Department of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria 3010, Australia.
Florey Institute of Neuroscience and Mental Health and Centre for Neuroscience, The University of Melbourne, Victoria 3010, Australia.
J Biol Chem. 2014 Mar 7;289(10):6669-6680. doi: 10.1074/jbc.M113.520189. Epub 2014 Jan 14.
Protein aggregation into intracellular inclusions is a key feature of many neurodegenerative disorders. A common theme has emerged that inappropriate self-aggregation of misfolded or mutant polypeptide sequences is detrimental to cell health. Yet protein quality control mechanisms may also deliberately cluster them together into distinct inclusion subtypes, including the insoluble protein deposit (IPOD) and the juxtanuclear quality control (JUNQ). Here we investigated how the intrinsic oligomeric state of three model systems of disease-relevant mutant protein and peptide sequences relates to the IPOD and JUNQ patterns of aggregation using sedimentation velocity analysis. Two of the models (polyalanine (37A) and superoxide dismutase 1 (SOD1) mutants A4V and G85R) accumulated into the same JUNQ-like inclusion whereas the other, polyglutamine (72Q), formed spatially distinct IPOD-like inclusions. Using flow cytometry pulse shape analysis (PulSA) to separate cells with inclusions from those without revealed the SOD1 mutants and 37A to have abruptly altered oligomeric states with respect to the nonaggregating forms, regardless of whether cells had inclusions or not, whereas 72Q was almost exclusively monomeric until inclusions formed. We propose that mutations leading to JUNQ inclusions induce a constitutively "misfolded" state exposing hydrophobic side chains that attract and ultimately overextend protein quality capacity, which leads to aggregation into JUNQ inclusions. Poly(Q) is not misfolded in this same sense due to universal polar side chains, but is highly prone to forming amyloid fibrils that we propose invoke a different engagement mechanism with quality control.
蛋白质聚集成细胞内包涵体是许多神经退行性疾病的一个关键特征。一个共同的主题已经出现，即错误折叠或突变多肽序列的不当自我聚集对细胞健康是有害的。然而，蛋白质质量控制机制也可能故意将它们聚集在一起形成不同的包涵体亚型，包括不溶性蛋白沉积物 (IPOD) 和核周质量控制 (JUNQ)。在这里，我们使用沉降速度分析研究了三种与疾病相关的突变蛋白和肽序列的模型系统的固有寡聚状态如何与 IPOD 和 JUNQ 聚集模式相关。其中两个模型（聚丙氨酸 (37A) 和超氧化物歧化酶 1 (SOD1) 突变体 A4V 和 G85R）聚集成相同的 JUNQ 样包涵体，而另一个，多聚谷氨酰胺 (72Q)，形成空间上不同的 IPOD 样包涵体。使用流式细胞术脉冲形状分析 (PulSA) 将含有包涵体的细胞与不含包涵体的细胞分离，结果显示 SOD1 突变体和 37A 的寡聚状态相对于非聚集形式发生了突然改变，无论细胞是否含有包涵体，而 72Q 在形成包涵体之前几乎完全是单体。我们提出，导致 JUNQ 包涵体形成的突变导致蛋白质处于持续的“错误折叠”状态，暴露出疏水性侧链，吸引并最终过度扩展蛋白质质量容量，导致聚集成 JUNQ 包涵体。多聚 (Q) 在这种意义上没有错误折叠，因为它具有普遍的极性侧链，但非常容易形成淀粉样纤维，我们提出它们与质量控制具有不同的结合机制。
