Investigating the importance of liver-specific glycans on NPC1 proteostasis - ABSTRACT Niemann-Pick disease type C is a fatal, autosomal recessive lipid storage disease affecting all ages. Before neurological defects appear, ~85% of patients develop hepatomegaly which can progress into hepatic steatosis, cirrhosis, hepatocellular carcinoma, and liver failure. Liver defects are particularly detrimental in patients with neonatal-onset, 10% of whom die from liver failure by 6 months of age. Although the liver is a significant contributor to disease, few Niemann-Pick C liver therapeutics are being developed. Niemann-Pick C is commonly caused by loss-of-function mutations in the NPC1 gene (95% of cases), encoding a multipass transmembrane glycoprotein required for exporting unesterified cholesterol from late endosomes and lysosomes. The most common disease-causing mutation (~20% of cases) is an isoleucine to threonine substitution at position 1061 (I1061T). I1061T-NPC1 misfolds in the endoplasmic reticulum (ER) and is rapidly degraded by the proteasome and ER-autophagy. Importantly, I1061T-NPC1 is functional if trafficked to the lysosome. This observation spurred interest in understanding NPC1 degradation for the development of brain proteostasis modulators. However, it is unknown if neuronal NPC1 proteostasis modulators will work in the liver due to the limited understanding of liver NPC1 regulation. Furthermore, our strong preliminary data indicate that liver NPC1 is more heavily glycosylated than brain NPC1. Given the critical role of glycans in protein folding, trafficking, function, and degradation there is a need to understand the role of NPC1 liver- specific glycosylation. The next step in addressing this need is to pursue the overall objective of this application: (i) produce pilot data for an RO1 which demonstrate that liver specific glycan sites are critical for NPC1 proteostasis. Here we will test the central hypothesis that liver-specific glycans alter NPC1 proteostasis. We will test our hypothesis using LC-MS/MS to identify NPC1 glycan sites in human liver and brain. Next, we will take advantage of NPC1 null iPSC derived hepatocytes and add back constructs containing WT, WT without liver specific glycans, I1061T, and I1061T without liver specific glycans. We will leverage biochemical and genetic assays to establish the extent to which liver-specific glycan sites impact NPC1 proteostasis (Aim 1). The rationale for this project is that defining the location and influence of tissue-specific NPC1 glycans will provide deeper insight into NPC1 proteostasis and a strong scientific framework for the development of new Niemann-Pick C liver proteostatic therapeutics. In addition to moving my research into a highly understudied research topic, we anticipate this work will provide a strong scientific framework to investigate he how liver- specific NPC1 glycans regulate proteostasis as a future aim in an R01 grant from NIDDK.
