Structural basis of Cu, Zn-superoxide dismutase amyloid fibril formation involves interaction of multiple peptide core regions

Abstract

Cu, Zn-superoxide dismutase (SOD1), an enzyme implicated in the progression of familial amyotrophic lateral sclerosis (fALS), forms amyloid fibrils under certain experimental conditions. As part of our efforts to understand ALS pathogenesis, in this study we found that reduction of the intramolecular disulfide bond destabilized the tertiary structure of metal free wildtype SOD1 and greatly enhanced fibril formation in vitro. We also identified fibril core peptides that are resistant to protease digestion by using mass spectroscopy and Edman degradation analyses. Three regions dispersed throughout the sequence were detected as fibril core sequences of SOD1. Interestingly, by using three synthetic peptides that correspond to these identified regions, we determined that each region was capable of fibril formation, either alone or in a mixture containing multiple peptides. It was also revealed that by reducing the disulfide bond and causing a decrease in the structural stability, the amyloid fibril formation of a familial mutant SOD1 G93A was accelerated even under physiological conditions. These results demonstrate that by destabilizing the structure of SOD1 by removing metal ions and breaking the intramolecular disulfide bridge, multiple fibril-forming core regions are exposed, which then interact with each another and form amyloid fibrils under physiological conditions.

The molecular tweezer CLR01 inhibits aberrant superoxide dismutase 1 (SOD1) self-assembly in vitro and in the G93A-SOD1 mouse model of ALS

“…These experiments suggest that CLR01 binds preferentially to the Lys residues at positions 70 and 75. Previously, several regions in SOD1 have been reported to be important for the aggregation process, including residues 14 -21, 30 -38, 101-107, and 147-153 (44) and 10 -23, 92-122, and 137-153 (45). In addition, residues 28 -38 have been suggested to be involved in mediating SOD1 toxicity, presumably in an amyloidogenic oligomer structure.…”

Cu/Zn Superoxide Dismutase Forms Amyloid Fibrils under Near-Physiological Quiescent Conditions: The Roles of Disulfide Bonds and Effects of Denaturant

“…Many proteins linked to neurodegenerative diseases form amyloid fibrils through a nucleation-dependent polymerization mechanism. , In the case of SOD1, which is a relatively stable protein, in vitro studies of fibril formation have typically involved extreme experimental conditions, such as elevated temperature, low pH, the presence of denaturant or organic solvents, , or oxidative cross-linking of cysteines to form disulfide bridges . It has also been shown that forceful agitation of the sample under otherwise physiological conditions triggers apo, disulfide-reduced SOD1 to form ordered fibrillar structures similar to those found in other amyloidogenic diseases. ,− Thus, it has been established that SOD1 forms fibrils under a range of different conditions that are all rather harsh and nonphysiological, but the question remains whether SOD1 can also form fibrils under quiescent and otherwise physiological conditions.…”

MIF homolog d-dopachrome tautomerase (D-DT/MIF-2) does not inhibit accumulation and toxicity of misfolded SOD1

“…As mentioned previously, mutant and wild type SOD1 might misfold and aggregate into amyloid fibrils under certain conditions 63 – 65 . Several studies have demonstrated the general toxicity of amyloid aggregates 66 , 67 .…”