Ashkaran Faezeh, Seyedalipour Bagher, Baziyar Payam, Hosseinkhani Saman
Department of Molecular and Cell Biology, Faculty of Basic Science, University of Mazandaran, Babolsar, Iran.
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
BMC Chem. 2024 Sep 19;18(1):177. doi: 10.1186/s13065-024-01289-x.
Despite the many mechanisms it has created to prevent unfolding and aggregation of proteins, many diseases are caused by abnormal folding of proteins, which are called misfolding diseases. During this process, proteins undergo structural changes and become stable, insoluble beta-sheet aggregates called amyloid fibrils. Mutations/disruptions in metal ion homeostasis in the ALS-associated metalloenzyme superoxide dismutase (SOD1) reduce conformational stability, consistent with the protein aggregation hypothesis for neurodegenerative diseases. However, the exact mechanism of involvement is not well understood. Hence, to understand the role of mutation/ metal deficiency in SOD1 misfolding and aggregation, we investigated the effects of apo/holo SOD1 variants on structural properties using biophysical/experimental techniques. The MD results support the idea that the mutation/metal deficiency can lead to a change in conformation. The increased content of β-sheet structures in apo/holo SOD1 variants can be attributed to the aggregation tendency, which was confirmed by FTIR spectroscopy and dictionary of secondary structure in proteins (DSSP) results. Thermodynamic studies of GdnHCl showed that metal deficiency/mutation/intramolecular S-S reduction together are required to initiate misfolding/aggregation of SOD1. The results showed that apo/holo SOD1 variants under destabilizing conditions induced amyloid aggregates at physiological pH, which were detected by ThT/ANS fluorescence, as well as further confirmation of amyloid/amorphous species by TEM. This study confirms that mutations in the electrostatic loop of SOD1 lead to structural abnormalities, including changes in hydrophobicity, reduced disulfide bonds, and an increased propensity for protein denaturation. This process facilitates the formation of amyloid/amorphous aggregates ALS-associated.
尽管机体已经产生了许多机制来防止蛋白质解折叠和聚集，但许多疾病仍是由蛋白质异常折叠引起的，这类疾病被称为错误折叠疾病。在此过程中，蛋白质会发生结构变化，形成稳定的、不溶性的β-折叠聚集体，即淀粉样纤维。与肌萎缩侧索硬化症（ALS）相关的金属酶超氧化物歧化酶（SOD1）中金属离子稳态的突变/破坏会降低其构象稳定性，这与神经退行性疾病的蛋白质聚集假说一致。然而，具体的参与机制尚不清楚。因此，为了了解SOD1错误折叠和聚集过程中突变/金属缺乏的作用，我们使用生物物理/实验技术研究了脱辅基/结合金属的SOD1变体对结构特性的影响。分子动力学（MD）结果支持了突变/金属缺乏会导致构象变化这一观点。脱辅基/结合金属的SOD1变体中β-折叠结构含量的增加可归因于聚集倾向，傅里叶变换红外光谱（FTIR）和蛋白质二级结构字典（DSSP）结果证实了这一点。盐酸胍（GdnHCl）的热力学研究表明，金属缺乏/突变/分子内二硫键还原共同作用才会引发SOD1的错误折叠/聚集。结果表明，在不稳定条件下，脱辅基/结合金属的SOD1变体在生理pH值下会诱导淀粉样聚集体的形成，这可通过硫黄素T（ThT）/8-苯胺基-1-萘磺酸（ANS）荧光检测到，透射电子显微镜（TEM）进一步证实了淀粉样/无定形聚集体的存在。这项研究证实，SOD1静电环中的突变会导致结构异常，包括疏水性变化、二硫键减少以及蛋白质变性倾向增加。这一过程促进了与ALS相关的淀粉样/无定形聚集体的形成。
