Function and regulation of the Cdc48-20S proteasome

蛋白酶体是真核生物和古细菌生命领域中主要的atp依赖蛋白酶，在蛋白质质量控制过程中降解受损或错误折叠的蛋白质以及调节多种细胞信号通路是必需的。为了进行蛋白质降解，20S肽酶与真核生物中19S调节颗粒的六聚体AAA+酶或与古细菌PAN的AAA+环结合。在初步研究中，我发现Cdc48-20S复合物是古细菌嗜酸热原体中的一种新型蛋白酶体。Cdc48（别名p97/VCP/VAT）是一种丰富而重要的双环AAA+分子机器，普遍存在于古细菌和真核生物中。古细菌Cdc48的生理作用长期以来一直不确定，但真核生物Cdc48以很大程度上未知的机制参与无数生物过程，包括膜融合、细胞周期调节和泛素-蛋白酶体系统。有趣的是，允许古细菌Cdc48和20S协同降解蛋白质的序列和结构特征在真核酶中也是保守的。因此，与20S肽酶合作的蛋白质水解可能是Cdc48家族的祖先功能，该家族已经在数十亿年的进化中保存下来。关于这种新型蛋白酶体的运作方式、生理功能和细胞调控的关键问题仍有待解答。解决古细菌Cdc48-20S蛋白酶体的高分辨率低温电镜结构将有助于了解这两个大分子如何相互作用。我还将探讨适配器蛋白如何与Cdc48相互作用，影响其构象，并通过生化相互作用研究，机械酶学和生物物理表征来变构调节其功能。最后，我将测试一种令人兴奋的可能性，即另一种Cdc48-20S蛋白酶体也在真核生物领域发挥作用。我的目标是了解天然和错误折叠的蛋白质是如何被Cdc48-20S蛋白酶体识别、展开、易位并最终降解的。我的研究将回答有关蛋白酶体进化起源的问题，并将为这种atp依赖性蛋白酶和相关酶的运作原理提供见解。我的工作也将有助于阐明真核生物Cdc48在泛素依赖性蛋白水解、细胞周期调节和蛋白质错误折叠疾病中的作用。

Proteasomes, the major ATP-dependent proteases in the eukaryotic and archaeal domains of life, are required for degradation of damaged or misfolded proteins during protein-quality control and for regulation of diverse cell-signalling pathways. To execute protein degradation, the 20S peptidase combines with the hexameric AAA+ enzymes of the 19S regulatory particle in eukarya or with the AAA+ ring of archaeal PAN.In pilot studies, I identified the Cdc48-20S complex as a novel proteasome in the archaeon Thermoplasma acidophilum. Cdc48 (alias p97/VCP/VAT) is an abundant and essential double-ring AAA+ molecular machine ubiquitously present in archaea and eukarya. The physiological role of archaeal Cdc48 has long been uncertain, but eukaryotic Cdc48 participates by largely unknown mechanism in a myriad of biological processes, including membrane fusion, cell-cycle regulation, and the ubiquitin-proteasome-system. Intriguingly, the sequence and structural features that allow archaeal Cdc48 and 20S to collaborate in protein degradation are also conserved in the eukaryotic enzymes. Proteolysis in collaboration with the 20S peptidase may therefore be an ancestral function of the Cdc48 family that has have been preserved over billions of years of evolution.Key questions about the modus operandi, physiological function, and cellular regulation of this novel proteasome remain to be answered. Solving a high-resolution cryo-EM structure of the archaeal Cdc48-20S proteasome will help understanding how both macromolecules interact. I will also explore how adapter proteins interact with Cdc48, affect its conformation, and allosterically regulate its function using biochemical interaction studies, mechanistic enzymology, and biophysical characterization. Finally, I will test the exciting possibility that an alternative Cdc48-20S proteasome also functions in the eukaryotic domain of life.I aim to understand how native and misfolded proteins are recognized, unfolded, translocated, and finally degraded by Cdc48-20S proteasome. My research will answer questions about the evolutionary origins of proteasomes and will provide insights into the operational principles of this ATP-dependent protease and related enzymes. My work will also help to clarify the role of eukaryotic Cdc48 during ubiquitin-dependent proteolysis, cell cycle regulation, and protein-misfolding diseases.