Proteasomal degradation of ubiquitylated proteins is sophisticatedly regulated at multiple levels^1–3. A primary regulatory checkpoint is the removal of ubiquitin chains from substrates by the deubiquitylating enzyme USP14 that associates reversibly with the proteasome. How USP14 is activated and regulates the proteasome function remains unknown^4–7. Here we report cryo-electron microscopy (cryo-EM) structures of human USP14 in complex with the 26S proteasome in nine conformational states at 3.0-3.6 Å resolution, captured during polyubiquitylated protein degradation. Time-resolved cryo-EM analysis of the conformational continuum revealed two parallel pathways of proteasome state transitions induced by USP14 and captured transient conversion of substrate-engaged intermediates into substrate-inhibited intermediates. On the substrate-engaged pathway, USP14 activation allosterically reprograms conformational landscape of the AAA-ATPase motor and stimulates opening of the core particle gate^8–10, enabling observation of a near-complete cycle of asymmetric ATP hydrolysis around the ATPase ring during processive substrate unfolding. Dynamic USP14-ATPase interactions decouple the ATPase activity from RPN11-catalysed deubiquitylation^11–13 and kinetically introduce three regulatory checkpoints on the proteasome, at the steps of ubiquitin recognition, substrate translocation initiation and ubiquitin chain recycling. These findings provide unprecedented insights into the complete functional cycles of USP14-regulated proteasome and of USP14 activation-deubiquitylation-disassembly and establish mechanistic foundations for USP14-targeted therapeutic discovery.

中文翻译：

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USP14和蛋白酶体动态相互调节的机制见解

泛素化蛋白质的蛋白酶体降解在多个水平^1-3受到精密调节。一个主要的监管检查点是通过与蛋白酶体可逆结合的去泛素化酶 USP14 从底物中去除泛素链。USP14 如何被激活并调节蛋白酶体功能仍然未知^4–7. 在这里，我们报告了人类 USP14 与 26S 蛋白酶体在 9 种构象状态下以 3.0-3.6 Å 分辨率形成复合物的冷冻电子显微镜 (cryo-EM) 结构，在多泛素化蛋白质降解过程中捕获。构象连续体的时间分辨冷冻电镜分析揭示了由 USP14 诱导的蛋白酶体状态转变的两条平行途径，并捕获了底物参与中间体向底物抑制中间体的瞬时转化。在底物参与通路上，USP14 激活变构地重新编程 AAA-ATPase 马达的构象景观并刺激核心粒子门的打开^8-10，能够在进行性底物解折叠过程中观察到围绕 ATPase 环的不对称 ATP​​ 水解的近乎完整的循环。动态 USP14-ATPase 相互作用将 ATPase 活性从 RPN11 催化的去泛素化^{11-13 中}解耦，并在泛素识别、底物易位起始和泛素链回收的步骤中在蛋白酶体上动力学引入三个调节检查点。这些发现为 USP14 调节的蛋白酶体和 USP14 激活-去泛素化-分解的完整功能周期提供了前所未有的见解，并为 USP14 靶向治疗发现建立了机制基础。