Search for inhibitory factor for polyglutamine protein ubioquitylation

今年，我们测试了泛素类蛋白是否可以修饰聚谷氨酰胺蛋白。我们从cDNA文库中克隆了FAT10、ubl5、FubI、Isg15和urm1，并构建了过表达构建体供哺乳动物表达。我们将这些结构与聚谷氨酰胺蛋白共表达，并用免疫沉淀法降低pQ。对附着蛋白的免疫检测显示，FAT10共价附着于下拉的pQ蛋白上。通过在变性条件下拉下pQ以检测与pQ共价结合的FAT10进一步证实了这一点。已知FAT10与泛素同源，并且已知与赖氨酸残基共价偶联。我们测试了不同量的泛素共表达的FAT10，发现FAT10影响PQ蛋白的泛素化。通过过表达FAT10，加速了pQ蛋白的降解。这些数据提示FAT10可能在pQ发病机制中发挥重要作用。我们现在正在确认数据并准备发表。

This year, we tested if any of the ubiquitin like proteins could modify polyglutamine proteins. We cloned FAT10, ubl5, FubI, Isg15 and urm1 from cDNA library and made overexpression constructs for mammalian expression. We co-expressed these constructs with polyglutamine proteins and pulled down pQ with immunoprecipitation method. Immunodetection of attached proteins revealed that FAT10 was covalently attached to pulled-down pQ proteins. This was further confirmed by pulling down pQ under denatured condition to detect FAT10 covalently bound to pQs. FAT10 is known to be homologous to ubiquitin abd known to covalently conjugated to lysine residues. We tested various amounts of FAT10 with ubiquitin co-expressed to find FAT10 affects ubiquitylation of PQ protein. By overexpressing FAT10, degradation of pQ proteins are accelerated. These data suggested that FAT10 might play and important role in pQ pathogenesis. We are now confirming the data and preparing for publication.