Structural and biochemical studies elucidating the biogenesis pathway of 20S proteasomes

在这项建议中，我们解决了在伴侣引导的组装和成熟的蛋白酶体20S核心粒子(CP)复合体中事件的空间和时间顺序。我们的目标是通过确定高分辨率的低温EM重建来从结构上表征CP生物发生的后期步骤，以确定蛋白酶体伴侣蛋白和20S CP亚基之间的确切相互作用。同时，我们要分析是什么机制(S)启动了β亚基的自催化激活。同时，我们想通过对单个组装中间体的表征，从生化和结构上研究到目前为止知之甚少的蛋白酶体CP生物发生的早期步骤，并且将被用来概括不同的体外组装步骤，目的是确定事件的顺序和它们的检查点。

In this proposal we address the spatial and temporal order of events in the chaperone-guided assembly and maturation of proteasomal 20S core particle (CP) complexes. We aim to structurally characterise the late steps of CP biogenesis by determining high-resolution cryo EM reconstructions in order to identify the exact interactions between proteasomal chaperones and 20S CP subunits. At the same time, we want to analyse by which mechanism(s) autocatalytic activation of the beta subunits is initiated. In parallel, we want to investigate the so far poorly understood early steps of proteasome CP biogenesis biochemically and structurally through the characterization of individual assembly intermediates, which will moreover be used to recapitulate distinct assembly steps in vitro with the aim to determine the order of events and their checkpoints.