唐氏综合征关键区域编码蛋白TTC3影响个体发育的机制研究

The TTC3 gene is located within the Down syndrome critical region (DSCR) on human chromosome 21, the trisomic locus responsible for Down syndrome (DS). Although dosage imbalance of the genes located within the DSCR is believed to play a role in the molecular pathogenesis of DS, due to the complexities of the clinical manifestation of DS, the molecular mechanisms remain to be determined. The TTC3 gene encodes several Tetratricopeptide repeat (TRP) motifs at N-terminal end and canonical E3 RING finger motif at the C-terminal end. Our previous results showed that TTC3 interacts with Hsp90, Hsp70 and PSMB6 through its N-terminus, which indicate TTC3 may play an important role in ubiquitinating those misfolded client proteins binding to molecular chaperone, and bring them to proteasome. We also identified a protein (TTC3 Interactive Protein in Mitochondria, TIPIM) interacted with TTC3 by using yeast two hybrid, this protein locates in mitochondria and may play a role in the DNA damage repairmen in the mitochondria, which suggests that overexpression of TTC3 may cause dysfunction of mitochondria and further induce apoptosis. The TTC3 transgenic mice we generated have shown serious retardation during the development with phenotypes such as kyphosis and short neck, which are similar to the observation in other Down syndrome mice model and the phenotypes of DS patients. In this application, we propose to further study the molecular mechanism underlying the abnormal development of TTC3 transgenic mice. We are going to thoroughly analysis the phenotypes of the mice, mostly focus on the development of nervous system, bone and heart. We are interested in (1) does the transgenic mouse have other phenotypes mimic the DS patients; whether the TTC3 transgenic mice have learning and memory deficiency; (2) how TTC3 affect the function of those proteins interact with it, such as TIPIM, Akt and Citron kinase; (3) whether the overexpression of TTC3 may cause the mitochondria dysfunction. Due to the transgenic mice we have now are hard to breed, we also plan to generate TTC3 conditional transgenic mouse for future research.

TTC3基因位于人类21号染色体的21q22.2，该区域内的基因被认为与大部分唐氏综合征表型有关，所以被称为唐氏综合征关键区域。有关TTC3蛋白的具体功能目前少有报道。在前一个国家自然科学基金（30970610）的资助下，我们发现TTC3可与分子伴侣Hsp90、Hsp70及蛋白酶体β6亚基（PSMB6）等蛋白相互作用，提示TTC3可能是连接分子伴侣和蛋白酶体之间的一个桥梁；我们还发现了一个定位于线粒体中的TTC3相互作用蛋白，该蛋白的泛素化和降解受到TTC3的调控，提示TTC3高表达可能影响线粒体的正常功能，继而导致细胞增殖与分化的异常；我们制备的TTC3转基因小鼠发育缓慢，而且表现出明显的脊柱后凸等表型，与其它唐氏综合征模型小鼠有相似之处。本项目中我们计划在此基础上从分子机制研究和动物模型解析两个方面着手，进一步深入研究TTC3的正常生理功能，以及当其过表达时导致个体发育异常的分子机制。

人TTC3（Tetratricopeptide repeat domain 3）基因位于第21号染色体，编码全长2025个氨基酸残基的TTC3蛋白。先前研究表明，TTC3在胚胎发育过程中呈现出时序性和组织特异性的表达，暗示其可能在胚胎发育过程中发挥了重要的调控作用。与此同时，该基因位于染色体唐氏综合征关键区域（DSCR）上，提示其可能与唐氏综合征的发病存在关联。我们的研究发现TTC3可与分子伴侣Hsp90、Hsp70及蛋白酶体β6亚基（PSMB6）等蛋白相互作用，提示TTC3可能是连接分子伴侣和蛋白酶体之间的一个桥梁；我们还发现了定位于线粒体中DNA聚合酶gamma和TTC3相互作用，该蛋白的泛素化和降解受到TTC3的调控，提示TTC3高表达可能影响线粒体的正常功能，继而导致细胞增殖与分化的异常；我们构建了多株TTC3转基因小鼠，但是在后代中均检测不到该蛋白的表达，我们推测可能是TTC3蛋白易形成聚集体导致内源性Ttc3蛋白失活，使得高表达外源TTC3的胚胎早期死亡造成的，因为我们制备的Ttc3基因敲除小鼠在出生后很快死亡，Ttc3-/-的MEF细胞增殖缓慢。