Biophysical Chemistry, Center for Molecular Protein Science, Department of Chemistry, Lund University , 221 00 Lund, Sweden.
Department of Chemistry, Indian Institute of Technology Delhi , New Delhi 110016, India.
ACS Chem Neurosci. 2017 Sep 20;8(9):2019-2026. doi: 10.1021/acschemneuro.7b00162. Epub 2017 Jun 20.
Cu/Zn superoxide dismutase (SOD1) forms intracellular aggregates that are pathological indicators of amyotrophic lateral sclerosis. A large body of research indicates that the entry point to aggregate formation is a monomeric, metal-ion free (apo), and disulfide-reduced species. Fibril formation by SOD1 in vitro has typically been reported only for harsh solvent conditions or mechanical agitation. Here we show that monomeric apo-SOD1 in the disulfide-reduced state forms fibrillar aggregates under near-physiological quiescent conditions. Monomeric apo-SOD1 with an intact intramolecular disulfide bond is highly resistant to aggregation under the same conditions. A cysteine-free variant of SOD1 exhibits fibrillization behavior and fibril morphology identical to those of disulfide-reduced SOD1, firmly establishing that intermolecular disulfide bonds or intramolecular disulfide shuffling are not required for aggregation and fibril formation. The decreased lag time for fibril formation resulting from reduction of the intramolecular disulfide bond thus primarily reflects the decreased stability of the folded state relative to partially unfolded states, rather than an active role of free sulfhydryl groups in mediating aggregation. Addition of urea to increase the amount of fully unfolded SOD1 increases the lag time for fibril formation, indicating that the population of this species does not dominate over other factors in determining the onset of aggregation. Our results contrast with previous results obtained for agitated samples, in which case amyloid formation was accelerated by denaturant. We reconcile these observations by suggesting that denaturants destabilize monomeric and aggregated species to different extents and thus affect nucleation and growth.
铜锌超氧化物歧化酶（SOD1）形成细胞内聚集体，这些聚集体是肌萎缩侧索硬化症的病理标志。大量研究表明，聚合形成的切入点是单体、无金属离子（apo）和二硫键还原的物种。SOD1 在体外的纤维形成通常仅在苛刻的溶剂条件下或机械搅拌下报道。在这里，我们表明在近生理静止条件下，还原状态的二硫键还原的单体 apo-SOD1 形成纤维状聚集体。在相同条件下，具有完整分子内二硫键的单体 apo-SOD1 高度抵抗聚集。SOD1 的无半胱氨酸变体表现出与还原的 SOD1 相同的纤维化行为和纤维形态，这有力地证明了分子间二硫键或分子内二硫键重排对于聚集和纤维形成不是必需的。由于分子内二硫键的还原导致纤维形成的滞后时间缩短，这主要反映了折叠态相对于部分展开态的稳定性降低，而不是游离巯基在介导聚集中的主动作用。添加脲以增加完全展开的 SOD1 的量会增加纤维形成的滞后时间，这表明该物种的群体在决定聚集的起始时并没有超过其他因素的主导地位。我们的结果与以前对搅拌样品获得的结果形成对比，在这种情况下，变性剂加速了淀粉样蛋白的形成。我们通过提出变性剂以不同程度使单体和聚集物种失稳，从而影响成核和生长来调和这些观察结果。
