Abstract
Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease associated with aggregation of superoxide dismutase 1 (SOD1) protein. More than 160 mutations in human SOD1 have been identified in familial ALS and extensively characterized in previous studies. Here, we investigated the effects of T18S and E40K mutations on protein aggregation of canine SOD1. These two mutations are exclusively found in canine degenerative myelopathy (an ALS-like neurodegenerative disease in dogs), whose phenotype is unknown at the level of protein folding. Interestingly, the T18S and E40K mutations did not alter far-UV CD spectrum, enzymatic activity, or global structural stability of canine SOD1. However, thioflavin-T assay and transmission electron microscopy analysis revealed that these mutations promote formation of fibrous aggregates, in particular in the Cu(2+)/Zn(2+)-unbound state. These evidence suggested that the T18S and E40K mutations promote protein aggregation through a unique mechanism, possibly involving destabilization of the local structure, reduction of net negative charge, or production of disulfide-linked oligomers.