Cytotoxicity of Superoxide Dismutase 1 in Cultured Cells Is Linked to Zn2+ Chelation

Neurodegeneration in protein-misfolding disease is generally assigned to toxic function of small, soluble protein aggregates. Largely, these assignments are based on observations of cultured neural cells where the suspect protein material is titrated directly into the growth medium. In the present study, we use this approach to shed light on the cytotoxic action of the metalloenzyme Cu/Zn superoxide dismutase 1 (SOD1), associated with misfolding and aggregation in amyotrophic lateral sclerosis (ALS). The results show, somewhat unexpectedly, that the toxic species of SOD1 in this type of experimental setting is not an aggregate, as typically observed for proteins implicated in other neuro-degenerative diseases, but the folded and fully soluble apo protein. Moreover, we demonstrate that the toxic action of apoSOD1 relies on the protein's ability to chelate Zn2+ ions from the growth medium. The decreased cell viability that accompanies this extraction is presumably based on disturbed Zn2+ homeostasis. Consistently, mutations that cause global unfolding of the apoSOD1 molecule or otherwise reduce its Zn2+ affinity abolish completely the cytotoxic response. So does the addition of surplus Zn2+. Taken together, these observations point at a case where the toxic response of cultured cells might not be related to human pathology but stems from the intrinsic limitations of a simplified cell model. There are several ways proteins can kill cultured neural cells but all of these need not to be relevant for neurodegenerative disease.蛋白质错误折叠疾病相关的神经退行性变，通常被归因于小型可溶性蛋白质聚集体的毒性功能。此类归因大多基于培养神经细胞的实验研究：即将疑似致病的蛋白质物质直接滴定至生长培养基中。本研究采用该实验方法，探究与肌萎缩侧索硬化症（amyotrophic lateral sclerosis, ALS）中蛋白质错误折叠及聚集相关的金属酶铜锌超氧化物歧化酶1（Cu/Zn superoxide dismutase 1, SOD1）的细胞毒性作用。实验结果出人意料地显示，在该实验体系中，SOD1的毒性物种并非其他神经退行性疾病中常见的蛋白质聚集体，而是折叠完整且完全可溶性的脱辅基蛋白（apo protein）。此外，本研究证实，脱辅基SOD1（apoSOD1）的细胞毒性作用，依赖于该蛋白从生长培养基中螯合锌离子（Zn²+）的能力。由此引发的细胞活力下降，推测源于锌离子稳态失衡。与之相符的是，导致脱辅基SOD1分子全局解折叠或降低其锌离子结合亲和力的突变，会完全消除细胞毒性反应；额外添加过量锌离子也可达到相同效果。综上，这些观察结果表明，培养细胞的毒性反应或许与人类病理无关，而是源于简化细胞模型的固有局限性。蛋白质可通过多种途径杀伤培养神经细胞，但并非所有途径都与神经退行性疾病相关。