Identification of the factors mediating ubiquitination of inclusion body in neurodegenerative diseases

据报道，在神经退行性疾病患者的神经元中发现的大多数包涵体是泛素化的。本研究项目的目的是确定介导组成包涵体的蛋白质泛素化的因素。作为模型系统，我们选择了Machado-Joseph病（MJD），其中MJD 1蛋白的多聚谷氨酰胺延伸异常扩展。我们发现MJD 1蛋白在体内和体外都经历了泛素化。利用体外泛素化系统作为检测泛素化活性的方法，我们用几种柱层析纯化了MJD 1泛素化所需的因子。最后，通过凝胶过滤柱层析，在大于1,000 kDa的组分中回收活性。我们最后通过氨基酸序列测定鉴定了泛素化酶复合物的一些组分，并分离了编码这些蛋白质的cDNA。目前，我们研究了包涵体形成的生物学意义及其在神经退行性疾病中的参与。

It has been reported that most of inclusion bodies which are found in patients' neurons of neurodegenerative diseases are ubiquitinated. The aim of this research project is to identify the factors to mediate ubiquitination of the proteins constituting the inclusion body. As a model system, we chose Machado-Joseph disease (MJD), in which polyglutamine stretch of MJD1 protein is abnormally expanded. We found that MJD1 protein undergoes ubiquitination both in vivo and in vitro. Using the in vitro ubiquitination system as an assay for detection of ubiquitinating activity, we purified the factors required for ubiquitination of MJD1 with several column chromatography. Finally, the activity was recovered in the fraction > 1,000 kDa with gel-filtration column chromatography.We finally identified some components of the ubiquitinating enzyme complex by amino acid sequencing, and isolated the cDNA encoding the proteins. Currently we examined the biological significance and involvement of the formation of inclusion body in neurodegenerative diseases.