Mutations of Profilin-1 Associated with Amyotrophic Lateral Sclerosis Promote Aggregation Due to Structural Changes of Its Native State
Mutations of Profilin-1 Associated with Amyotrophic Lateral Sclerosis Promote Aggregation Due to Structural Changes of Its Native State
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DOI：
10.1021/acschembio.5b00598
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发表时间：
2015-11-01
影响因子：
4
通讯作者：
Chiti, Fabrizio
中科院分区：
文献类型：
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作者：
Del Poggetto, Edoardo;Bemporad, Francesco;Chiti, Fabrizio
编码profin -1的PFN1基因最近与家族性肌萎缩性侧索硬化症（fALS）有关，因为在家族性肌萎缩性侧索硬化症患者中发现了三种突变，即C71G、M114T和G118V，另一种突变E117G被认为是疾病发病的中等危险因素。在这项工作中，我们纯化了四种profin -1变体以及野生型蛋白。所得到的聚集体似乎是纤维状的，与ThT结合较弱，并具有大量的分子间β -片结构。利用ThT荧光分析、远紫外圆二色性和动态光散射，我们发现所有四种变体都比野生型具有更高的聚集倾向。其中，C71G突变诱导的聚集变化最为显著，其次是G118V和M114T突变，最后是E117G突变。这种倾向被发现与这组profin -1变异的构象稳定性没有严格相关，这是通过尿素诱导的平衡变性和折叠/展开动力学来确定的。然而，通过远紫外圆二色性、本征荧光光谱、ANS结合、丙烯酰胺猝灭和动态光散射等手段监测到，它与折叠态的结构变化相关。总的来说，结果表明，所有四种突变都增加了profin -1的聚集倾向，这种聚集行为在很大程度上取决于突变诱导的蛋白质折叠状态的结构变化。
The PFN1 gene, coding for profilin-1, has recently been associated with familial amyotrophic lateral sclerosis (fALS), as three mutations, namely C71G, M114T, and G118V, have been found in patients with familial forms of the disease and another, E117G, has been proposed to be a moderate risk factor for disease onset. In this work, we have purified the four profilin-1 variants along with the wild-type protein. The resulting aggregates appear to be fibrillar, to have a weak binding to ThT, and to possess a significant amount of intermolecular beta-sheet structure. Using ThT fluorescence assays, far-UV circular dichroism, and dynamic light scattering, we found that all four variants have an aggregation propensity higher than that of the wild-type counterpart. In particular, the C71G mutation was found to induce the most dramatic change in aggregation, followed by the G118V and M114T substitutions and then the E117G mutation. Such a propensity was found not to strictly correlate with the conformational stability in this group of profilin-1 variants, determined using both urea-induced denaturation at equilibrium and folding/unfolding kinetics. However, it correlated with structural changes of the folded states, as monitored with far-UV circular dichroism, intrinsic fluorescence spectroscopy, ANS binding, acrylamide quenching, and dynamic light scattering. Overall, the results suggest that all four mutations increase the tendency of profilin-1 to aggregate and that such aggregation behavior is largely determined by the mutation-induced structural changes occurring in the folded state of the protein.