The new obesity-associated protein, neuronal growth regulator 1 (NEGR1), is implicated in Niemann-Pick disease Type C (NPC2)-mediated cholesterol trafficking

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Highlights

  • NEGR1 interacts with NPC2, a key player in intracellular cholesterol trafficking.

  • NEGR1 co-localizes with NPC2 in the late endosomes and increases NPC2 stability.

  • NEGR1 overexpression relieved cholesterol accumulation in endosomal compartments.

  • These findings provide the first insight into the role of NEGR1 in intracellular cholesterol homeostasis and human obesity.

Abstract

Neuronal growth regulator 1 (NEGR1) is a newly identified raft-associated protein, which has recently been spotlighted as a new locus related to human obesity. Niemann-Pick disease Type C2 (NPC2) protein functions as a key player in the intracellular cholesterol trafficking, and its defect is linked to a fatal human neurodegenerative disease, NPC. In this study, we identified that NEGR1 interacts with NPC2 and increases its protein stability. Ectopically expressed NEGR1 proteins relieved an abnormal cholesterol accumulation in endosomal compartments. Importantly, NEGR1-defective mouse embryonic fibroblast cells exhibit increased cholesterol levels and triglyceride contents. These findings provide the first insight into the role of NEGR1 in intracellular cholesterol homeostasis, possibly explaining the missing link between NEGR1 with human obesity.

Introduction

As an important cell membrane constituent, cellular cholesterol level is well balanced via cellular intake, intracellular storage, and extracellular efflux [1]. For this reason, deregulation of cholesterol is implicated in diverse human diseases, such as atherosclerosis, diabetes, and several neurological diseases including Niemann-Pick disease and Alzheimer's disease [2]. Niemann-Pick disease type C (NPC) is an autosomal recessive disorder characterized by marked intracellular accumulation of unesterified cholesterol in the late endosome/lysosome compartments and severe deterioration of central nerve system with premature death [3]. About 5% of NPC cases are linked to NPC2, a soluble 151-amino acid glycoprotein, found in lysosomes and secretory fluids such as epididymal fluid and plasma [4]. Although NPC2 is closely implicated in cholesterol trafficking, the exact molecular mechanisms regarding the egress of endosomal cholesterol is still unknown [5].

In the cell membrane, cholesterol is mainly found in a detergent-insoluble membrane sub-domain called lipid raft [6]. These highly dynamic structures are also enriched in various membrane-associated proteins including glycosylphosphatidylinositol (GPI)-anchored proteins, which are responsible for important cellular processes such as cell-cell interaction, cell migration, and signal transduction [7].

Human neuronal growth regulator 1 (NEGR1) is a homolog of rat kilon (kindred of IgLON) that was identified as a new raft component from rat brain [8]. Protein analysis revealed that NEGR1 is a GPI-anchored protein with three C2 type immunoglobulin domains. Our recent study identified NEGR1 down-regulation in various human cancers, which may function in cell-cell adhesion [9]. Recently, NEGR1 has gained high attention as a new locus responsible for human body weight control [10]. Several genetic studies have revealed the close association between NEGR1 and human obesity, however, no functional clues have been discovered so far. In this study, we propose that human NEGR1 may function in the intracellular cholesterol trafficking, thereby providing the first insight into the molecular mechanisms of NEGR1 involvement in human obesity.

Section snippets

Cell culture, yeast two-hybrid screening, and cloning

HEK293, HeLa, and U178 cells were grown in DMEM media containing 10% FBS (Invitrogen), while CRT-MG and MEF cells were cultured in Ham's F-12 media. Transient transfection was performed using Effectene (Qiagen). Yeast two-hybrid screening was conducted using GAL4-based Matchmaker™ Two-hybrid System (Clontech) according to manufacturer's instructions. The truncated N-terminal region (amino acids 40-215) of NEGR1 was sub-cloned into pGBKT7 vector and used as a bait. Positive clones were selected

Human NEGR1 interacts with NPC2

To discover the as yet-unidentified role of human NEGR1, we performed yeast two-hybrid screening using human testis library. For convenience sake, we termed each C2 domain of NEGR1 as D1, D2, and D3 from the N-terminus (Fig. 1A). Since the entire NEGR1 protein displayed auto-activation in GAL4 promoter system, we used the truncated form constituting the N-terminal two C2 domains (D1-2) as bait. We isolated a positive clone, which was subsequently identified as NPC2 by sequence analysis. When we

Discussion

Human brain is highly enriched in cholesterol as compared to other body tissues [19]. It harbors approximately 25% of the total body cholesterol, since cholesterol is the main lipid component of myelin sheath and cell membrane lipid rafts in neuron and astrocytes [20]. For that reason, deregulation of cholesterol may provoke profound consequences in the function of the central nervous system (CNS). Since plasma lipoproteins are not able to cross the blood-brain barrier, nearly all cholesterol

Acknowledgements

This research was supported by Basic Science Research Program by the Ministry of Education, Science and Technology of Korea (NRF-2016R1A2B4009244) and research fund of Chungnam National University.

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