Abstract
Niemann-Pick C (NPC) disease is an autosomal recessive disorder that leads to excessive storage of cholesterol and other lipids in late endosomes and lysosomes. The large majority of NPC disease is caused by mutations in NPC1, a large polytopic membrane protein that functions in late endosomes. There are many disease-associated mutations in NPC1, and most patients are compound heterozygotes. The most common mutation, NPC1I1061T, has been shown to cause endoplasmic reticulum-associated degradation of the NPC1 protein. Treatment of patient-derived NPC1I1061T fibroblasts with histone deacetylase inhibitors (HDACis) vorinostat or panobinostat increases expression of the mutant NPC1 protein and leads to correction of the cholesterol storage. Here, we show that several other human NPC1 mutant fibroblast cell lines can also be corrected by vorinostat or panobinostat and that treatment with vorinostat extends the lifetime of the NPC1I1061T protein. To test effects of HDACi on a large number of NPC1 mutants, we engineered a U2OS cell line to suppress NPC1 expression by shRNA and then transiently transfected these cells with 60 different NPC1 mutant constructs. The mutant NPC1 did not significantly reduce cholesterol accumulation, but approximately 85% of the mutants showed reduced cholesterol accumulation when treated with vorinostat or panobinostat.
Highlights
Niemann-Pick C (NPC) disease is an autosomal recessive disorder that leads to excessive storage of cholesterol and other lipids in late endosomes and lysosomes
Using procedures described in detail in Materials and Methods, we measured the effect of treatment with vorinostat on the lifetime of newly synthesized Niemann-Pick C1 (NPC1) protein in fibroblasts with a homozygous NPC1I1061T mutation
It has been shown previously that nearly half of the WT NPC1 protein is degraded by an endoplasmic reticulum-associated degradation (ERAD) pathway, and essentially all of the newly synthesized NPC1I1061T protein is degraded by ERAD [26]
Summary
Niemann-Pick C (NPC) disease is an autosomal recessive disorder that leads to excessive storage of cholesterol and other lipids in late endosomes and lysosomes. Treatment of patient-derived NPC1I1061T fibroblasts with histone deacetylase inhibitors (HDACis) vorinostat or panobinostat increases expression of the mutant NPC1 protein and leads to correction of the cholesterol storage. Lipoprotein-derived cholesterol is normally transported out of late endosomes and lysosome (LE/Ly) by a process that requires the Niemann-Pick C1 (NPC1) and NPC2 proteins [1, 2]. The cholesterol can be transferred to the N-terminal domain of NPC1, a LE/Ly membrane protein with 13 transmembrane segments [4] This model is supported by recent structural studies of NPC1 [5, 6] and NPC2 bound to the middle lumenal domain of NPC1 [7].
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