NPC1L1 (Niemann-Pick C1-like 1) mediates sterol-specific unidirectional transport of non-esterified cholesterol in McArdle-RH7777 hepatoma cells

Recent evidence suggests that NPC1L1 (Niemann-Pick C1-like 1) is critical for intestinal sterol absorption in mice, yet mechanisms by which NPC1L1 regulates cellular sterol transport are lacking. In the study we used a McArdle-RH7777 rat hepatoma cell line stably expressing NPC1L1 to examine the sterol-specificity and directionality of NPC1L1-mediated sterol transport. As previously described, cholesterol-depletion-driven recycling of NPC1L1 to the cell surface facilitates cellular uptake of non-esterified (free) cholesterol. However, it has no impact on the uptake of esterified cholesterol, indicating free sterol specificity. Interestingly, the endocytic recycling of NPC1L1 was also without effect on beta-sitosterol uptake, indicating that NPC1L1 can differentiate between free sterols of animal and plant origin in hepatoma cells. Furthermore, NPC1L1-driven free cholesterol transport was unidirectional, since cellular cholesterol efflux to apolipoprotein A-I, high-density lipoprotein or serum was unaffected by NPC1L1 expression or localization. Additionally, NPC1L1 facilitates mass non-esterified-cholesterol uptake only when it is located on the cell surface and not when it resides intracellularly. Finally, NPC1L1-dependent cholesterol uptake required adequate intracellular K(+), yet did not rely on intracellular Ca(2+), the cytoskeleton or signalling downstream of protein kinase A, protein kinase C or pertussis-toxin-sensitive G-protein-coupled receptors. Collectively, these findings support the notion that NPC1L1 can selectively recognize non-esterified cholesterol and promote its unidirectional transport into hepatoma cells.     

Kinetic imaging of NPC1L1 and sterol trafficking between plasma membrane and recycling endosomes in hepatoma cells

Niemann-Pick C1-like 1 (NPC1L1) is a recently identified protein that mediates intestinal cholesterol absorption and regulates biliary cholesterol excretion. The itineraries and kinetics of NPC1L1 trafficking remain uncertain. In this study, we have visualized movement of NPC1L1-enhanced green fluorescent protein (NPC1L1-EGFP) and cholesterol analogs in hepatoma cells. At steady state, about 42% of NPC1L1 resided in the transferrin (Tf)-positive, sterol-enriched endocytic recycling compartment (ERC), whereas time-lapse microscopy demonstrated NPC1L1 traffic between the plasma membrane and the ERC. Fluorescence recovery after photobleaching revealed rapid recovery (half-time approximately 2.5 min) of about 35% of NPC1L1 in the ERC, probably replenished from peripheral sorting endosomes. Acute cholesterol depletion blocked internalization of NPC1L1-EGFP and Tf and stimulated recycling of NPC1L1-EGFP from the ERC to the plasma membrane. NPC1L1-EGFP facilitated transport of fluorescent sterols from the plasma membrane to the ERC. Insulin induced translocation of vesicles containing NPC1L1 and fluorescent sterol from the ERC to the cell membrane. Upon polarization of hepatoma cells, NPC1L1 resided almost exclusively in the canalicular membrane, where the protein is highly mobile. Our study demonstrates dynamic trafficking of NPC1L1 between the cell surface and intracellular compartments and suggests that this transport is involved in NPC1L1-mediated cellular sterol uptake.     

NPC1L1 and cholesterol transport

The polytopic transmembrane protein, Niemann-Pick C1-Like 1 (NPC1L1), is enriched in the apical membrane of small intestine absorptive enterocytes where it mediates extracellular sterol transport across the brush border membrane. It is essential for intestinal sterol absorption and is the molecular target of ezetimibe, a potent cholesterol absorption inhibitor that lowers blood cholesterol in humans. NPC1L1 is also highly expressed in human liver. The hepatic function of NPC1L1 may be to limit excessive biliary cholesterol loss. NPC1L1-dependent sterol uptake seems to be a clathrin-mediated endocytic process and is regulated by cellular cholesterol content. Recently, NPC1L1 inhibition has been shown to have beneficial effects on components of the metabolic syndrome, such as obesity, insulin resistance, and fatty liver, in addition to atherosclerosis.     

The Clathrin Adaptor Proteins ARH,Dab2, and Numb Play Distinct Roles in Niemann-Pick C1-Like 1 Versus Low Density Lipoprotein Receptor-mediated Cholesterol Uptake

The uptake of circulating low density lipoproteins (LDL) is mediated by LDL receptor (LDLR) through clathrin-dependent endocytosis. At the early stage of this process, adaptor proteins ARH and Dab2 specifically bind the endocytic signal motif in LDLR and recruit clathrin/AP2 to initiate internalization. On the other hand, intestinal cholesterol is absorbed by Niemann-Pick C1-Like 1 (NPC1L1) through clathrin-dependent endocytosis. Another adaptor protein, Numb recognizes the endocytic motif in NPC1L1 C terminus and couples NPC1L1 to endocytic machinery. The ARH, Dab2, and Numb proteins contain a homogeneous phosphotyrosine binding (PTB) domain that directly binds endocytic motifs. Because ARH, Dab2, and Numb are all PTB domain family members, the emerging mystery is whether these adaptors act complementally in LDLR and NPC1L1 endocytosis. Here, we found that ARH and Dab2 did not bind NPC1L1 and were not required for NPC1L1 internalization. Similarly, Numb lacked the ability to interact with the LDLR C terminus and was dispensable for LDL uptake. Only the Numb isoforms with shorter PTB domain could facilitate NPC1L1 endocytosis. Besides the reported function in intestinal cholesterol absorption, Numb also mediated cholesterol reabsorption from bile in liver. We further identified a Numb variant with G595D substitution in humans of low blood LDL-cholesterol. The G595D substitution impaired NPC1L1 internalization and cholesterol reabsorption, due to attenuating affinity of Numb to clathrin/AP2. These results demonstrate that Numb specifically regulates NPC1L1-mediated cholesterol absorption both in human intestine and liver, distinct from ARH and Dab2, which selectively participate in LDLR-mediated LDL uptake.     

Molecular characterization of the NPC1L1 variants identified from cholesterol low absorbers

Niemann-Pick C1-like 1 (NPC1L1) is an essential protein for dietary cholesterol absorption. Nonsynonymous (NS) variants of NPC1L1 in humans have been suggested to associate with cholesterol absorption variations. However, information concerning the characteristics and mechanism of these variants in cholesterol uptake is limited. In this study, we analyzed the cholesterol uptake ability of the 19 reported NS variants of NPC1L1 identified from cholesterol low absorbers. Among these variants, L110F, R306C, A395V, G402S, T413M, R693C, R1214H, and R1268H could partially mediate cellular cholesterol uptake and were categorized as partially dysfunctional variants. The other 11 variants including T61M, N132S, D398G, R417W, G434R, T499M, S620C, I647N, G672R, S881L, and R1108W could barely facilitate cholesterol uptake, and were classified into the severely dysfunctional group. The partially dysfunctional variants showed mild defects in one or multiple aspects of cholesterol-regulated recycling, subcellular localization, glycosylation, and protein stability. The severely dysfunctional ones displayed remarkable defects in all these aspects and were rapidly degraded through the ER-associated degradation (ERAD) pathway. In vivo analyses using adenovirus-mediated expression in mouse liver confirmed that the S881L variant failed to localize to liver canalicular membrane, and the mice showed defects in biliary cholesterol re-absorption, while the G402S variant appeared to be similar to wild-type NPC1L1 in mouse liver. This study suggests that the dysfunction of the 19 variants on cholesterol absorption is due to the impairment of recycling, subcellular localization, glycosylation, or stability of NPC1L1.     

Dietary cholesterol induces trafficking of intestinal Niemann-Pick Type C1 Like 1 from the brush border to endosomes

The transmembrane protein Niemann-Pick C1 Like 1 (NPC1L1) belongs to the Niemann-Pick C1 (NPC1) family of cholesterol transporters and is mainly expressed in the liver and the small intestine. NPC1L1 is believed to be the main transporter responsible for the absorption of dietary cholesterol. Like NPC1, NPC1L1 contains a sterol sensing domain, suggesting that it might be sensitive to dietary cholesterol. To test this hypothesis, mucosal explants were cultured in the presence or absence of cholesterol. In the absence of cholesterol NPC1L1 was localized mainly in the brush border of the enterocyte, colocalizing with the brush border enzyme aminopeptidase N (APN), and only a minor part was present in intracellular compartments. In contrast, following culture in the presence of cholesterol a major part of NPC1L1 was found in intracellular compartments positive for the early endosomal marker early endosome antigen 1, whereas only a minor fraction was left in the brush border. Neither APN, lactase, nor sucrase-isomaltase was endocytosed in parallel, demonstrating that this is a selective cholesterol-induced endocytosis of NPC1L1. Conceivably either the induced internalization could be due to NPC1L1 acting as an endocytic cholesterol receptor or it could be a mechanism to reduce the cholesterol uptake. The fluorescent cholesterol analog NBD-cholesterol readily labeled the cytoplasm also under conditions nonpermissible for endocytosis, arguing against a receptor-mediated uptake. We therefore propose that cholesterol is absorbed by NPC1L1 acting as a membrane transporter and that NPC1L1 is internalized to an endosomal compartment to reduce the absorption of cholesterol.     

The small GTPase Cdc42 interacts with Niemann-Pick C1-like 1 (NPC1L1) and controls its movement from endocytic recycling compartment to plasma membrane in a cholesterol-dependent manner

Niemann-Pick C1-like 1 (NPC1L1) is a multi-transmembrane protein that mediates the absorption of dietary and biliary cholesterol through vesicular endocytosis. The subcellular localization of NPC1L1 is regulated by cholesterol. Cholesterol depletion induces the transport of NPC1L1 to plasma membrane (PM) from endocytic recycling compartment that requires MyoVb·Rab11a·Rab11-FIP2 triple complex, and cholesterol-replenishment renders the internalization of NPC1L1 together with cholesterol. Here, we find that GTP-bound Cdc42 interacts with NPC1L1. Cholesterol depletion regulates the activation of Cdc42 and enhances NPC1L1-Cdc42 interaction. Overexpression of constitutive GTP-bound Cdc42 mutant form or knockdown of Cdc42 inhibits the transport of NPC1L1 to the PM and disturbs the cholesterol-regulated binding of NPC1L1 to Rab11a, MyoVb, and actin. Knockdown of Cdc42 downstream effectors N-WASP or Arp3 also leads to the similar results. In liver-specific Cdc42 knock-out (Cdc42 LKO) mice, NPC1L1 fails to localize to bile canaliculi, and the biliary cholesterol cannot be efficiently reabsorbed. These results indicate that Cdc42 controls the cholesterol-regulated transport and localization of NPC1L1, and plays a role in cholesterol absorption.     

The cholesterol absorption inhibitor ezetimibe acts by blocking the sterol-induced internalization of NPC1L1

Niemann-Pick C1-like 1 (NPC1L1) is a polytopic transmembrane protein that plays a critical role in cholesterol absorption. Ezetimibe, a hypocholesterolemic drug, has been reported to bind NPC1L1 and block cholesterol absorption. However, the molecular mechanism of NPC1L1-mediated cholesterol uptake and how ezetimibe inhibits this process are poorly defined. Here we find that cholesterol specifically promotes the internalization of NPC1L1 and that this process requires microfilaments and the clathrin/AP2 complex. Blocking NPC1L1 endocytosis dramatically decreases cholesterol internalization, indicating that NPC1L1 mediates cholesterol uptake via its vesicular endocytosis. Ezetimibe prevents NPC1L1 from incorporating into clathrin-coated vesicles and thus inhibits cholesterol uptake. Together, our data suggest a model wherein cholesterol is internalized into cells with NPC1L1 through clathrin/AP2-mediated endocytosis and ezetimibe inhibits cholesterol absorption by blocking the internalization of NPC1L1.     

The N-terminal domain of NPC1L1 protein binds cholesterol and plays essential roles in cholesterol uptake

Niemann-Pick C1-like 1 (NPC1L1) is a multitransmembrane protein playing a crucial role in dietary and biliary cholesterol absorption. Cholesterol promotes the formation and endocytosis of NPC1L1-flotillin-cholesterol membrane microdomains, which is an early step in cholesterol uptake. How cholesterol is sensed in this step is unknown. Here, we find that the N-terminal domain (NTD) of NPC1L1 binds cholesterol. Mutation of residue Leu-216 in NPC1L1-NTD eliminates cholesterol binding, decreases the formation of NPC1L1-flotillin-cholesterol membrane microdomains, and prevents NPC1L1-mediated cholesterol uptake in culture cells and mice livers. NPC1L1-NTD specifically binds cholesterol but not plant sterols, which may account for the selective cholesterol absorption in intestine. Furthermore, 25- or 27-hydroxycholesterol competes with cholesterol to bind NPC1L1-NTD and inhibits the cholesterol induced endocytosis of NPC1L1. Together, these results demonstrate that plasma membrane-localized NPC1L1 binds exogenous cholesterol via its NTD, and facilitates the formation of NPC1L1-flotillin-cholesterol membrane microdomains that are then internalized into cells through the clathrin-AP2 pathway. Our study uncovers the mechanism of cholesterol sensing by NPC1L1 and proposes a mechanism for selective cholesterol absorption.     

Multiple plasma membrane receptors but not NPC1L1 mediate high-affinity, ezetimibe-sensitive cholesterol uptake into the intestinal brush border membrane

We compared cholesterol uptake into brush border membrane vesicles (BBMV) made from the small intestines of either wild-type or Niemann-Pick C1-like 1 (NPC1L1) knockout mice to elucidate the contribution of NPC1L1 to facilitated uptake; this uptake involves cholesterol transport from lipid donor particles into the BBM of enterocytes. The lack of NPC1L1 in the BBM of the knockout mice had no effect on the rate of cholesterol uptake. It follows that NPC1L1 cannot be the putative high-affinity, ezetimibe-sensitive cholesterol transporter in the brush border membrane (BBM) as has been proposed by others. The following findings substantiate this conclusion: (I) NPC1L1 is not a brush border membrane protein but very likely localized to intracellular membranes; (II) the cholesterol absorption inhibitor ezetimibe and its analogues reduce cholesterol uptake to the same extent in wild-type and NPC1L1 knockout mouse BBMV. These findings indicate that the prevailing belief that NPC1L1 facilitates intestinal cholesterol uptake into the BBM and its interaction with ezetimibe is responsible for the inhibition of this process can no longer be sustained.     

The ABCA1 transporter modulates late endocytic trafficking: insights from the correction of the genetic defect in Tangier disease

We have previously established that the ABCA1 transporter, which plays a critical role in the lipidation of extracellular apolipoprotein acceptors, traffics between late endocytic vesicles and the cell surface (Neufeld, E. B., Remaley, A. T., Demosky, S. J., Jr., Stonik, J. A., Cooney, A. M., Comly, M., Dwyer, N. K., Zhang, M., Blanchette-Mackie, J., Santamarina-Fojo, S., and Brewer, H. B., Jr. (2001) J. Biol. Chem. 276, 27584-27590). The present study provides evidence that ABCA1 in late endocytic vesicles plays a role in cellular lipid efflux. Late endocytic trafficking was defective in Tangier disease fibroblasts that lack functional ABCA1. Consistent with a late endocytic protein trafficking defect, the hydrophobic amine U18666A retained NPC1 in abnormally tubulated, cholesterol-poor, Tangier disease late endosomes, rather than cholesterol-laden lysosomes, as in wild type fibroblasts. Consistent with a lipid trafficking defect, Tangier disease late endocytic vesicles accumulated both cholesterol and sphingomyelin and were immobilized in a perinuclear localization. The excess cholesterol in Tangier disease late endocytic vesicles retained massive amounts of NPC1, which traffics lysosomal cholesterol to other cellular sites. Exogenous apoA-I abrogated the cholesterol-induced retention of NPC1 in wild type but not in Tangier disease late endosomes. Adenovirally mediated ABCA1-GFP expression in Tangier disease fibroblasts corrected the late endocytic trafficking defects and restored apoA-I-mediated cholesterol efflux. ABCA1-GFP expression in wild type fibroblasts also reduced late endosome-associated NPC1, induced a marked uptake of fluorescent apoA-I into ABCA1-GFP-containing endosomes (that shuttled between late endosomes and the cell surface), and enhanced apoA-I-mediated cholesterol efflux. The combined results of this study suggest that ABCA1 converts pools of late endocytic lipids that retain NPC1 to pools that can associate with endocytosed apoA-I, and be released from the cell as nascent high density lipoprotein.     

Ezetimibe blocks the internalization of NPC1L1 and cholesterol in mouse small intestine

The multiple transmembrane protein Niemann-Pick C1 like1 (NPC1L1) is essential for intestinal cholesterol absorption. Ezetimibe binds to NPC1L1 and is a clinically used cholesterol absorption inhibitor. Recent studies in cultured cells have shown that NPC1L1 mediates cholesterol uptake through vesicular endocytosis that can be blocked by ezetimibe. However, how NPC1L1 and ezetimibe work in the small intestine is unknown. In this study, we found that NPC1L1 distributed in enterocytes of villi and transit-amplifying cells of crypts. Acyl-CoA cholesterol acyltransferase 2 (ACAT2), another important protein for cholesterol absorption by providing cholesteryl esters to chylomicrons, was mainly presented in the apical cytoplasm of enterocytes. NPC1L1 and ACAT2 were highly expressed in jejunum and ileum. ACAT1 presented in the Paneth cells of crypts and mesenchymal cells of villi. In the absence of cholesterol, NPC1L1 was localized on the brush border of enterocytes. Dietary cholesterol induced the internalization of NPC1L1 to the subapical layer beneath the brush border and became partially colocalized with the endosome marker Rab11. Ezetimibe blocked the internalization of NPC1L1 and cholesterol and caused their retention in the plasma membrane. This study demonstrates that NPC1L1 mediates cholesterol entering enterocytes through vesicular endocytosis and that ezetimibe blocks this step in vivo.     

Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis

Niemann-Pick C1 Like 1 (NPC1L1) is a protein localized in jejunal enterocytes that is critical for intestinal cholesterol absorption. The uptake of intestinal phytosterols and cholesterol into absorptive enterocytes in the intestine is not fully defined on a molecular level, and the role of NPC1L1 in maintaining whole body cholesterol homeostasis is not known. NPC1L1 null mice had substantially reduced intestinal uptake of cholesterol and sitosterol, with dramatically reduced plasma phytosterol levels. The NPC1L1 null mice were completely resistant to diet-induced hypercholesterolemia, with plasma lipoprotein and hepatic cholesterol profiles similar to those of wild type mice treated with the cholesterol absorption inhibitor ezetimibe. Cholesterol/cholate feeding resulted in down-regulation of intestinal NPC1L1 mRNA expression in wild type mice. NPC1L1 deficiency resulted in up-regulation of intestinal hydroxymethylglutaryl-CoA synthase mRNA and an increase in intestinal cholesterol synthesis, down-regulation of ABCA1 mRNA, and no change in ABCG5 and ABCG8 mRNA expression. NPC1L1 is required for intestinal uptake of both cholesterol and phytosterols and plays a major role in cholesterol homeostasis. Thus, NPC1L1 may be a useful drug target for the treatment of hypercholesterolemia and sitosterolemia.     

Requirement of Myosin Vb��Rab11a��Rab11-FIP2 Complex in Cholesterol-regulated Translocation of NPC1L1 to the Cell Surface

Niemann-Pick C1-like 1 (NPC1L1) plays a critical role in the enterohepatic absorption of free cholesterol. Cellular cholesterol depletion induces the transport of NPC1L1 from the endocytic recycling compartment to the plasma membrane (PM), and cholesterol replenishment causes the internalization of NPC1L1 together with cholesterol via clathrin-mediated endocytosis. Although NPC1L1 has been characterized, the other proteins involved in cholesterol absorption and the endocytic recycling of NPC1L1 are largely unknown. Most of the vesicular trafficking events are dependent on the cytoskeleton and motor proteins. Here, we investigated the roles of the microfilament and microfilament-associated triple complex composed of myosin Vb, Rab11a, and Rab11-FIP2 in the transport of NPC1L1 from the endocytic recycling compartment to the PM. Interfering with the dynamics of the microfilament by pharmacological treatment delayed the transport of NPC1L1 to the cell surface. Meanwhile, inactivation of any component of the myosin Vb·Rab11a·Rab11-FIP2 triple complex inhibited the export of NPC1L1. Expression of the dominant-negative mutants of myosin Vb, Rab11a, or Rab11-FIP2 decreased the cellular cholesterol uptake by blocking the transport of NPC1L1 to the PM. These results suggest that the efficient transport of NPC1L1 to the PM is dependent on the microfilament-associated myosin Vb·Rab11a·Rab11-FIP2 triple complex.Cholesterol homeostasis in human bodies is maintained through regulated cholesterol synthesis, absorption, and excretion. Intestinal cholesterol absorption is one of the major pathways to maintain cholesterol balance. NPC1L1 (Niemann-Pick C1-like protein 1), a polytopic transmembrane protein highly expressed in the intestine and liver, is required for dietary cholesterol uptake and biliary cholesterol reabsorption (1–4). Genetic or pharmaceutical inactivation of NPC1L1 significantly inhibits cholesterol absorption and confers the resistance to diet-induced hypercholesterolemia (1, 2, 4). Ezetimibe, an NPC1L1-specific inhibitor, is currently used to prevent and treat cardiovascular diseases (5).Human NPC1L1 contains 1,332 residues with 13 transmembrane domains (6). The third to seventh transmembrane helices constitute a conserved sterol-sensing domain (4, 7). NPC1L1 recycles between the endocytic recycling compartment (ERC)³ and the plasma membrane (PM) in response to the changes of cholesterol level (8). ERC is a part of early endosomes that is involved in the recycling of many transmembrane proteins. It is also reported that ERC is a pool for free cholesterol storage (9). When cellular cholesterol concentration is low, NPC1L1 moves from the ERC to the PM (8, 10). Under cholesterol-replenishing conditions, NPC1L1 and cholesterol are internalized together and transported to the ERC (8). Disruption of microfilament, depletion of the clathrin·AP2 complex, or ezetimibe treatment can impede the endocytosis of NPC1L1, thereby decreasing cholesterol internalization (8, 10, 11).The microfilament (MF) system, part of the cytoskeleton network, is required for multiple cellular functions such as cell shape maintenance, cell motility, mitosis, protein secretion, and endocytosis (12, 13). The major players in the microfilament system are actin fibers and motor proteins (14). Actin fibers form a network that serves as the tracks for vesicular transport (15, 16). Meanwhile, the dynamic assembly and disassembly of actin fibers and the motor proteins provides the driving force for a multitude of membrane dynamics including endocytosis, exocytosis, and vesicular trafficking between compartments (15, 16).Myosins are a large family of motor proteins that are responsible for actin-based mobility (14). Class V myosins (17, 18), comprising myosin Va, Vb, and Vc, are involved in a wide range of vesicular trafficking events in different mammalian tissues. Myosin Va is expressed mainly in neuronal tissues (19, 20), whereas myosins Vb and Vc are universally expressed with enrichment in epithelial cells (21, 22). Class V myosins are recruited to their targeting vesicles by small GTPase proteins (Rab) (23). Rab11a and Rab11 family-interacting protein 2 (Rab11-FIP2) facilitate the binding of myosin Vb to the cargo proteins of endocytic recycling vesicles (24–28).Myosin Vb binds Rab11a and Rab11-FIP2 through the C-terminal tail (CT) domain. The triple complex of myosin Vb, Rab11a, and Rab11-FIP2 is critical for endocytic vesicular transport and the recycling of many proteins including transferrin receptor (29), AMPA receptors (30), CFTR (28), GLUT4 (31, 32), aquaporin-2 (26), and β2-adrenergic receptors (33). The myosin Vb-CT domain (24) competes for binding to Rab11a and Rab11-FIP2 and functions as a dominant-negative form. Expression of the CT domain substantially impairs the transport of vesicles. Deficient endocytic trafficking is also observed in cells expressing the GDP-locked form of Rab11a (S25N) (34) or a truncated Rab11-FIP2, which competes for the rab11a binding (35).Here we investigated the roles of actin fibers and motor proteins in the cholesterol-regulated endocytic recycling of NPC1L1. Using pharmaceutical inactivation, dominant-negative forms, and an siRNA technique, we demonstrated that actin fibers and myosin Vb·Rab11a·Rab11-FIP2 triple complex are involved in the export of NPC1L1 to the PM and that this intact MF-associated triple complex is required for efficient cholesterol uptake. Characterization of the molecules involved in the recycling of NPC1L1 may shed new light upon the mechanism of cholesterol absorption.     

Niemann-Pick C1-Like 1 and cholesterol uptake

Niemann-Pick C1-Like 1 (NPC1L1) is a polytopic transmembrane protein responsible for dietary cholesterol and biliary cholesterol absorption. Consistent with its functions, NPC1L1 distributes on the brush border membrane of enterocytes and the canalicular membrane of hepatocytes in humans. As the molecular target of ezetimibe, a hypocholesterolemic drug, its physiological and pathological significance has been recognized and intensively studied for years. Recently, plenty of new findings reveal the molecular mechanism of NPC1L1's role in cholesterol uptake, which may provide new insights on our understanding of cholesterol absorption. In this review, we summarized recent progress in these studies and proposed a working model, hoping to provide new perspectives on the regulation of cholesterol transport and metabolism.     

A sharper instrument for dissecting signalling events: a specific AGC kinase inhibitor

Dietary and biliary cholesterol are taken up by intestinal epithelial cells and transported to the endoplasmic reticulum. At the endoplasmic reticulum, cholesterol is esterified, packaged into chylomicrons and secreted into the lymph for delivery to the bloodstream. NPC1L1 (Niemann-Pick C1-like 1) is a protein on the enterocyte brush-border membrane that facilitates cholesterol absorption. Cholesterol's itinerary as it moves to the endoplasmic reticulum is unknown, as is the identity of any cellular proteins that facilitate the movement. Two proteins that play an important role in intracellular cholesterol transport and could potentially influence NPC1L1-mediated cholesterol uptake are NPC1 and NPC2 (Niemann-Pick type C disease proteins 1 and 2). In this issue of the Biochemical Journal, Dixit and colleagues show that the absence or presence of NPC1 and NPC2 has no effect on intestinal cholesterol absorption in the mouse. Thus neither protein fills the gap in our knowledge of intra-enterocyte cholesterol transport. Furthermore, the NPC1/NPC2 pathway would not be a good target for limiting the uptake of dietary cholesterol.     

Cholesterol-depletion corrects APP and BACE1 misstrafficking in NPC1-deficient cells

Cholesterol accumulation in Niemann-Pick type C disease (NPC) causes increased levels of the amyloid-precursor-protein C-terminal fragments (APP-CTFs) and intracellular amyloid-β peptide (Aβ), the two central molecules in Alzheimer's disease (AD) pathogenesis. We previously reported that cholesterol accumulation in NPC-cells leads to cholesterol-dependent increased APP processing by β-secretase (BACE1) and decreased APP expression at the cell surface (Malnar et al. Biochim Biophys Acta. 1802 (2010) 682-691.). We hypothesized that increased formation of APP-CTFs and Aβ in NPC disease is due to cholesterol-mediated altered endocytic trafficking of APP and/or BACE1. Here, we show that APP endocytosis is prerequisite for enhanced Aβ levels in NPC-cells. Moreover, we observed that NPC cells show cholesterol dependent sequestration and colocalization of APP and BACE1 within enlarged early/recycling endosomes which can lead to increased β-secretase processing of APP. We demonstrated that increased endocytic localization of APP in NPC-cells is likely due to both its increased internalization and its decreased recycling to the cell surface. Our findings suggest that increased cholesterol levels, such as in NPC disease and sporadic AD, may be the upstream effector that drives amyloidogenic APP processing characteristic for Alzheimer's disease by altering endocytic trafficking of APP and BACE1.     

Sterol-modulated glycolipid sorting occurs in niemann-pick C1 late endosomes

The Niemann-Pick C1 (NPC1) protein and endocytosed low density lipoprotein (LDL)-derived cholesterol were shown to enrich separate subsets of vesicles containing lysosomal associated membrane protein 2. Localization of Rab7 in the NPC1-containing vesicles and enrichment of lysosomal hydrolases in the cholesterol-containing vesicles confirmed that these organelles were late endosomes and lysosomes, respectively. Lysobisphosphatidic acid, a lipid marker of the late endosomal pathway, was found in the cholesterol-enriched lysosomes. Recruitment of NPC1 to Rab7 compartments was stimulated by cellular uptake of cholesterol. The NPC1 compartment was shown to be enriched in glycolipids, and internalization of GalNAcbeta1-4[NeuAcalpha2-3]Galbeta1-4Glcbeta1-1'-ceramide (G(M2)) into endocytic vesicles depends on the presence of NPC1 protein. The glycolipid profiles of the NPC1 compartment could be modulated by LDL uptake and accumulation of lysosomal cholesterol. Expression in cells of biologically active NPC1 protein fused to green fluorescent protein revealed rapidly moving and flexible tubular extensions emanating from the NPC1-containing vesicles. We conclude that the NPC1 compartment is a dynamic, sterol-modulated sorting organelle involved in the trafficking of plasma membrane-derived glycolipids as well as plasma membrane and endocytosed LDL cholesterol.