鼠疫杆菌效应蛋白YopJ与MAPK激酶的作用机制研究

The emerging of novel pathogens poise great urgency to a comprehensive understanding of the mechanism underlying their pathogenesis. Most Gram nagtive bacteria employ the type III secretion system(T3SS) to translocate virulence proteins, collectively known as effectors. Once arriving at the host cell, the effectors manipulate specific host cellular process, thereby promoting bacterial invasion and proliferation. The YopJ superfamily effectors are the most widely distributed T3SS effectors, as well the only known enzymes that acetylate Ser and Thr residues in proteins. In mammalian cells, YopJ protein produced by Yersinia spp interacts with MAPK kinases and IKKβ, which play critical roles in MAPK and NF-κB pathways, respectively. YopJ cetylates important Ser and Thr residues on the activation loop of these kinases to inhibit their activation. However, the mechanism by which YopJ binds and acetylate aubstrates remains unclear. We are going to crystal YopJ in complex with MAPK kinase, and solve the complex structures to reveal how YopJ recognizes and acetylates its substrate, thereby providing foundation for further development of YopJ in curing tumor and immune-related deseases.

目前，各种新型致病菌层出不穷，使得深入研究其致病机理显得尤为重要。革兰氏阴性菌通常借助于三型分泌系统（T3SS）向宿主细胞中输送多种效应蛋白以调控某些特定的信号通道，从而达到抑制宿主细胞免疫反应的目的。YopJ家族效应蛋白是分布最为广泛的一类T3SS效应蛋白，也是目前唯一已知的可以修饰蛋白肽链中的丝氨酸（Ser）和苏氨酸（Thr）的乙酰转移酶。由鼠疫杆菌产生的YopJ蛋白可以识别在MAPK和NF-κB通路中起关键作用的MAPK激酶和IKKβ，并乙酰化其活化环（activation loop）上保守的丝氨酸和苏氨酸，从而阻止这些残基被磷酸化以激活激酶。但YopJ识别这些激酶的机制还不明确，我们将通过解析YopJ与MAPK激酶成员的复合物结构，揭示YopJ识别靶标蛋白的模式，阐述YopJ乙酰化活化环的作用机制，为进一步开发YopJ成为免疫抑制和肿瘤治疗的潜在药物分子提供理论基础。

YopJ家族效应蛋白是目前已经发现的分布最为广泛的一类T3SS效应蛋白，其家族成员广泛分布在动物致病菌和植物致病菌中，并在致病过程中发挥着重要的作用。在本项目中，我们通过对YopJ家族蛋白的结构研究证实植酸InsP6可以异构调节YopJ家族蛋白的底物的结合。结合与不结合InsP6的PopP2结构对比揭示InsP6结合过程中可以诱导fold- switching区域的二级结构从一段α-螺旋转变成一个反向β-折叠片层，并通过连接β-折叠的环区来稳定底物识别螺旋，从而实现对底物结合的精确调控。虽然fold-switching区域在其他YopJ家族蛋白中的存在形式不一样，但它们都可能在InsP6的调控下实现较大的构象变化，进而改变底物识别螺旋的位置。所以我们的研究结果突破了原计划书中的单个蛋白与底物复合物的作用机制阐述的局限，而是从更高层面更深入的揭示了整个蛋白家族的一个新颖的工作机制。