脊髓小脑性共济失调新亚型（SCA35）致病基因TGM6的功能研究

In our previous study we have cloned TGM6, a new causative gene of autosomal dominant spinocerebellar ataxia type 35. Transglutaminase 6 protein is a member of the transglutaminase family whose most common function is to catalyze the formation of isopeptide linkages between the carboxamide group of protein bound glutamine residues and the ε-amino group of protein-bound lysine residue. TGs may contribute to the pathogenesis of many neurodegenerative disorders, including spinocerebellar ataxia，Alzheimer's disease, Parkinson's disease， and Huntington's disease, but the details of its mechanism is not clear. First of all, we intend to explore TG6 physiological function by screening and identifying its interplay proteins and analyzing its expression and localization in central nervous system. Secondly, we want to figure out whether mutational TG6 proteins have some effects on its subcellular localization, protein stability and enzyme activity and whether mutational TG6 proteins have effects on aggregation and degradation of PolyQ protein or cell viability. Meanwhile, we plan to establish TGM6 transgenic mouse models and to explore TGM6 pathogenesis from several aspects， such as ethology, histochemistry and pathophysiology, so as to provide some theoretical clues for diagnosis, prevention and therapy of SCA.

前期我们克隆了一新的脊髓小脑性共济失调35亚型（SCA35）致病基因- TGM6。TGM6编码蛋白TG6是转谷氨酰胺酶（TG）家族成员之一，TGs主要催化谷氨酰胺残基的咪唑羧酰胺基团与赖氨酸残基的ε氨基基团反应，参与了SCA、阿尔兹海默病、帕金森病及亨廷顿病等多种神经退行性疾病的发病过程，但其具体发病机制仍不明确。本课题拟在前期工作的基础上，首先了解TG6蛋白的正常生理功能，筛选和鉴定其互作蛋白，研究TG6在神经系统的分布及表达TG6神经元的特性；然后在细胞水平探讨致病突变对TG6的亚细胞定位、蛋白稳定性、细胞活性及其突变后对PolyQ蛋白的聚集、降解等特性的影响；同时构建野生型和突变型TGM6转基因小鼠，从行为学、组织化学和病理生理学等多角度探讨TG6突变导致SCA的发病机制，为此类疾病的诊断、预防和治疗提供理论依据。

前期我们克隆了一新的脊髓小脑性共济失调35亚型（SCA35）致病基因- TGM6。TGM6参与了SCA等多种神经退行性疾病的发病过程，本课题在前期工作的基础上，成功构建了野生型及两种突变型TGM6重组真核、原核表达载体，并在细胞水平发现野生型及突变型TG6蛋白的亚细胞定位没有明显改变，主要位于细胞浆中；野生型及突变型TG6蛋白的表达量没有明显差异；蛋白稳定性实验发现，突变型TG6蛋白结构稳定性降低，在体内很容易被降解，半衰期明显缩短；TG活性检测发现，突变型TG6蛋白的TG活性较野生型明显下降，尤其以L517W突变体的活性降低最为显著；野生型及突变型TG6蛋白与polyQ蛋白之间存在着相互作用，且均能通过促进polyQ蛋白由可溶性转变为不可溶进而促使其形成细胞内聚集体；过表达突变型TG6可促进星状孢子素（STS）或钙离子载体（A23187）诱导的NIH3T3细胞发生凋亡, 提示突变型TG6蛋白可能通过促进凋亡相关蛋白的激活加速了细胞凋亡。以上研究为后续的SCA35分子机制研究奠定了基础。