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

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.Misfolding of specific proteins can lead to the formation of aggregates, which is associated with most neurodegenerative disorders. Protein aggregates are extracellular in Alzheimer's disease, cytosolic in Parkinson's disease and amyotrophic lateral sclerosis, both cytosolic and intranuclear in polyglutamine (polyQ) 3 diseases, and localize to the endoplasmic reticulum (ER) in familial encephalopathy with neuroserpin inclusion bodies (FENIB), which is caused by neuroserpin mutations (1). Although the role of the protein aggregates in the pathogenesis of these diseases is still poorly understood, a common feature of these aggregates is that they are all marked by ubiquitin. Neurodegenerative protein aggregation is closely related to the ubiquitin-proteasome system, a major cellular turnover system (2). Mutant neuroserpin aggregates have been observed in brain and cultured cells (3, 4). Neuroserpin is a secretory glycoprotein (5) that is a member of the serine protease inhibitor serpin family, and it is mainly expressed in the CNS (6). Mutations in neuroserpin result in its misfolding and accumulation in the ER (3, 4, 7). To date, four mutants of neuroserpin, S49P, S52R, H338R, and G392E, have been identified in association with FENIB (8). In FENIB-diseased brains, G392E, the most disruptive mutant, forms more inclusion bodies and at an earlier age of onset (13 years) than the other neuroserpin mutants (9). Because the earlier onset of FENIB is strongly correlated with the level of intracellular neuroserpin accumulation, neuroserpin accumulation appears to play a central role in FENIB pathology.Many accumulated proteins that are caused by mutations or ER stress are targeted by a protein quality control system, termed ER-associated degradation (ERAD). ERAD is a degradation system in which proteins that are misfolded in the ER are exported to the cytosol for proteasomal degradation (10, 11). ER-associated E3 ubiquitin ligases, together with the VCPUfd1-Npl4 complex, are key components of the ERAD machinery. During the ERAD process, selected proteins are ubiquitinated by E3 ubiquitin lig...

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