Abstract
Wild-type human SOD1 forms a highly conserved intra-molecular disulfide bond between C57-C146, and in its native state is greatly stabilized by binding one copper and one zinc atom per monomer rendering the protein dimeric. Loss of copper extinguishes dismutase activity and destabilizes the protein, increasing accessibility of the disulfide with monomerization accompanying disulfide reduction. A further pair of free thiols exist at C6 and C111 distant from metal binding sites, raising the question of their function. Here we investigate their role in misfolding of SOD1 along a pathway that leads to formation of amyloid fibrils. We present the seeding reaction of a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1) to exclude variables caused by these free cysteines. Completely reduced fibril seeds decreasing the kinetic barrier to cleave the highly conserved intramolecular disulfide bond, and accelerating SOD1 reduction and initiation of fibrillation. Presence or absence of the pair of free thiols affects kinetics of fibrillation. Previously, we showed full maturation with both Cu and Zn prevents this behavior while lack of Cu renders sensitivity to fibrillation, with presence of the native disulfide bond modulating this propensity much more strongly than presence of Zn or dimerization. Here we further investigate the role of reduction of the native C57-C146 disulfide bond in fibrillation of wild-type hSOD1, firstly through removal of free thiols by paired mutations C6A, C111S (AS-SOD1), and secondly in seeded fibrillation reactions modulated by reductant tris (2-carboxyethyl) phosphine (TCEP). Fibrillation of AS-SOD1 was dependent upon disulfide reduction and showed classic lag and exponential growth phases compared with wild-type hSOD1 whose fibrillation trajectories were typically somewhat perturbed. Electron microscopy showed that AS-SOD1 formed classic fibrils while wild-type fibrillation reactions showed the presence of smaller “sausage-like” oligomers in addition to fibrils, highlighting the potential for mixed disulfides involving C6/C111 to disrupt efficient fibrillation. Seeding by addition of sonicated fibrils lowered the TCEP concentration needed for fibrillation in both wild-type and AS-SOD1 providing evidence for template-driven structural disturbance that elevated susceptibility to reduction and thus propensity to fibrillate.
Highlights
Over 100 different mutations in SOD1, the antioxidant enzyme, have been identified to account for fALS, a fatal neurodegenerative disorder caused by formation of abnormal protein aggregates in neuronal cells
Across the TfT fibrillation experiment, AS had shorter lag times than WT at comparable tris (2-carboxyethyl) phosphine (TCEP) concentrations, and the slope of AS was consistently steeper during the growth phase compared with WT
At the beginning SOD1 fibril was denatured in GuHCl for making amyloid seeds, but later we found that GuHCl can affect SOD1 fibrillation at much lower concentrations
Summary
Over 100 different mutations in SOD1 (an abundant copperand zinc-containing superoxide dismutase 1), the antioxidant enzyme, have been identified to account for fALS (familial amyotrophic lateral sclerosis), a fatal neurodegenerative disorder caused by formation of abnormal protein aggregates in neuronal cells. Once the nucleus forms, it functions as “seed” (a structural template) to convert native dimeric proteins into β-sheet-rich monomeric structures and subsequently elongate the protein fibril. This mechanism, which accelerates and even triggers protein aggregation, is called the seeding reaction (Szedberg and Pedersen, 1940; LeVine, 1999; Bouldin et al, 2012). Aggregation and amyloid fibril formation can be monitored based on spectroscopic and biophysical methods such as fluorescence spectroscopy, analytical ultracentrifugation, and so on (Doucette et al, 2004; Cristovao et al, 2019)
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