Identification of interacting molecules with α-synuclein and of their function in the pathology of neurodegenerative diseases

在研究包括帕金森病和弥漫性路易体病在内的一组路易体疾病的发病机制时，α-突触核蛋白被认为是关键分子之一，因为该蛋白的一些突变首先引起家族性帕金森病，其次它是路易体的主要蛋白成分。鉴定和鉴定与该蛋白相互作用的分子对于阐明LBD的发病机制是必不可少的。本研究主要集中在以下两个方面：第一，鉴定与α-突触核蛋白相互作用的新分子；第二，详细分析突触核蛋白与已知相互作用分子之间的相互作用。关于第一点，我们以A53T PD致病突变型突变体synuclein为诱饵，筛选了一个人胎脑cDNA文库，获得了一些候选克隆，目前正在进行鉴定。此外，通过用GST-突触核蛋白探测代谢性放射性同位素标记的COS-1蛋白，我们鉴定了一个表观分子量为72 kDa的多肽。它与谷胱甘肽-α-突触核蛋白相互作用，但不只与谷胱甘肽相互作用。关于后一点，我们主要集中在SynPhilin，它是在我们的项目期间作为一种与α-突触核蛋白相互作用的蛋白质而被报道的。与先前报道的结果相反，我们的结果表明，与N-末端和C-末端截短的突触蛋白与突触核蛋白的相互作用相比，全长野生型突触蛋白的相互作用非常弱，这表明N-末端或C-末端区域的存在对相互作用起到了抑制作用。

α-Synuclein is regarded as one of the key molecules when considering the pathogenic mechanisms of a group of Lewy body diseases (LBD), including Parkinson's disease (PD) and diffuse Lewy body disease, in that, first, some mutations of this protein cause familial PD and, second, it is a main proteineous component of the Lewy bodies. Identification and characterization of molecules interacting with this protein is indispensable for the elucidation of the pathogenic mechanisms of LBD.This project focused on the following two points ; first, identification of new molecules interacting with α-synuclein, and second, detailed analyses of the interaction between synuclein and the known interacting molecules. In regard to the first point, we screened a human fetal brain cDNA library using A53T PD-pathogenic mutant synuclein as a bait and obtained some candidate clones, characterization of which is now under way. In addition, by probing metabolically radioisotope-labeled COS-1 proteins with GST-synucleins, we identified a polypeptide with the apparent molecular weight of 72 kDa. which interacts with GST-α-synuclein but not with GST alone. Concerning the latter point, we mainly concentrated on synphilin, which was reported during the period of our project as a protein interacting with α-synuclein. In contradiction to the previously reported results, our results indicate that, as compared with the interaction of an N- and C-terminally truncated synphilin with synuclein, that of full-length wild type one is very week if any, suggesting that presence of either of the N-terminal or C-terminal region acts inhibitory on the interaction.