Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208, USA.
Biochem J. 2012 Aug 15;446(1):59-67. doi: 10.1042/BJ20120075.
The intramolecular disulfide bond in hSOD1 [human SOD1 (Cu,Zn superoxide dismutase 1)] plays a key role in maintaining the protein's stability and quaternary structure. In mutant forms of SOD1 that cause familial ALS (amyotrophic lateral sclerosis), this disulfide bond is more susceptible to chemical reduction, which may lead to destabilization of the dimer and aggregation. During hSOD1 maturation, disulfide formation is catalysed by CCS1 (copper chaperone for SOD1). Previous studies in yeast demonstrate that the yeast GSH/Grx (glutaredoxin) redox system promotes reduction of the hSOD1 disulfide in the absence of CCS1. In the present study, we probe further the interaction between hSOD1, GSH and Grxs to provide mechanistic insight into the redox kinetics and thermodynamics of the hSOD1 disulfide. We demonstrate that hGrx1 (human Grx1) uses a monothiol mechanism to reduce the hSOD1 disulfide, and the GSH/hGrx1 system reduces ALS mutant SOD1 at a faster rate than WT (wild-type) hSOD1. However, redox potential measurements demonstrate that the thermodynamic stability of the disulfide is not consistently lower in ALS mutants compared with WT hSOD1. Furthermore, the presence of metal cofactors does not influence the disulfide redox potential. Overall, these studies suggest that differences in the GSH/hGrx1 reaction rate with WT compared with ALS mutant hSOD1 and not the inherent thermodynamic stability of the hSOD1 disulfide bond may contribute to the greater pathogenicity of ALS mutant hSOD1.
hSOD1 [人 SOD1（Cu，Zn 超氧化物歧化酶 1）]中的分子内二硫键在维持蛋白质稳定性和四级结构方面起着关键作用。在导致家族性 ALS（肌萎缩侧索硬化症）的 SOD1 突变体中，该二硫键更容易受到化学还原的影响，这可能导致二聚体的不稳定性和聚集。在 hSOD1 成熟过程中，二硫键的形成由 CCS1（SOD1 的铜伴侣）催化。先前在酵母中的研究表明，酵母 GSH/Grx（谷胱甘肽过氧化物酶）氧化还原系统在没有 CCS1 的情况下促进 hSOD1 二硫键的还原。在本研究中，我们进一步研究了 hSOD1、GSH 和 Grxs 之间的相互作用，为 hSOD1 二硫键的氧化还原动力学和热力学提供了机制见解。我们证明 hGrx1（人 Grx1）使用单硫醇机制还原 hSOD1 二硫键，并且 GSH/hGrx1 系统还原 ALS 突变 SOD1 的速度比 WT（野生型）hSOD1 更快。然而，氧化还原电位测量表明，与 WT hSOD1 相比，ALS 突变体中二硫键的热力学稳定性并不总是较低。此外，金属辅因子的存在并不影响二硫键的氧化还原电位。总的来说，这些研究表明，与 WT 相比，GSH/hGrx1 与 ALS 突变 hSOD1 的反应速率的差异，而不是 hSOD1 二硫键的固有热力学稳定性，可能导致 ALS 突变 hSOD1 的更高致病性。
