Niemann-Pick type C1 I1061T mutant encodes a functional protein that is selected for endoplasmic reticulum-associated degradation due to protein misfolding

  • Gelsthorpe M
  • Baumann N
  • Millard E
 et al. 
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Abstract

Over 200 disease-causing mutations have been identified in the NPC1 gene. The most prevalent mutation, NPC1 I1061T , is predicted to lie within the cysteine-rich luminal domain and is associated with the classic juvenile-onset phenotype of Niemann-Pick type C disease. To gain insight into the molecular mechanism by which the NPC1 I1061T mutation causes disease, we examined expression of the mutant protein in human fibro-blasts homozygous for the NPC1 I1061T mutation. Despite similar NPC1 mRNA levels between wild type and NPC1 I1061T fibro-blasts, NPC1 protein levels are decreased by 85% in NPC1 I1061T cells. Metabolic labeling studies demonstrate that unlike wild type protein, which undergoes a glycosylation pattern shift from Endo H-sensitive to Endo H-resistant species, NPC1 I1061T pro-tein remains almost exclusively Endo H-sensitive and exhibits a reduced half-life (t1 ⁄ 2 6.5 h) versus wild type Endo H-resistant species (t1 ⁄ 2 42 h). Treatment with chemical chaperones, growth at permissive temperature, or inhibition of proteasomal degra-dation increases NPC1 I1061T protein levels, indicating that the mutant protein is likely targeted for endoplasmic reticulum-as-sociated degradation (ERAD) due to protein misfolding. Over-expression of NPC1 I1061T in NPC1-deficient cells results in late endosomal localization of the mutant protein and complementa-tion of the NPC mutant phenotype, likely due to a small proportion of the nascent NPC1 I1061T protein that is able to fold correctly and escape the endoplasmic reticulum quality control checkpoints. Our findings provide the first description of an endoplasmic retic-ulum trafficking defect as a mechanism for human NPC disease, shedding light on the mechanism by which the NPC1 I1061T muta-tion causes disease and suggesting novel approaches to treat NPC disease caused by the NPC1 I1061T mutation. Niemann-Pick type C (NPC) 3 disease is a fatal neurodegen-erative disease characterized by neuronal lipid storage and

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Authors

  • Mark E. Gelsthorpe

  • Nikola Baumann

  • Elizabeth Millard

  • Sarah E. Gale

  • S. Joshua Langmade

  • Jean E. Schaffer

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