Conformational switches of AXIN in Wnt/catenin signalling

Wnt/β-catenin信号网络高度保守，控制着胚胎发育中的重要事件以及整个生命过程中的组织动态平衡。Wnt/β-catenin信号的大小由转录共激活因子β-catenin的水平和活性决定，在其第二个突出作用中，它还介导细胞间的黏附，在这种情况下作用于质膜。转录活性的β-连环蛋白的水平一直受到β-连环蛋白破坏复合体的作用的限制，这是一种多蛋白复合体，它捕获β-连环蛋白，然后依次将其磷酸化和泛素化，使其成为蛋白酶体降解的靶点。AXIN是破坏复合体的中央支架蛋白。它包含两个结构域，N-端的RGS(G蛋白信号转导调节因子)结构域和C-末端的DIX(蓬乱和轴蛋白)结构域，这两个结构域之间有一个延伸的连接子。轴蛋白的构象状态、它们的调控转换以及构象对轴蛋白与其他信号成分相互作用的影响还知之甚少。此外，目前尚不清楚构象转换是否会影响轴蛋白的聚合能力。利用昆虫细胞/杆状病毒系统，我们成功地表达和纯化了包括AXIN1在内的所有β-连环蛋白破坏复合体的全长成分。这为研究破坏复杂组件之间的相互作用以及这些结合事件的机制细节提供了可能。该项目的目的是了解Axin的翻译后修饰如何控制Axin的构象和寡聚状态，从而通过与β-连环蛋白破坏复合体和Wnt信号体的组件结合来组装蛋白质复合体的能力。该项目旨在评估Axin的构象是如何通过磷酸化和PAR化来确定的，以及这些翻译后修饰如何决定Axin与其他Wnt/β-catenin途径成分的相互作用，从而控制β-连环蛋白依赖的转录。

The Wnt/beta-catenin signalling network is highly conserved and controls important events in embryonic development as well as tissue homeostasis throughout life. The magnitude of Wnt/beta-catenin signalling is determined by the levels and activity of the transcriptional coactivator beta-catenin which also, in its second prominent role, mediates cell-cell adhesion, in this case acting at the plasma membrane. The levels of transcriptionally active beta-catenin are constantly being limited through the action of the beta-catenin destruction complex, a multi-protein complex that captures beta-catenin and then sequentially phosphorylates and ubiquitinates it to target it for proteasomal degradation.AXIN is the central scaffolding protein of the destruction complex. It contains two domains, an N-terminal RGS (regulator of G-protein signalling) domain and a C-terminal DIX (Dishevelled and AXIN) domain with an extended linker connecting the two domains. The conformational states of AXIN, their regulated transition and the impact the conformations have on interactions of AXIN with other signalling components are poorly understood. Moreover, it remains unknown whether conformational switching affects the ability of AXIN to polymerise. Using the insect cell/baculovirus system, we have successfully expressed and purified all full-length components of the beta-catenin destruction complex, including AXIN1. This opens up the possibility to study the mutual interactions of destruction complex components and the regulation of these binding events in mechanistic detail. The aim of this project is to understand how post-translational modification of AXIN controls AXIN conformation and oligomerisation status and thereby its ability to assemble protein complexes by binding to components of the beta-catenin destruction complex and Wnt signalosome.The project aims to assess how AXIN conformation is determined by phosphorylation and PARylation, and how in turn these post-translational modifications determine interactions of AXIN with other Wnt/beta-catenin pathway components to control beta-catenin-dependent transcription.