Abstract
Familial encephalopathy with neuroserpin inclusion bodies is a neurodegenerative disorder characterized by the accumulation of neuroserpin polymers in the endoplasmic reticulum (ER) of cortical and subcortical neurons in the CNS because of neuroserpin point mutations. ER-associated degradation (ERAD) is involved in mutant neuroserpin degradation. In this study, we demonstrate that two ER-associated E3 ligases, Hrd1 and gp78, are involved in the ubiquitination and degradation of mutant neuroserpin. Overexpression of Hrd1 and gp78 decreases the mutant neuroserpin protein level, whereas Hrd1 and gp78 knockdown increases mutant neuroserpin stability. Moreover, ERAD impairment by mutant valosin-containing protein increases the mutant neuroserpin protein level and aggregate formation. Thus, these findings identify mutant neuroserpin as an ERAD target and show that Hrd1 and gp78 mediate mutant neuroserpin turnover through the ERAD pathway.
Highlights
Misfolding of specific proteins can lead to the formation of aggregates, which is associated with most neurodegenerative disorders
We examined the effects of the proteasome on mutant neuroserpin aggregate formation using the proteasome inhibitor MG132
Using immunoprecipitation with anti-GFP antibodies, we found that VCP/p97 was coimmunoprecipitated with G392E neuroserpin (Fig. 2B), suggesting that ER-associated degradation (ERAD) may be involved in mutant neuroserpin degradation
Summary
Plasmid Constructs—Full-length human neuroserpin cDNA was first amplified using PCR from a human fetal brain cDNA library (Clontech) using the primers 5Ј-GAAGATCTATATGGCTTTCCTTGGAC-3Ј and 5Ј-CGGAATTCTTAAAGTTCTTCGAAATC-3Ј. FLAG-neuroserpin was constructed by subcloning the amplified PCR product using the primers 5Ј-CCCAAGCTTATGGCTTTCCTTGGAC-3Ј and 5Ј-GCTCTAGAAAGTTCTTCGAAATC-3Ј from the pEGFP-C2-neuroserpin construct and inserting it into the p3xFLAG-Myc-CMV-24 (Sigma) vector at the HindIII and XbaI sites. Mouse monoclonal anti-FLAG, antiFLAG conjugated with HRP, and anti-tubulin and rabbit polyclonal anti-calnexin antibodies were all from Sigma. The cells were treated with 0.25% Triton X-100 for 10 min, blocked with 0.5% FBS in PBS, and incubated with the primary antibody followed by Rho (red)- or FITC (green)-conjugated donkey anti-mouse or antirabbit secondary antibodies (Santa Cruz Biotechnology) or Alexa Fluor 350 (blue)-labeled goat anti-mouse IgG (Invitrogen). Cycloheximide Chase Analysis—Twenty-four hours after transfection, the 293 cells expressing neuroserpin were treated with 150 g/ml CHX to inhibit protein synthesis. Pulse-chase Analysis—293 cells stably expressing EGFPtagged G392E neuroserpin were starved in methionine-free medium and incubated for 30 min. 20836 JOURNAL OF BIOLOGICAL CHEMISTRY gation at 105,000 ϫ g for 60 min, and the fractions were further processed for immunoblot analysis
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