Htet Zaw Min, Dong Ken C, Martin Andreas
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720, USA.
California Institute for Quantitative Biosciences, University of California at Berkeley, Berkeley, CA 94720, USA.
bioRxiv. 2024 Oct 24:2024.10.24.620116. doi: 10.1101/2024.10.24.620116.
The 26S proteasome is the major compartmental protease in eukaryotic cells, responsible for the ATP-dependent turnover of obsolete, damaged, or misfolded proteins that are delivered for degradation through attached ubiquitin modifications. In addition to targeting substrates to the proteasome, ubiquitin was recently shown to promote degradation initiation by directly modulating the conformational switching of the proteasome, yet the underlying mechanisms are unknown. Here, we used biochemical, mutational, and single-molecule FRET-based approaches to show that the proteasomal deubiquitinase Rpn11 functions as an allosteric sensor and facilitates the early steps of degradation. After substrate recruitment to the proteasome, ubiquitin binding to Rpn11 interferes with conformation-specific interactions of the ubiquitin-receptor subunit Rpn10, thereby stabilizing the engagement-competent state of the proteasome and expediting substrate insertion into the ATPase motor for mechanical translocation, unfolding, and Rpn11-mediated deubiquitination. These findings explain how modifications with poly-ubiquitin chains or multiple mono-ubiquitins allosterically promote substrate degradation and allow up to four-fold faster turnover by the proteasome.
26S蛋白酶体是真核细胞中的主要区室蛋白酶，负责通过附着的泛素修饰对过时、受损或错误折叠的蛋白质进行ATP依赖性的周转降解。除了将底物靶向蛋白酶体之外，最近研究表明泛素还可通过直接调节蛋白酶体的构象转换来促进降解起始，但其潜在机制尚不清楚。在此，我们使用生化、突变和基于单分子荧光共振能量转移的方法表明，蛋白酶体去泛素化酶Rpn11作为变构传感器发挥作用，并促进降解的早期步骤。在底物募集到蛋白酶体后，泛素与Rpn11的结合会干扰泛素受体亚基Rpn10的构象特异性相互作用，从而稳定蛋白酶体的参与活性状态，并加速底物插入ATP酶马达进行机械转运、展开以及Rpn11介导的去泛素化。这些发现解释了多聚泛素链或多个单泛素的修饰如何变构促进底物降解，并使蛋白酶体的周转速度加快四倍。
