-synuclein binding protein that is ubiquitinated by Parkin. Recently, a mutation in the synphilin-1 gene is reported in patients with sporadic Parkinson's disease. Although synphilin-1 localizes close to synaptic vesicles, its function remains unknown. To investigate the proteins that interact with synphilin-1, the present study performed a yeast two-hybrid screening and identified a novel interacting protein, Siah-1 ubiquitin ligase. Synphilin-1 and Siah-1 proteins were endogenously expressed in the central nervous system, and coimmunoprecipitated each other in rat brain homogenate. Confocal microscopic analysis revealed colocalization of both proteins in cells. Siah-1 was found to interact with the N-terminus of synphilin-1 through its substrate-binding domain, and specifically ubiquitinate synphilin-1 via its RING finger domain. Siah-1 facilitated synphilin-1 degradation via the ubiquitin-proteasome pathway more efficiently than Parkin. Siah-1 was found not to facilitate ubiquitination and degradation of wild type or mutant α-synuclein. Synphilin-1 inhibited high K^+-induced dopamine release from PC12 cells. Siah-1 was found to abrogate the inhibitory effect of synphilin-1 on dopamine release. Such findings suggest that Siah-1 might play a role in regulation of synphilin-1 function.