title: Endoplasmic reticulum-associated degradation of Niemann-Pick C1: evidence for the role of heat shock proteins and identification of lysine residues that accept ubiquitin.
abstract: Most cases with Niemann-Pick disease type C carry mutations in NPC1. Some of the mutations, including the most frequent I1061T, give rise to unstable proteins selected for endoplasmic reticulum-associated degradation. The purpose of the current study was to shed mechanistic insights into the degradation process. A proteasome inhibitor MG132 prolonged the life span of the wild-type NPC1 expressed in COS cells. The expressed protein associated with multiple chaperones including heat shock protein 90 (Hsp90), Hsp70, heat shock cognate protein 70 (Hsc70), and calnexin. Accordingly, expression of an E3 ligase CHIP (carboxyl terminus of Hsp70-interacting protein) enhanced MG132-induced accumulation of ubiquitylated NPC1. Co-expression and RNAi knockdown experiments in HEK cells indicated that Hsp70/Hsp90 stabilized NPC1, whereas Hsc70 destabilized it. In human fibroblasts carrying the I1061T mutation, adenovirus-mediated expression of Hsp70 or treatment with an HSP-inducer geranylgeranylacetone (GGA) increased the level of the mutant protein. In GGA-treated cells, the rescued protein was localized in the late endosome and ameliorated cholesterol accumulation. MALDI-TOF mass spectrometry revealed three lysine residues at amino acids 318, 792, and 1180 as potential ubiquitin-conjugation sites. Substitutions of the three residues with alanine yielded a mutant protein with a steady-state level more than three times higher than that of the wild-type. Introduction of the same substitutions to the I1061T mutant resulted in an increase in its protein level and functional restoration. These findings indicated the role of HSPs in quality control of NPC1 and revealed the role of three lysine residues as ubiquitin-conjugation sites.
authors.affiliation: From the Departments of Biological Regulations and.
authors.forename: Naoe
authors.initials: N
authors.lastname: Nakasone
authors.affiliation: Applied Biotechnology, Tottori Institute of Industrial Technology, Yonago 684-0041.
authors.forename: Yuko S
authors.initials: YS
authors.lastname: Nakamura
authors.affiliation: the Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University, Yonago 683-8503, and.
authors.forename: Katsumi
authors.initials: K
authors.lastname: Higaki
authors.affiliation: the Tottori University Hospital Cancer Center, Yonago 683-8504, Japan.
authors.forename: Nao
authors.initials: N
authors.lastname: Oumi
authors.affiliation: Child Neurology, Tottori University Faculty of Medicine, Yonago 683-8503.
authors.forename: Kousaku
authors.initials: K
authors.lastname: Ohno
authors.affiliation: From the Departments of Biological Regulations and ninomiya@med.tottori-u.ac.jp.
authors.forename: Haruaki
authors.initials: H
authors.lastname: Ninomiya
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D050883:HSC70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D020125:Mutation, Missense; D009542:Niemann-Pick Diseases / Q000235:genetics / Q000378:metabolism*; D013729:Terpenes / Q000494:pharmacology; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects / Q000235:genetics
chemical_list: D015853:Cysteine Proteinase Inhibitors; D050883:HSC70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; C494371:HSPA8 protein, human; D007976:Leupeptins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C410800:NPC1L1 protein, human; D013729:Terpenes; D025801:Ubiquitin; C009884:geranylacetone; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde
keywords: Chaperone; Cholesterol; ER-associated Degradation; Heat Shock Protein (HSP); Niemann-Pick C1; Ubiquitin