Department of Biochemistry and Biophysics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA; Biochemistry and Molecular Biophysics Graduate Group, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA.
Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL 35487, USA.
Mol Cell. 2023 Sep 21;83(18):3314-3332.e9. doi: 10.1016/j.molcel.2023.07.029. Epub 2023 Aug 24.
Hsp104 is an AAA+ protein disaggregase that solubilizes and reactivates proteins trapped in aggregated states. We have engineered potentiated Hsp104 variants to mitigate toxic misfolding of α-synuclein, TDP-43, and FUS implicated in fatal neurodegenerative disorders. Though potent disaggregases, these enhanced Hsp104 variants lack substrate specificity and can have unfavorable off-target effects. Here, to lessen off-target effects, we engineer substrate-specific Hsp104 variants. By altering Hsp104 pore loops that engage substrate, we disambiguate Hsp104 variants that selectively suppress α-synuclein toxicity but not TDP-43 or FUS toxicity. Remarkably, α-synuclein-specific Hsp104 variants emerge that mitigate α-synuclein toxicity via distinct ATPase-dependent mechanisms involving α-synuclein disaggregation or detoxification of soluble α-synuclein conformers. Importantly, both types of α-synuclein-specific Hsp104 variant reduce dopaminergic neurodegeneration in a C. elegans model of Parkinson's disease more effectively than non-specific variants. We suggest that increasing the substrate specificity of enhanced disaggregases could be applied broadly to tailor therapeutics for neurodegenerative disease.
Hsp104 是一种 AAA+ 蛋白解聚酶，可溶解和重新激活被困在聚集状态下的蛋白质。我们已经设计了增强型 Hsp104 变体，以减轻与致命神经退行性疾病有关的 α-突触核蛋白、TDP-43 和 FUS 的毒性错误折叠。虽然这些增强型 Hsp104 变体是有效的解聚酶，但它们缺乏底物特异性，可能会产生不利的非靶向效应。在这里，为了减轻非靶向效应，我们设计了具有底物特异性的 Hsp104 变体。通过改变与底物结合的 Hsp104 孔环，我们区分了能够选择性抑制 α-突触核蛋白毒性但不抑制 TDP-43 或 FUS 毒性的 Hsp104 变体。值得注意的是，出现了针对 α-突触核蛋白的特异性 Hsp104 变体，它们通过涉及 α-突触核蛋白解聚或可溶性 α-突触核蛋白构象解毒的不同 ATP 酶依赖性机制来减轻 α-突触核蛋白毒性。重要的是，两种类型的针对 α-突触核蛋白的 Hsp104 变体都比非特异性变体更有效地减少了秀丽隐杆线虫帕金森病模型中的多巴胺能神经退行性病变。我们认为，提高增强型解聚酶的底物特异性可以广泛应用于为神经退行性疾病定制治疗方法。
