端粒与端粒酶联系的分子调控机制及其在抗肿瘤中的潜在应用

Telomearse is the key enzyme in the maintenance of telomere length. Its function in extending telomere relies on the recognition of telomere DNA substrate at telomere end. However, for a long time the molecular basis for telomere-telomerase connection was elusive, nor was the regulation of this key process of telomerase function. Only until recently has the key molecule linking telomerase to telomere been discovered by us (Nature 2007). For the first time we demonstrated that an indispensable bridging/adaptor protein in the assembly of telomere protein complex, TPP1, is the long-sought-after key molecule in recruiting telomerase to telomere. It is now well accepted that TPP1 serves as the molecular linker between telomere and telomerase, and furthermore, we found that through direct protein-protein interaction TPP1 enhances the telomerase activity. The discovery that a telomere structural protein serves also the recruiter of telomerase solved the long-term question that compact telomere structure may "impede" the access of telomerase. However, how this process is regulated within cells is still not known. We propose to search for the possible regulator(s) and modification(s) of TPP1 in modulating telomerase association and activity, and testify the multi-factor regulation hypothesis in the control of TPP1-telomerase interaction. Based on this we will examine the anti-tumor effect of blocking TPP1-telomerase interaction using transgenic mouse model. This study will shed light on new strategies and validate the concept of blocking telomerase action for anti-tumor treatment.

端粒酶是真核细胞（包括人和各种哺乳动物）维护端粒长度的关键酶，其催化端粒延伸的功能依赖于对端粒底物的识别；然而，在过去很长的时间内，对其端粒定位的分子基础及其调节机制还缺乏了解。直到最近，连接端粒和端粒酶的关键纽带分子才由我们首次发现并报道（Nature，2007）。在我们的前期工作中，我们发现端粒紧密结构单位 - 端粒蛋白复合体的关键组装蛋白TPP1是负责招募端粒酶定位于端粒、并可促进端粒酶的活性的关键分子。这一发现解决了人们对于端粒结构"阻碍"端粒酶识别端粒末端DNA底物的疑问，但对于细胞如何调节TPP1和端粒酶之间的的动态结合机制还不清楚。我们拟从搜索这一过程的可能的调节因子和蛋白质修饰作用等入手，探索影响TPP1-端粒酶联系的多因子调节机制，并探讨阻断TPP1-端粒酶之间的联系在抗肿瘤中的潜在应用，为以端粒酶为靶标的抗肿瘤药物筛选和治疗提供新的思路和实验依据。

在本项工作中，我们重点分析了端粒损伤条件下、端粒蛋白TPP1的结合蛋白的差异、及应用新的BirA生物素标记技术搜索TPP1新互作蛋白、及建立TPP1 OB fold结构域缺失小鼠的工作。1）应用TPP1与TIN2结合缺陷的TPP1-dC突变体蛋白进行蛋白质组学分析发现，TPP1仍有少量与Shelterin复合体组分结合的数据，结合细胞学分析，TPP1-dC突变体可有少量端粒定位，说明突变体并没有完全丧失端粒定位的功能，但端粒DNA损伤的信号TIF的出现说明，端粒DNA部分受到损伤，蛋白质组学的结果也证明TPP1-dC突变体可与DNA-PKc、Ku86/ku70等结合，但Mre11-Rad50-Nbs1复合体基本未出现，表明TPP1-dC复合体主要集中在端粒单链DNA部分。2）应用BirA生物素标记方法、分析TPP1的互作蛋白的组学分析发现，端粒及DNA损伤修复、染色质重塑蛋白及DNA拓扑异构酶、DNA复制相关蛋白出现在分析结果中，表明与TPP1有稳定或瞬时的相互作用。3）通过经典的同源重组方法、建立并获得TPP1 OB折叠结构域缺失杂合小鼠，但未能获得纯合子小鼠，分析原因，可能突变体影响端粒维护，与文献中其它端粒Shelterin组分的缺失突变体小鼠的情形类似，暗示TPP1的端粒维护与端粒酶募集功能的不可分割性。通过本项工作，对于TPP1在端粒中的双重作用的认识得到了进一步深入。