Kinetic effects in directional proteasomal degradation of the green fluorescent protein
Kinetic effects in directional proteasomal degradation of the green fluorescent protein
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DOI：
10.1063/5.0015191
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发表时间：
2020-09-14
影响因子：
4.4
通讯作者：
Stan, George
中科院分区：
文献类型：
--
作者：
Avestan, Mohammad Sadegh;Javidi, Alex;Stan, George
26 S真核蛋白酶体是位于泛素-蛋白酶体系统中心的ATP依赖性降解机器，其通过泛素化蛋白的解折叠和降解来维持细胞活力。其19 S调节颗粒使用强大的异六聚体AAA+ ATP酶马达，该马达展开底物蛋白并将其穿过狭窄的中心孔，以便在相关的20 S肽酶内降解。在这项研究中，我们探测的ATP酶马达的展开和易位机制进行粗粒度模拟机械拉的绿色荧光蛋白底物通过孔。为了辨别控制底物蛋白质的N-C或C-N定向加工的因素，我们使用三种不同的模型，包括连续拉动，以恒定速度或恒定力，或重复力的不连续拉动。我们的研究结果揭示了不对称的展开要求，在N-和C-末端的连续施加力的牵引雅阁符合较软的机械界面附近的N-末端和非均匀的孔隙表面施加的限制。相比之下，当力施加在较软的N-末端时，通过AAA+马达模拟可变夹持的重复力施加导致较慢的展开动力学。这种行为可归因于一方面重折叠与另一方面未折叠的N-末端α-螺旋的旋转柔性和易位之间的动态竞争。这些结果突出了机械，热力学和动力学效应之间的相互作用，在定向降解的蛋白酶体。
The 26S eukaryotic proteasome is an ATP-dependent degradation machine at the center of the ubiquitin-proteasome system that maintains cell viability through unfolding and degradation of ubiquitinated proteins. Its 19S regulatory particle uses a powerful heterohexameric AAA+ ATPase motor that unfolds substrate proteins and threads them through the narrow central pore for degradation within the associated 20S peptidase. In this study, we probe unfolding and translocation mechanisms of the ATPase motor by performing coarse-grained simulations of mechanical pulling of the green fluorescent protein substrate through the pore. To discern factors controlling the N-C or C-N directional processing of the substrate protein, we use three distinct models involving continuous pulling, at constant velocity or constant force, or discontinuous pulling with repetitive forces. Our results reveal asymmetric unfolding requirements in N- and C-terminal pulling upon continuous application of force in accord with the softer mechanical interface near the N-terminal and restraints imposed by the heterogeneous pore surface. By contrast, repetitive force application that mimics variable gripping by the AAA+ motor results in slower unfolding kinetics when the force is applied at the softer N-terminal. This behavior can be attributed to the dynamic competition between, on the one hand, refolding and, on the other, rotational flexibility and translocation of the unfolded N-terminal alpha -helix. These results highlight the interplay between mechanical, thermodynamic, and kinetic effects in directional degradation by the proteasome.