The importance of proteasome grip depends on substrate stability
The importance of proteasome grip depends on substrate stability
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DOI：
10.1016/j.bbrc.2023.08.025
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发表时间：
2023-08-15
影响因子：
3.1
通讯作者：
Kraut，Daniel A.
中科院分区：
文献类型：
--
作者：
Stanton，Destini A.;Ellis，Emily A.;Kraut，Daniel A.
26 S蛋白酶体是真核生物细胞内蛋白质解折叠和降解的主要酶。ATP酶（Rpt 1-Rpt 6）的六聚环抓住底物，通过中心孔将其拉入20 S降解室，从而将其展开。在每个Rpt亚基中含有所谓的芳香桨基序的一组孔环被认为对于蛋白酶体的解折叠和易位底物的能力是重要的。基于结构和机械实验，来自相邻Rpt子单元的桨状物（其以螺旋楼梯构造排列）以手对手型机构抓紧并拉动基底，在楼梯的底部脱离并在顶部重新接合。我们测试的贡献的芳香桨展开基板的不同的稳定性突变桨单独或组合。对于易于展开的底物（绿色荧光蛋白的环状排列体; GFP），突变对降解速率几乎没有影响。对于具有中等稳定性（增强的GFP）的底物，个体突变对GFP展开速率有适度的影响，并且交替芳香桨突变体比连续突变体对展开有更大的不利影响。对于更稳定的底物（超折叠GFP），解折叠总体上较慢，并且多个同时突变基本上阻止解折叠。我们的研究结果强调了上下文相关的需要抓地力展开过程中，支持基板展开和易位的手在手上的模型，并建议，对于难以展开基板，重要的是要有同时强大的接触基板展开发生。结果还表明了一个动力学校正模型，其中不能容易地展开的底物被剪切，去除起始区域并防止徒劳的展开尝试。
The 26S proteasome is responsible for the unfolding and degradation of intracellular proteins in eukaryotes. A hexameric ring of ATPases (Rpt1-Rpt6) grabs onto substrates and unfolds them by pulling them through a central pore and translocating them into the 20S degradation chamber. A set of pore loops containing a so-called aromatic paddle motif in each Rpt subunit is believed to be important for the proteasome's ability to unfold and translocate substrates. Based on structural and mechanistic experiments, paddles from adjacent Rpt subunits, which are arrayed in a spiral staircase conformation, grip and pull on the substrate in a hand-over-hand type mechanism, disengaging at the bottom of the staircase and re-engaging at the top. We tested the contribution of the aromatic paddles to unfolding substrates of differing stabilities by mutating the paddles singly or in combination. For an easy-to-unfold substrate (a circular permutant of green fluorescent protein; GFP), mutations had little effect on degradation rates. For a substrate with moderate stability (enhanced GFP), there were modest effects of individual mutations on GFP unfolding rates, and alternating aromatic paddle mutants had a larger detrimental effect on unfolding than sequential mutants. For a more stable substrate (superfolder GFP), unfolding is overall slower, and multiple simultaneous mutations essentially prevent unfolding. Our results highlight the context-dependent need for grip during unfolding, support the hand-over-hand model for substrate unfolding and translocation, and suggest that for hard-to-unfold substrates, it is important to have simultaneous strong contacts to the substrate for unfolding to occur. The results also suggest a kinetic proofreading model, where substrates that cannot be easily unfolded are instead clipped, removing the initiation region and preventing futile unfolding attempts.