Streltsov Victor A, Ganio Katherine E, Nuttall Stewart D, Hernandez J Andres, Dennys Cassandra N, Crouch Peter J, Estevez Alvaro G, Franco Maria Clara, Beckman Joseph S, Roberts Blaine R
bioRxiv. 2025 Sep 10:2025.09.10.675162. doi: 10.1101/2025.09.10.675162.
A subset of familial cases of amyotrophic lateral sclerosis (fALS) are caused by mutations to copper, zinc superoxide dismutase (Cu, Zn SOD1). There are over 200 mutations to SOD1 that have been associated with fALS and the majority of these mutations are dominantly inherited. Thus, individuals are heterozygous and express both wild-type SOD1 and the mutant form of the protein. Paradoxically, when rodent models are produced that mimic the co-expression of wild-type SOD1 with mutant fALS SOD1 the motor neuron disease accelerates. Previously, we have shown that the loss of zinc from an SOD1 kills cultured motor neurons due to a gained, redox activity catalyzed by the active-site copper. Furthermore, motor neuron toxicity of zinc-deficient SOD1 is enhanced by wild-type Cu, Zn SOD1. Because SOD1 exists as a non-covalent dimer, the enhanced toxicity might result from stabilization of the heterodimeric interface between zinc-deficient SOD1 and Cu, Zn-SOD1. However, experimentation with the heterodimer is difficult because SOD1 subunits exchange in minutes. To better characterize the role of dimer stabilization on the enhanced toxicity of fALS mutant SOD1 by wild type SOD1, we genetically tethered a zinc-deficient SOD1 subunit with a Cu, Zn SOD1 subunit with a 16-residue linker. The x-ray structure of the tethered heterodimer shows that zinc-deficient subunit adopts a wild-type-like conformation and is not misfolded. The heterodimer intermediate also produced peroxynitrite from nitric oxide, and the tethered SOD1 was strikingly toxic to primary cultures of motor neurons. This work supports the concept that zinc-deficient SOD1 is a likely toxic intermediate in ALS. Furthermore, the wild-type allele in human familial-SOD1 ALS patients may physically contribute to the dominant inheritance of SOD1 mutations through heterodimer formation.
家族性肌萎缩侧索硬化症（fALS）的一部分病例是由铜锌超氧化物歧化酶（Cu，Zn SOD1）的突变引起的。SOD1有200多种与fALS相关的突变，其中大多数突变是显性遗传的。因此，个体是杂合子，同时表达野生型SOD1和该蛋白的突变形式。矛盾的是，当构建出模拟野生型SOD1与突变型fALS SOD1共表达的啮齿动物模型时，运动神经元疾病会加速发展。此前，我们已经表明，SOD1中锌的缺失会导致培养的运动神经元死亡，这是由于活性位点铜催化产生了一种新获得的氧化还原活性。此外，野生型Cu，Zn SOD1会增强锌缺乏型SOD1对运动神经元的毒性。由于SOD1以非共价二聚体形式存在，毒性增强可能是由于锌缺乏型SOD1与Cu，Zn - SOD1之间异二聚体界面的稳定。然而，对异二聚体进行实验很困难，因为SOD1亚基在几分钟内就会交换。为了更好地表征二聚体稳定对野生型SOD1增强fALS突变型SOD1毒性的作用，我们通过一个16个残基的接头将一个锌缺乏型SOD1亚基与一个Cu，Zn SOD1亚基进行基因连接。连接的异二聚体的X射线结构表明，锌缺乏型亚基采用了类似野生型的构象，没有错误折叠。该异二聚体中间体还能由一氧化氮产生过氧亚硝酸盐，并且连接的SOD1对运动神经元原代培养物具有显著毒性。这项工作支持了锌缺乏型SOD1可能是ALS中一种毒性中间体的概念。此外，人类家族性SOD1 ALS患者中的野生型等位基因可能通过异二聚体的形成在物理上促成了SOD1突变的显性遗传。
