Arkinson Connor, Dong Ken C, Gee Christine L, Costello Shawn M, Soe Aimee Chi, Hura Greg L, Marqusee Susan, Martin Andreas
California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA, USA.
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
Nat Struct Mol Biol. 2025 Apr 11. doi: 10.1038/s41594-025-01527-3.
The ubiquitin-like modifier FAT10 targets hundreds of proteins in the mammalian immune system to the 26S proteasome for degradation. This degradation pathway requires the cofactor NUB1, yet the underlying mechanisms remain unknown. Here, we reconstituted a minimal in vitro system with human components and revealed that NUB1 uses the intrinsic instability of FAT10 to trap its N-terminal ubiquitin-like domain in an unfolded state and deliver it to the 26S proteasome for engagement, allowing the degradation of FAT10-ylated substrates in a ubiquitin-independent and p97-independent manner. Using hydrogen-deuterium exchange, structural modeling and site-directed mutagenesis, we identified the formation of an intricate complex with FAT10 that activates NUB1 for docking to the 26S proteasome, and our cryo-EM studies visualized the highly dynamic NUB1 complex bound to the proteasomal Rpn1 subunit during FAT10 delivery and the early stages of ATP-dependent degradation. These findings identified a previously unknown mode of cofactor-mediated, ubiquitin-independent substrate delivery to the 26S proteasome that relies on trapping partially unfolded states for engagement by the proteasomal ATPase motor.
类泛素修饰因子FAT10将哺乳动物免疫系统中的数百种蛋白质靶向26S蛋白酶体进行降解。这种降解途径需要辅因子NUB1，但其潜在机制仍不清楚。在这里，我们用人源成分重建了一个最小的体外系统，发现NUB1利用FAT10的内在不稳定性将其N端类泛素结构域捕获在未折叠状态，并将其递送至26S蛋白酶体进行结合，从而以不依赖泛素和不依赖p97的方式降解FAT10修饰的底物。通过氢-氘交换、结构建模和定点诱变，我们确定了与FAT10形成的一种复杂复合物，该复合物激活NUB1以对接至26S蛋白酶体，并且我们的冷冻电镜研究观察到在FAT10递送和ATP依赖性降解的早期阶段，高度动态的NUB1复合物与蛋白酶体Rpn1亚基结合。这些发现确定了一种以前未知的辅因子介导的、不依赖泛素的底物递送至26S蛋白酶体的模式，该模式依赖于捕获部分未折叠状态以供蛋白酶体ATP酶马达结合。
