Department Biochemistry of Neurodegenerative Diseases, Institute of Biochemistry and Pathobiochemistry, Ruhr University, Bochum, Germany.
Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany.
J Mol Biol. 2024 Dec 1;436(23):168839. doi: 10.1016/j.jmb.2024.168839. Epub 2024 Oct 28.
Several proteins associated with neurodegenerative diseases, such as the mammalian prion protein (PrP), undergo liquid-liquid phase separation (LLPS), which led to the hypothesis that condensates represent precursors in the formation of neurotoxic protein aggregates. However, the mechanisms that trigger aberrant phase separation are incompletely understood. In prion diseases, protease-resistant and infectious amyloid fibrils are composed of N-terminally truncated PrP, termed C2-PrP. C2-PrP is generated by regulated proteolysis (β-cleavage) of the cellular prion protein (PrP) specifically upon prion infection, suggesting that C2-PrP is a misfolding-prone substrate for the propagation of prions. Here we developed a novel assay to investigate the role of both LLPS and β-cleavage in the formation of C2-PrP aggregates. We show that β-cleavage induces the formation of C2-PrP aggregates, but only when full-length PrP had formed biomolecular condensates via LLPS before proteolysis. In contrast, C2-PrP remains soluble after β-cleavage of non-phase-separated PrP. To investigate whether extracellular molecular chaperones modulate LLPS of PrP and/or misfolding of C2-PrP, we focused on Clusterin. Clusterin does not inhibit LLPS of full-length PrP, however, it prevents aggregation of C2-PrP after β-cleavage of phase-separated PrP. Furthermore, Clusterin interferes with the in vitro amplification of infectious human prions isolated from Creutzfeldt-Jakob disease patients. Our study revealed that regulated proteolysis triggers aberrant phase transition of biomolecular condensates into aggregates and identified Clusterin as a component of the extracellular quality control pathway to prevent the formation and propagation of pathogenic PrP conformers.
几种与神经退行性疾病相关的蛋白质，如哺乳动物朊病毒蛋白（PrP），会发生液-液相分离（LLPS），这导致了一个假说，即凝聚物代表了形成神经毒性蛋白聚集体的前体。然而，触发异常相分离的机制尚未完全理解。在朊病毒病中，蛋白酶抗性和感染性的淀粉样纤维由 N 端截断的 PrP 组成，称为 C2-PrP。C2-PrP 是通过细胞朊病毒蛋白（PrP）的受调控的蛋白水解（β-切割）产生的，特别是在朊病毒感染时，这表明 C2-PrP 是朊病毒传播的易错误折叠底物。在这里，我们开发了一种新的测定法来研究 LLPS 和β-切割在 C2-PrP 聚集体形成中的作用。我们表明，β-切割诱导 C2-PrP 聚集体的形成，但只有当全长 PrP 通过 LLPS 形成生物分子凝聚物后，β-切割才会发生。相比之下，C2-PrP 在非相分离的 PrP 经过β-切割后仍保持可溶性。为了研究细胞外分子伴侣是否调节 PrP 的 LLPS 和/或 C2-PrP 的错误折叠，我们专注于 Clusterin。Clusterin 不会抑制全长 PrP 的 LLPS，然而，它可以防止相分离的 PrP 经β-切割后 C2-PrP 的聚集。此外，Clusterin 干扰从克雅氏病患者中分离出的感染性人类朊病毒的体外扩增。我们的研究表明，受调控的蛋白水解触发生物分子凝聚物异常相变形成聚集体，并确定 Clusterin 为细胞外质量控制途径的组成部分，以防止致病性 PrP 构象的形成和传播。
