Characterization of the NPC1L1 gene and proteome from an exceptional responder to ezetimibe

Highlights

• NPC1L1 exon 2 amino acid mutations identified in an exceptional ezetimibe responder.

• Molecular modeling showed altered cholesterol entry and cholesterol binding.

• Amino acid changes reduced cholesterol uptake when ezetimibe was present.

• Major amino acids changes involved are L52P and I300T.

• NPC1L1 protein:protein interactions are altered.

Abstract

Background

Strategies to reduce LDL-cholesterol involve reductions in cholesterol synthesis or absorption. We identified a familial hypercholesterolemia patient with an exceptional response to the cholesterol absorption inhibitor, ezetimibe. Niemann-Pick C 1-like 1 (NPC1L1) is the molecular target of ezetimibe.

Methods and results

Sequencing identified nucleotide changes predicted to change amino acids 52 (L52P), 300 (I300T) and 489 (S489G) in exceptional NPC1L1. In silico analyses identified increased stability and cholesterol binding affinity in L52P-NPC1L1 versus WT-NPC1L1. HEK293 cells overexpressing WT-NPC1L1 or NPC1L1 harboring amino acid changes singly or in combination (Comb-NPC1L1) had reduced cholesterol uptake in Comb-NPC1L1 when ezetimibe was present. Cholesterol uptake was reduced by ezetimibe in L52P-NPC1L1, I300T-NPC1L1, but increased in S489G-NPC1L1 overexpressing cells. Immunolocalization studies found preferential plasma membrane localization of mutant NPC1L1 independent of ezetimibe. Flotillin 1 and 2 expression was reduced and binding to Comb-NPC1L1 was reduced independent of ezetimibe exposure. Proteomic analyses identified increased association with proteins that modulate intermediate filament proteins in Comb-NPC1L1 versus WT-NPC1L1 treated with ezetimibe.

Conclusion

This is the first detailed analysis of the role of NPC1L1 mutations in an exceptional responder to ezetimibe. The results point to a complex set of events in which the combined mutations were shown to affect cholesterol uptake in the presence of ezetimibe. Proteomic analysis suggests that the exceptional response may also lie in the nature of interactions with cytosolic proteins.

Introduction

Elevated LDL-cholesterol is a major risk factor for cardiovascular disease and lowering of LDL-cholesterol will decrease the relative risk for cardiovascular disease by 25–35% [1]. Cholesterol homeostasis is achieved by de novo biosynthesis, intestinal absorption and biliary excretion [2]. Cholesterol absorption varies among healthy individuals and ranges from 29 to 80% [3]. Whereas inhibitors of HMGCoA reductase, the statins reduce cholesterol synthesis, ezetimibe inhibits cholesterol absorption across the intestinal cell wall [4]. Patients with familial hypercholesterolemia are at a high risk for atherosclerosis. When ezetimibe was administered to statin-treated familial hypercholesterolemic patients, it further decreased LDL-cholesterol by an average of 19.9% ± 8.0% [5]. However, when the patients were stratified by response, it became clear that some familial hypercholesterolemic patients were at the extremes of response. Exceptional responders, those in the top 10%, had large ≥35% reductions and low-responders, those in the bottom 10%, had very small ≤10% reductions in their LDL-cholesterol.

Cholesterol uptake in the intestine is an active process involving the action of transporters (reviewed in [6]). Niemann-Pick C 1-like 1 (NPC1L1) gene was identified as related to the Niemann-Pick C1 gene [7]. Both proteins are involved in cholesterol transport and show homology to the Patched family of proteins. NPC1L1 was later shown to be responsible for intestinal absorption of dietary cholesterol [8]. Further research identified NPC1L1 as the protein target for ezetimibe [9], [10], [11] and the extracellular loop C of NPC1L1 as the site of ezetimibe binding [12].

Sequencing and characterization of genes in exceptional responders has proven useful to identify genetic variations that change function. In particular, this has become an important focus of oncologists in the search for more effective and efficient treatment regimens, as illustrated by a major new initiative recently announced by the National Cancer Institute of the United States [13]. While this approach to understanding and refining the effectiveness of potential pharmacological agents has yet to be considered for cardiovascular disease, it is clearly likely to become an important weapon in treating cardiovascular disease. Accordingly, we sequenced the NPC1L1 gene in an exceptional responder to ezetimibe.

NPC1L1 has 20 exons spread over a ∼29 kb on chromosome 7p13 [7]. NPC1L1 is a 1332 amino acid protein with 13 transmembrane domains. Its second exon is unusually large at 1526 bp and encodes the cholesterol and ezetimibe binding sites. There are numerous sequence changes predicted to produce both synonymous and non-synonymous amino acid changes in the NPC1L1 protein. Within exon 2, the www.ncbi.nlm.nih/gov/projects/SNP/snp database shows many non-synonymous, synonymous, frame shift and missense amino acid changes. The role(s) of these changes in protein function or stability are unclear, but some sequence variation within the promoter region and within the gene have been correlated with improved or reduced uptake of cholesterol [14], [15], [16], [17], [18], [19], [20]. Similarly, patient response to ezetimibe has varied with NPC1L1 sequence variations [5], [21].

To answer these questions, we have characterized the structure of the N-terminal region in silico, examined the impact of over-expression on cell proliferation, its effect on cholesterol absorption, and on ezetimibe action, as well as identified new proteomic partners of the variant NPC1L1 genes in vitro.