黄牛Sho疏水结构域多态对疯牛病调控蛋白功能的影响

Bovine spongiform encephalopathy is one of prion diseases,regulated by two proteins prion protein (PrP) and Shadow of prion protein (Shadoo, Sho). Sho contains an internal hydrophobic domain (HD) which displays exceptionally high conservation across a wide range of species, implying that it plays a vital role in exercising Sho's physiological function. Like the HD of cellular prion protein (PrPC), Sho's HD has stress-protective activity. Furthermore, an interaction between Sho's HD and PrPC has been identified, suggesting Sho may play a role in the physiological function of PrPC and prion pathogenesis. In our previous work at population level, a 12-bp insertion/deletion (indel) polymorphism of Sho's HD was only found in BSE-susceptible cattle breeds but never in BSE-unsusceptible buffalo breeds. In addition, several indel polymorphisms have also been reported in other BSE-susceptible animals, such as sheep and human. These results suggest that Sho's HD is an important domain and the indel polymorphisms may be associated with the susceptibility of prion diseases. Unfortunately, the influences of the indel polymorphisms on their functions of Sho and PrPC have rarely been studied. Further studies are needed to address these questions. In this study, we focus on several biological processes, which are relative to prion pathogenesis, including stress-protective activity, the interaction between Sho and PrPC, and the C-terminal fragments of PrPC and Sho produced by endogenous protease. We will investigate the influences on these biological processes due to the 12-bp indel polymorphism of Sho's HD in cattle, and discuss the role of the indel polymorphism in bovine spongiform encephalopathy and a possible regulation mechanism. This study will provide new insights into clarifying the unresolved pathogenic mechanism of prion diseases.

疯牛病是一种朊病毒疾病，受到朊病毒和Shadoo（Sho）两种蛋白调控。Sho含有一个高度保守的疏水结构域（HD），HD具有神经保护能力，能与正常型朊病毒（PrPC）相互作用。申请人前期研究发现：在群体水平疯牛病易感的黄牛Sho HD中有12-bp的插入/缺失(indel)多态，但在不易感的水牛中未发现。同时，朊病毒疾病易感物种中也发现有不同程度的indel现象，提示Sho HD中indel多态可能与该病的易感性相关。然而，这些多态对Sho和PrPC功能的影响知之甚少。本项目拟从与该病的感染和发展有着紧密联系的几个生物学问题入手，包括胁迫神经保护能力、Sho与PrPC的相互作用、Sho和PrPC C末端片段剪切方式，考察黄牛Sho HD中12-bp indel多态对这些生物学过程的影响，探讨这些多态在疯牛病中的作用和可能的调控机制，从新的视角为朊病毒疾病的致病机理的解析提供有意义的线索。

疯牛病是一种朊病毒疾病，受朊病毒蛋白PrP和类朊病毒蛋白Sho 两种蛋白的调控。Sho和正常型的朊病毒蛋白（PrPC）在结构和功能上有许多相似性，且两个蛋白都是在神经系统中高表达。有意思的是，感染疾病型朊病毒蛋白（PrPSc）的小鼠和神经细胞中都会出现Sho急剧下降的情况。这些都暗示了Sho可能参与了朊病毒疾病的调控。我们之前的研究发现疯牛病易感的黄牛Sho蛋白的HD结构域有12-bp的插入/缺失（insertion/deletion, indel）多态现象，引起4个氨基酸的插入/缺失，但是疯牛病不易感的水牛中均未发现。本项目我们深入研究了Sho HD结构域indel多态对其蛋白结构、功能、及其与其它蛋白相互作用的影响。结果显示：1）indel多态对Sho蛋白的表达，糖苷键的形成，GPI锚的形成和蛋白的胁迫保护能力均无明显影响；2）indel多态会影响Sho和PrP的结合，酵母双杂交结果显示野生型Sho(wt) 以及12-bp插入的突变体Sho(+12-bp) 能够与PrP结合，然而HD结构域缺失Sho(△HD) 蛋白，以及12-bp缺失Sho(-12-bp) 蛋白会丧失与PrP的结合能力，提示Sho 与PrP蛋白的互作是通过HD结构域实现的。3）indel多态会对Sho C末端淀粉核心片段的形成产生的影响。首先，Sho野生型及其三种突变体蛋白在蛋白酶K（PK）的消化下，均会产生Sho的C1和C2片段，且在pH7.5的缓冲溶液中处理14天后的蛋白，更易形成这些短片段。其次，Sho(△HD)突变体蛋白对PK的消化抗性最强；4）通过非标记（Label-free）质谱定量检测技术，我们分析了与Sho(wt)或Sho(△HD) 相互作用的蛋白质组的差异蛋白，发现有80个蛋白能与Sho(wt)互作但不能与Sho(△HD) 相互作用，表明这些蛋白是通过HD结构域和Sho相互作用的。另外有35个蛋白在Sho(wt) 互作蛋白组样品中的含量高出Sho(△HD) 10倍以上。这些数据说明Sho的HD结构域中的GXXXG元件在Sho和其它蛋白相互作用中起到了重要作用。我们的这些结果揭示了Sho蛋白HD结构域会影响Sho蛋白的生化学性质，也会影响与其它蛋白的相互作用，这些为朊病毒生化学的研究提供了有力的数据支持。研究成果发表论文5篇，其中SCI论文4篇，正在整理和撰写论文1篇。