Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520.
Section of Infections of the Nervous System, National Institute for Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 2018 Dec 18;115(51):E11924-E11932. doi: 10.1073/pnas.1817477115. Epub 2018 Dec 3.
The human chaperonin Hsp60 is thought to play a role in the progression of Alzheimer's disease by mitigating against intracellular β-amyloid stress. Here, we show that the bacterial homolog GroEL (51% sequence identity) reduces the neurotoxic effects of amyloid-β(1-42) (Aβ42) on human neural stem cell-derived neuronal cultures. To understand the mechanism of GroEL-mediated abrogation of neurotoxicity, we studied the interaction of Aβ42 with GroEL using a variety of biophysical techniques. Aβ42 binds to GroEL as a monomer with a lifetime of ∼1 ms, as determined from global analysis of multiple relaxation-based NMR experiments. Dynamic light scattering demonstrates that GroEL dissolves small amounts of high-molecular-weight polydisperse aggregates present in fresh soluble Aβ42 preparations. The residue-specific transverse relaxation rate profile for GroEL-bound Aβ42 reveals the presence of three anchor-binding regions (residues 16-21, 31-34, and 40-41) located within the hydrophobic GroEL-consensus binding sequences. Single-molecule FRET analysis of Aβ42 binding to GroEL results in no significant change in the FRET efficiency of a doubly labeled Aβ42 construct, indicating that Aβ42 samples a random coil ensemble when bound to GroEL. Finally, GroEL substantially slows down the disappearance of NMR visible Aβ42 species and the appearance of Aβ42 protofibrils and fibrils as monitored by electron and atomic force microscopies. The latter observations correlate with the effect of GroEL on the time course of Aβ42-induced neurotoxicity. These data provide a physical basis for understanding how Hsp60 may serve to slow down the progression of Alzheimer's disease.
人们认为，热休克蛋白 60（Hsp60）通过减轻细胞内β-淀粉样蛋白应激，在阿尔茨海默病的进展中发挥作用。在这里，我们表明，细菌同源物 GroEL（51%序列同一性）减轻了淀粉样蛋白-β（1-42）（Aβ42）对人神经干细胞源性神经元培养物的神经毒性作用。为了了解 GroEL 介导的神经毒性阻断的机制，我们使用多种生物物理技术研究了 Aβ42 与 GroEL 的相互作用。通过对多个基于弛豫的 NMR 实验的全局分析，确定 Aβ42 作为单体与 GroEL 结合，寿命约为 1 ms。动态光散射表明，GroEL 溶解了新鲜可溶性 Aβ42 制剂中存在的少量高分子量多分散聚集物。GroEL 结合的 Aβ42 的残基特异性横向弛豫率谱揭示了存在三个锚定结合区域（残基 16-21、31-34 和 40-41），位于疏水性 GroEL 共识结合序列内。Aβ42 与 GroEL 结合的单分子 FRET 分析导致双标记 Aβ42 构建体的 FRET 效率没有明显变化，表明 Aβ42 在与 GroEL 结合时采样无规卷曲集合。最后，GroEL 大大减缓了 NMR 可见 Aβ42 物种的消失以及 Aβ42 原纤维和纤维的出现，如电子和原子力显微镜监测所示。后一种观察结果与 GroEL 对 Aβ42 诱导的神经毒性时间过程的影响相关。这些数据为理解 Hsp60 如何减缓阿尔茨海默病的进展提供了物理基础。
