Characterization of inhibitor-bound alpha-synuclein dimer: role of alpha-synuclein N-terminal region in dimerization and inhibitor binding.
Characterization of inhibitor-bound alpha-synuclein dimer: role of alpha-synuclein N-terminal region in dimerization and inhibitor binding.
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DOI：
10.1016/j.jmb.2009.10.068
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发表时间：
2010-01
影响因子：
5.6
通讯作者：
Y. Yamaguchi;Masami Masuda;H. Sasakawa;T. Nonaka;S. Hanashima;S. Hisanaga;Koichi Kato;M. Hasegawa
中科院分区：
文献类型：
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作者：
Y. Yamaguchi;Masami Masuda;H. Sasakawa;T. Nonaka;S. Hanashima;S. Hisanaga;Koichi Kato;M. Hasegawa
α-突触核蛋白是丝状包涵体的主要成分，丝状包涵体是帕金森病和其他α-突触核蛋白病的组织学标志。先前的分析表明，几种多酚抑制α-突触核蛋白的组装，具有较低的微摩尔IC 50值，并且在它们的存在下形成SDS稳定的、无细胞毒性的可溶性α-突触核蛋白寡聚体。为了更好地理解抑制机制，显然需要对结合于突触体的α-突触核蛋白寡聚体进行结构解析。为了详细地表征结合于底物的α-突触核蛋白，我们在多酚抑制剂、依昔苯酮、棉酚和多巴胺的存在下制备了α-突触核蛋白二聚体，并纯化了产物。肽图和质谱分析显示，艾昔苯酮处理的α-突触核蛋白单体和二聚体在α-突触核蛋白的所有四个甲硫氨酸残基处被氧化。对内切蛋白酶Asp-N消化产物的免疫印迹分析和氧化还原循环染色表明，N-末端区域（1-60）参与α-突触核蛋白的二聚化和exifone结合。对结合于α-突触核蛋白的二聚体的超高场NMR分析表明，来自α-突触核蛋白N-末端区域的信号表现出谱线增宽，证实N-末端区域参与了结合物诱导的二聚化。C-末端部分仍然主要表现出在单体α-突触核蛋白中观察到的无规卷曲特征。我们认为α-突触核蛋白的N端区域在α-突触核蛋白组装体的形成中起着关键作用。
α-Synuclein is a major component of filamentous inclusions that are histological hallmarks of Parkinson's disease and other α-synucleinopathies. Previous analyses have revealed that several polyphenols inhibit α-synuclein assembly with low micromolar IC50values, and that SDS-stable, noncytotoxic soluble α-synuclein oligomers are formed in their presence. Structural elucidation of inhibitor-bound α-synuclein oligomers is obviously required for the better understanding of the inhibitory mechanism. In order to characterize inhibitor-bound α-synucleins in detail, we have prepared α-synuclein dimers in the presence of polyphenol inhibitors, exifone, gossypetin, and dopamine, and purified the products. Peptide mapping and mass spectrometric analysis revealed that exifone-treated α-synuclein monomer and dimer were oxidized at all four methionine residues of α-synuclein. Immunoblot analysis and redox-cycling staining of endoproteinase Asp-N-digested products showed that the N-terminal region (1–60) is involved in the dimerization and exifone binding of α-synuclein. Ultra-high-field NMR analysis of inhibitor-bound α-synuclein dimers showed that the signals derived from the N-terminal region of α-synuclein exhibited line broadening, confirming that the N-terminal region is involved in inhibitor-induced dimerization. The C-terminal portion still predominantly exhibited the random-coil character observed in monomeric α-synuclein. We propose that the N-terminal region of α-synuclein plays a key role in the formation of α-synuclein assemblies.