Drivers of Hsp104 potentiation revealed by scanning mutagenesis of the middle domain.
Drivers of Hsp104 potentiation revealed by scanning mutagenesis of the middle domain.
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通过扫描中间结构域的诱变揭示了 Hsp104 增强的驱动因素。
DOI：
10.1002/pro.4126
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Jackrel,MeredithE
中科院分区：
文献类型：
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作者：
Ryan,JeremyJ;Bao,Aaron;Bell,Braxton;Ling,Cendi;Jackrel,MeredithE
Hsp104是一种酵母蛋白分解酶，它可以通过在整个卷曲-卷曲中间结构域（MD）的不同位置发生大量错义突变而增强。增强的Hsp104变体可以对抗TDP - 43、FUS和α -突触核蛋白的毒性和错误折叠，这些蛋白与神经退行性疾病有关。然而，增强的MD变异通常表现出脱靶毒性。此外，许多退化突变如何赋予增强作用仍然是一个困惑，这阻碍了治疗性Hsp104变体的工程设计。在这里，我们试图全面定义Hsp104增强的关键驱动因素。利用扫描诱变技术，我们反复研究了Hsp104 MD中每个位置的调制效应。筛选该文库以鉴定增强变体发现，MD中26%位置的错义突变产生了对抗FUS毒性的变体。螺旋2 -螺旋3/4 MD界面的调制会增强Hsp104，而类似的螺旋1-2界面的突变则不会。令人惊讶的是，我们发现增强Hsp104对人类疾病底物的活性比削弱Hsp104的天然功能更有可能。我们发现单个突变可以广泛地破坏MD结构并导致功能增强，这表明这可能是赋予Hsp104增强的共同机制。使用这种方法，我们已经证明了MD的调制可以产生具有减少脱靶效应的工程变体。
Hsp104, a yeast protein disaggregase, can be potentiated via numerous missense mutations at disparate locations throughout the coiled‐coil middle domain (MD). Potentiated Hsp104 variants can counter the toxicity and misfolding of TDP‐43, FUS, and α‐synuclein, proteins which are implicated in neurodegenerative disorders. However, potentiated MD variants typically exhibit off‐target toxicity. Further, it has remained confounding how numerous degenerate mutations confer potentiation, hampering engineering of therapeutic Hsp104 variants. Here, we sought to comprehensively define the key drivers of Hsp104 potentiation. Using scanning mutagenesis, we iteratively studied the effects of modulation at each position in the Hsp104 MD. Screening this library to identify enhanced variants reveals that missense mutations at 26% of positions in the MD yield variants that counter FUS toxicity. Modulation of the helix 2–helix 3/4 MD interface potentiates Hsp104, whereas mutations in the analogous helix 1–2 interface do not. Surprisingly, we find that there is a higher likelihood of enhancing Hsp104 activity against human disease substrates than impairing Hsp104 native function. We find that single mutations can broadly destabilize the MD structure and lead to functional potentiation, suggesting this may be a common mechanism conferring Hsp104 potentiation. Using this approach, we have demonstrated that modulation of the MD can yield engineered variants with decreased off‐target effects.