As a result of mutation, some fALS SOD1 proteins may be partially unfolded or mimic oxidatively modified SOD1 and thus adversely affect proteasome function in vivo. SOD1 has been used as a model system and it has been demonstrated that mildly oxidized SOD1 is one of the proteins that is specifically targeted for proteolysis by the 20 S proteasome as a consequence of the damage. The pathway for degradation of oxidized SOD1 requires no ubiquitination or ATP
and recognition of the mildly oxidized SOD1 protein is likely due to partial unfolding leading to exposure of hydrophobic portions of the protein .
Recent studies have strongly suggested that proteasome levels and/or activities are adversely affected by the presence of the ALS-mutant SOD1 proteins [144-147]. The level of 20 S proteasome activity was substantially reduced in lumbar spinal neurons relative to the surrounding neuropil in the G93A SOD1 transgenic mouse and impairment of the proteasome is an early event and contributes to ALS pathogenesis . We hypothesize that some of the fALS-mutant SOD1 proteins are likely to monomerize or be partially unfolded in vivo as a consequence of their loss of metal ion binding ability, destabilization of their apo-proteins, or because of enhanced oxidative damage to the mutant SOD1 that may or may not be mediated by bound copper ions. In any of these cases, the partially unfolded fALS-mutant SOD1 proteins are likely to expose hydrophobic portions of their structures and they may be abnormally targeted to the proteasome even without oxidation. If this is true, it could help explain the inhibition of proteasome function observed in the disease models.
To test directly the susceptibility of SOD1 to be digested by the 20 S protea-some, a number of in vitro proteasomal digestion assays were performed using wild-type and pathogenic SOD1 proteins . Contrary to the in vivo studies, none of the SOD1 proteins, metal-bound or metal-free, are found to be inhibitors of proteasomal activity in vitro. Furthermore, all the metal-bound, dimeric forms of SOD1 (wild-type and mutant) are not substrates for degradation. However, the metal-free forms of both wild type and mutant are substrates to varying degrees, with the disulfide-reduced, monomeric form of SOD1 being the best substrate by far. If the burden of misfolded SOD1 proteins exceeds the capacity of the protein degradation system in vivo, these monomeric proteins may accumulate and self-associate. All of the SOD1 proteins digested, including wild-type, are cleaved into four distinct fragments ranging from 1.7 to 7.8 kDa in size. It is possible that it is the products of this proteolytic digestion that may be toxic to motor neurons, either through direct aggregation of the fragments or through other as yet unknown toxic effects of these peptides.
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