Proteasomal conformation controls unfolding ability
Proteasomal conformation controls unfolding ability
复制标题
DOI：
10.1073/pnas.2101004118
复制
发表时间：
2021-06-22
影响因子：
11.1
通讯作者：
Kraut, Daniel A.
中科院分区：
文献类型：
--
作者：
Cresti, Julianna R.;Manfredonia, Abramo J.;Kraut, Daniel A.
26S蛋白酶体是真核细胞中负责大部分蛋白质降解的大分子机器。当蛋白酶体降解泛素化蛋白质时，它从底物接受构象（s1）转变为一组底物加工构象（s3样），每种构象都通过不同的分子内接触稳定。研究这些构象变化的工具仍然有限，尽管已提出几种相互作用对于稳定蛋白酶体的各种构象很重要，但很难在平衡条件下直接测试这些相互作用。在这里，我们描述了一个构象敏感的福斯特共振能量转移测定，其中荧光蛋白融合到Sem1和Rpn 6，这是更接近彼此在基板处理构象比在基板接受构象。使用此测定，我们发现，两组相互作用，一个涉及Rpn 5和另一个涉及Rpn 2，都是重要的稳定基板加工构象。破坏这些相互作用的突变既使底物加工构象相对于底物接受构象不稳定，又降低蛋白酶体成功解折叠和降解难以解折叠底物的能力，从而在蛋白酶体的构象状态与其解折叠能力之间提供联系。
The 26S proteasome is the macromolecular machine responsible for the bulk of protein degradation in eukaryotic cells. As it degrades a ubiquitinated protein, the proteasome transitions from a substrate-accepting conformation (s1) to a set of substrateprocessing conformations (s3 like), each stabilized by different intramolecular contacts. Tools to study these conformational changes remain limited, and although several interactions have been proposed to be important for stabilizing the proteasome's various conformations, it has been difficult to test these directly under equilibrium conditions. Here, we describe a conformationally sensitive Forster resonance energy transfer assay, in which fluorescent proteins are fused to Sem1 and Rpn6, which are nearer each other in substrate-processing conformations than in the substrate-accepting conformation. Using this assay, we find that two sets of interactions, one involving Rpn5 and another involving Rpn2, are both important for stabilizing substrate-processing conformations. Mutations that disrupt these interactions both destabilize substrate-processing conformations relative to the substrate-accepting conformation and diminish the proteasome's ability to successfully unfold and degrade hard-to-unfold substrates, providing a link between the proteasome's conformational state and its unfolding ability.