Gp78, an ER associated E3, promotes SOD1 and ataxin-3 degradation

Abstract

Superoxide dismutase-1 (SOD1) and ataxin-3 are two neurodegenerative disease proteins in association with familial amyotrophic lateral sclerosis and Machado-Joseph disease/spinocerebellar ataxia type 3. Both normal and mutant types of SOD1 and ataxin-3 are degraded by the proteasome. It was recently reported that these two proteins are associated with the endoplasmic reticulum (ER). Mammalian gp78 is an E3 ubiquitin ligase involved in ER-associated degradation (ERAD). Here, we show that gp78 interacts with both SOD1 and ataxin-3. Overexpression of gp78 promotes the ubiquitination and degradation of these two proteins, whereas knockdown of gp78 stabilizes them. Moreover, gp78 represses aggregate formation of mutant SOD1 and protect cells against mutant SOD1-induced cell death. Furthermore, gp78 is increased in cells transfected with these two mutant proteins as well as in ALS mice. Thus, our results suggest that gp78 functions in the regulation of SOD1 and ataxin-3 to target them for ERAD.

The Endoplasmic Reticulum (ER)-associated Degradation System Regulates Aggregation and Degradation of Mutant Neuroserpin

“…Subsequent studies showed that mutant neuroserpin is retained in the ER as accumulated polymers in a cellular model (3). Interestingly, ERAD E3s, such as Hrd1 and gp78, suppress the neurodegeneration mediated by aggregate-prone proteins, including Alzheimer's disease-linked amyloid precursor proteins (27), Parkinson's disease-linked ␣-synuclein (28), amyotrophic lateral sclerosis-linked SOD1 (20), polyglutamine disease-linked Nhtt and ataxin-3 (20,29,30), and prion diseaselinked prion protein (31,32). In this study, we found that Hrd1 and gp78 are involved in mutant neuroserpin ubiquitination and degradation and that the ERAD pathway contributes to the neuroserpin degradation and aggregation machinery, as indicated by the observations that impairment of ERAD by defective VCP and gp78 significantly increases mutant neuroserpin stability and aggregation.…”

“…Hrd1 and gp78 serve divergent ERAD substrate populations (20,24,36,37). gp78 differs from Hrd1 in its complex domain structure, which, in addition to its RING finger, includes the ubiquitin-binding Cue domain (23, 24, 36).…”

“…Hrd1, gp78, the FLAG-ubiquitin constructs, pEGFP-Mito, and ER markers were described previously (20). pCMV-VCPMyc (Myc-tagged VCP/p97) was obtained from Dr. Baoliang Song (Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China), and pcDNA3.1-His-p97 WT and pcDNA3.1-His-p97 QQ (His-tagged VCP/p97) was obtained from Dr. Yihong Ye (National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health).…”

“…Cells were transfected with either expression vectors or siRNAs using Lipofectamine 2000 reagent (Invitrogen) at ϳ50% confluence in DMEM without serum. Double-stranded oligonucleotides against gp78 named si-gp78 -1 and si-gp78 -2 were described previously (20). Double-stranded oligonucleotides designed against nucleotides 175-195 of human Hrd1 cDNA or 883-903 of human VCP cDNA were synthesized by Shanghai GenePharma (Shanghai, China).…”

“…Mouse monoclonal anti-FLAG, anti-FLAG conjugated with HRP, and anti-tubulin and rabbit polyclonal anti-calnexin antibodies were all from Sigma. Rabbit polyclonal anti-gp78 antibodies were described previously (20). The secondary antibodies sheep anti-mouse IgG-HRP and antirabbit IgG-HRP (Amersham Biosciences) were used.…”