Function and regulation of the Cdc48-20S proteasome

蛋白酶体是真核生物和古生物领域中主要的依赖于ATP的蛋白酶，在蛋白质质量控制过程中需要降解受损或错误折叠的蛋白质，并调节不同的细胞信号通路。为了执行蛋白质降解，20S多肽酶与真核生物19S调控颗粒的六聚体AAA+酶或古生菌PAN的AAA+环结合。在初步研究中，我确定CDC48-20S复合体是嗜酸热原体中的一个新的蛋白酶体。CDC48(别名p97/VCP/VAT)是古细菌和真核生物中普遍存在的一种含量丰富且必需的双环AAA+分子机器。古生菌CDC48的生理功能一直不确定，但真核细胞CDC48以一种未知的机制参与了许多生物学过程，包括膜融合、细胞周期调节和泛素-蛋白酶体系统。有趣的是，使古生菌CDC48和20S合作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多肽酶协同作用的蛋白分解可能是CDC48家族的祖先功能，该家族经过数十亿年的进化已经被保存下来。关于这个新的蛋白酶体的工作方式、生理功能和细胞调控的关键问题仍有待回答。解决古细菌CDC48-20S蛋白酶体的高分辨率冷冻EM结构将有助于理解这两个大分子是如何相互作用的。我还将利用生化相互作用研究、机制酶学和生物物理表征来探索接头蛋白如何与CDC48相互作用，影响其构象，并以变构方式调节其功能。最后，我将测试另一种CDC48-20S蛋白酶体在生命真核领域发挥作用的令人兴奋的可能性。我的目标是了解CDC48-20S蛋白酶体如何识别、展开、转位并最终降解天然和错误折叠的蛋白质。我的研究将回答有关蛋白酶体进化起源的问题，并将为这种依赖于ATP的蛋白酶和相关酶的工作原理提供洞察力。我的工作还将有助于阐明真核细胞CDC48在泛素依赖的蛋白分解、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.