去氢骆驼蓬碱靶向WWP1促进突变亨廷顿蛋白降解的分子机理研究

Huntington's disease (HD), one kind of neurodegenerative diseases, is mainly attributed to the mutant Huntingtin (mHtt), which can form aggregates in neurocytes thus resulting cytotoxicity. Cells remove misfolded proteins usually through the ubiquitin-proteasome system. The ubiquitin protein ligase WWP1, performs atypical ubiquitin modification to its substrate mHtt and inhibits mHtt degradation. The small molecular harmine's effects on some neurodegenerative diseases have been revealed. However, there has been no report about harmine's effect on HD. Here, the applicants screened and identified a natural small molecular compound, named harmine, down-regulating the expression of WWP1 thus inhibiting the formation of mHtt aggregates in neurocytes and relieving HD symptom. Molecular docking was used for the study of harmine and WWP1. Harmine has been found binding to WWP1 via hydrogen bond to Asn705, Glu697, His888 and Arg893 in the crystal structure and partially impedes the transferring of ubiquitin. However, the molecular mechanism of harmine's down-regulation of WWP1 and mediation the degradation of mHtt's ubiquitination is still unknown. Protein crystallography and molecular docking are applied in the project to explore binding sites of harmine and WWP1. Molecular mechanism could be explicitly clarified via WWP1 mutagenesis assay, circular dichroism spectroscopy and fluorescent spectrometry test. This research will provide theoretical evidence for harmine's clinical application in curing HD, all of which will serve as vital significance for HD new drug research and development.

亨廷顿病（Huntington's disease, HD）是突变亨廷顿蛋白（mutant huntingtin, mHtt）在神经元胞体内形成聚集物引发细胞毒性而导致的神经退行性罕见病。泛素-蛋白酶体系统是清除mHtt的重要途径，而泛素连接酶WWP1介导的非典型泛素化却抑制mHtt降解。小分子化合物去氢骆驼蓬碱在某些神经退行性病中已有研究，但在HD中尚未见报道。前期研究发现去氢骆驼蓬碱下调WWP1表达促进mHtt降解并缓解HD病症。分子对接结果显示，去氢骆驼蓬碱与WWP1半胱氨酸催化位点附近的Asn705、Glu697、His888和Arg893通过氢键结合并部分阻碍泛素转移，但其下调WWP1介导mHtt泛素化的分子机制尚不清楚。本项目拟采用结构生物学手段，通过突变体实验，圆二色谱及荧光光谱实验阐明其分子机制。这为去氢骆驼蓬碱治疗HD的临床应用提供理论依据，对HD新药研发具有重要意义。

为了研发小分子化合物Harmine对神经退行性疾病靶向治疗的功效及其促进突变亨廷顿蛋白降解的具体分子机制，我们开展了本项目的研究工作。项目主要研究内容包括：①确证Harmine降低WWP1对mHtt非典型泛素化水平的作用机制；②验证Harmine是否直接与WWP1作用并部分阻碍活化的泛素分子转移至WWP1所识别的底物mHtt 上； ③阐明Harmine与WWP1的具体结合方式，并揭示其促进mHtt降解的分子机制。 我们的研究结果发现：①小分子化合物Harmine具有在体外抑制mHtt聚集物形成的作用。②Harmine在HD小鼠体内具有缓解HD病症的效果。③Harmine可在转录水平调控WWP1表达。④Harmine可与WWP1的催化位点Cys890附近区域结合。本项目的科学意义在于通过多层次体内外各种模型，首次发现了小分子化合物Harmine靶向治疗神经退行性疾病的治疗功效及其促进突变亨廷顿蛋白降解的分子机制，揭示了小分子化合物Harmine抑制mHtt 聚集物形成的的药理学活性及其具体分子机制，本课题的研究补充和丰富了小分子化合物在HD中的治疗作用，具有重要的科学意义，同时又为寻找和开发治疗HD候选药物提供了基础研究和新的靶点。