Dorfin-CHIP chimeric proteins potently ubiquitylate and degrade familial ALS-related mutant SOD1 proteins and reduce their cellular toxicity

The ubiquitin–proteasome system (UPS) is involved in the pathogenetic mechanisms of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Dorfin is a ubiquitin ligase (E3) that degrades mutant SOD1 proteins, which are responsible for familial ALS. Although Dorfin has potential as an anti-ALS molecule, its life in cells is short. To improve its stability and enhance its E3 activity, we developed chimeric proteins containing the substrate-binding hydrophobic portion of Dorfin and the U-box domain of the carboxyl terminus of Hsc70-interacting protein (CHIP), which has strong E3 activity through the U-box domain. All the Dorfin-CHIP chimeric proteins were more stable in cells than was wild-type Dorfin (DorfinWT). One of the Dorfin-CHIP chimeric proteins, Dorfin-CHIPL, ubiquitylated mutant SOD1 more effectively than did DorfinWT and CHIP in vivo, and degraded mutant SOD1 protein more rapidly than DorfinWT does. Furthermore, Dorfin-CHIPL rescued neuronal cells from mutant SOD1-associated toxicity and reduced the aggresome formation induced by mutant SOD1 more effectively than did DorfinWT.