A Novel SOD1 Intermediate Oligomer, Role of Free Thiols and Disulfide Exchange

• Published in: Frontiers in neuroscience Vol. 14; p. 619279
• Main Authors: Koo, Bon-Kyung, Munroe, William, Gralla, Edith B, Valentine, Joan Selverstone, Whitelegge, Julian P
• Format: Journal Article
• Language: English
• Published: Switzerland Frontiers Research Foundation 18.02.2021; Frontiers Media S.A
• Subjects: Amyloid; Amyotrophic lateral sclerosis; Copper; Cu/Zn superoxide dismutase 1; Dimerization; disulfide reduction; Electron microscopy; Enzymes; fibril seeding; Fibrillation; Fibrillogenesis; Mixed disulfides; Molecular weight; Mutation; Neuroscience; oligomerization; Phosphine; Potassium; Protein seeding; Proteins; Sedimentation & deposition; Seeds; Sulfhydryl groups; Superoxide dismutase; Thiols; Transgenic animals; analytical ultracentrifugation; oligomerization; Cu/Zn superoxide dismutase 1; disulfide reduction; fibril seeding; amyotrophic lateral sclerosis; fibrillation
• ISSN: 1662-4548; 1662-453X; 1662-453X
• DOI: 10.3389/fnins.2020.619279

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.