Structural insights into the mechanism of human NPC1L1-mediated cholesterol uptake

Niemann-Pick C1-like 1 (NPC1L1) protein plays a central role in the intestinal cholesterol absorption and is the target of a drug, ezetimibe, which inhibits NPC1L1 to reduce cholesterol absorption. Here, we present cryo–electron microscopy structures of human NPC1L1 in apo state, cholesterol-enriched state, and ezetimibe-bound state to reveal molecular details of NPC1L1-mediated cholesterol uptake and ezetimibe inhibition. Comparison of these structures reveals that the sterol-sensing domain (SSD) could respond to the cholesterol level alteration by binding different number of cholesterol molecules. Upon increasing cholesterol level, SSD binds more cholesterol molecules, which, in turn, triggers the formation of a stable structural cluster in SSD, while binding of ezetimibe causes the deformation of the SSD and destroys the structural cluster, leading to the inhibition of NPC1L1 function. These results provide insights into mechanisms of NPC1L1 function and ezetimibe action and are of great significance for the development of new cholesterol absorption inhibitors.

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Do mammalian NPC1 and NPC2 play a role in intestinal cholesterol absorption?

Biochemical Journal ◽

10.1042/bj20071167 ◽

2007 ◽

Vol 408 (1) ◽

pp. 1-5 ◽

Cited By ~ 28

Author(s):

Sayali S. Dixit ◽

David E. Sleat ◽

Ann M. Stock ◽

Peter Lobel

Keyword(s):

Cholesterol Metabolism ◽

Cholesterol Absorption ◽

Intestinal Cholesterol Absorption ◽

Cholesterol Uptake ◽

Uptake Pathway ◽

Molecular Events ◽

Niemann Pick Disease ◽

Type C ◽

Niemann Pick ◽

Pick Disease

NPC1L1 (Niemann–Pick C1-like 1), the pharmacological target of the cholesterol-uptake inhibitor ezetimibe, is a transporter localized on the brush border of enterocytes. Although this protein plays a key role in intestinal uptake of sterols, multiple molecular events that underlie intestinal cholesterol absorption have not been fully characterized. Two proteins that might be involved in this process are NPC1 and NPC2 (Niemann–Pick disease type C proteins 1 and 2), which function in the endosomal/lysosomal cholesterol egress pathway and whose deficiency results in NPC (Niemann–Pick type C) disease. The involvement of these proteins in intestinal cholesterol absorption was examined in mutant mice lacking either NPC1 or NPC2. Our data indicate that deficiencies in either protein do not have an effect on cholesterol uptake or absorption. This contrasts with recent results obtained for the fruitfly Drosophila melanogaster, which indicate that a deficiency of NPC1 (dNPC1a being its Drosophila homologue) leads to activation of an NPC1L1 (Drosophila homologue dNPC1b)-independent cholesterol uptake pathway, underscoring fundamental differences in mammalian and non-mammalian cholesterol metabolism.

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CRYO-Electron Microscopy of the Acto-Myosin-ATP Complex

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100179993 ◽

1990 ◽

Vol 48 (1) ◽

pp. 248-249

Author(s):

John Trinickt ◽

Howard White

Keyword(s):

Electron Microscopy ◽

Atp Hydrolysis ◽

Myosin Head ◽

Bound State ◽

Cross Linking ◽

Weakly Bound ◽

Cryo Electron Microscopy ◽

Myosin Subfragment 1 ◽

Attached State ◽

High Fraction

The primary force of muscle contraction is thought to involve a change in the myosin head whilst attached to actin, the energy coming from ATP hydrolysis. This change in attached state could either be a conformational change in the head or an alteration in the binding angle made with actin. A considerable amount is known about one bound state, the so-called strongly attached state, which occurs in the presence of ADP or in the absence of nucleotide. In this state, which probably corresponds to the last attached state of the force-producing cycle, the angle between the long axis myosin head and the actin filament is roughly 45°. Details of other attached states before and during power production have been difficult to obtain because, even at very high protein concentration, the complex is almost completely dissociated by ATP. Electron micrographs of the complex in the presence of ATP have therefore been obtained only after chemically cross-linking myosin subfragment-1 (S1) to actin filaments to prevent dissociation. But it is unclear then whether the variability in attachment angle observed is due merely to the cross-link acting as a hinge.We have recently found low ionic-strength conditions under which, without resorting to cross-linking, a high fraction of S1 is bound to actin during steady state ATP hydrolysis. The structure of this complex is being studied by cryo-electron microscopy of hydrated specimens. Most advantages of frozen specimens over ambient temperature methods such as negative staining have already been documented. These include improved preservation and fixation rates and the ability to observe protein directly rather than a surrounding stain envelope. In the present experiments, hydrated specimens have the additional benefit that it is feasible to use protein concentrations roughly two orders of magnitude higher than in conventional specimens, thereby reducing dissociation of weakly bound complexes.

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Modulation of intestinal cholesterol absorption by high glucose levels: impact on cholesterol transporters, regulatory enzymes, and transcription factors

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.90376.2008 ◽

2008 ◽

Vol 295 (5) ◽

pp. G873-G885 ◽

Cited By ~ 45

Author(s):

Z. Ravid ◽

M. Bendayan ◽

E. Delvin ◽

A. T. Sane ◽

M. Elchebly ◽

...

Keyword(s):

Transcription Factors ◽

Protein Expression ◽

High Glucose ◽

Cholesterol Absorption ◽

Cholesterol Homeostasis ◽

Cholesterol Transport ◽

Intestinal Cholesterol Absorption ◽

Cholesterol Uptake ◽

Glucose Levels

Growing evidence suggests that the small intestine may contribute to excessive postprandial lipemia, which is highly prevalent in insulin-resistant/Type 2 diabetic individuals and substantially increases the risk of cardiovascular disease. The aim of the present study was to determine the role of high glucose levels on intestinal cholesterol absorption, cholesterol transporter expression, enzymes controlling cholesterol homeostasis, and the status of transcription factors. To this end, we employed highly differentiated and polarized cells (20 days of culture), plated on permeable polycarbonate filters. In the presence of [14C]cholesterol, glucose at 25 mM stimulated cholesterol uptake compared with Caco-2/15 cells supplemented with 5 mM glucose ( P < 0.04). Because combination of 5 mM glucose with 20 mM of the structurally related mannitol or sorbitol did not change cholesterol uptake, we conclude that extracellular glucose concentration is uniquely involved in the regulation of intestinal cholesterol transport. The high concentration of glucose enhanced the protein expression of the critical cholesterol transporter NPC1L1 and that of CD36 ( P < 0.02) and concomitantly decreased SR-BI protein mass ( P < 0.02). No significant changes were observed in the protein expression of ABCA1 and ABCG8, which act as efflux pumps favoring cholesterol export out of absorptive cells. At the same time, 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity was decreased ( P < 0.007), whereas ACAT activity remained unchanged. Finally, increases were noted in the transcription factors LXR-α, LXR-β, PPAR-β, and PPAR-γ along with a drop in the protein expression of SREBP-2. Collectively, our data indicate that glucose at high concentrations may regulate intestinal cholesterol transport and metabolism in Caco-2/15 cells, thus suggesting a potential influence on the cholesterol absorption process in Type 2 diabetes.

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A role for NPC1 and NPC2 in intestinal cholesterol absorption – the hypothesis gutted

Biochemical Journal ◽

10.1042/bj20071340 ◽

2007 ◽

Vol 408 (1) ◽

Cited By ~ 5

Author(s):

Laura Liscum

Keyword(s):

Endoplasmic Reticulum ◽

Dietary Cholesterol ◽

Cholesterol Absorption ◽

Cholesterol Transport ◽

Intestinal Epithelial ◽

Intestinal Cholesterol Absorption ◽

Biochemical Journal ◽

Good Target ◽

Type C ◽

Niemann Pick

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.

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Niemann-Pick C1 Like 1 Protein Is Critical for Intestinal Cholesterol Absorption

Science ◽

10.1126/science.1093131 ◽

2004 ◽

Vol 303 (5661) ◽

pp. 1201-1204 ◽

Cited By ~ 1161

Author(s):

S. W. Altmann

Keyword(s):

Cholesterol Absorption ◽

Intestinal Cholesterol Absorption ◽

Niemann Pick

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New Structural Insights into the Genome and Minor Capsid Proteins of BK Polyomavirus using Cryo-Electron Microscopy

Structure ◽

10.1016/j.str.2016.02.008 ◽

2016 ◽

Vol 24 (4) ◽

pp. 528-536 ◽

Cited By ~ 32

Author(s):

Daniel L. Hurdiss ◽

Ethan L. Morgan ◽

Rebecca F. Thompson ◽

Emma L. Prescott ◽

Margarita M. Panou ◽

...

Keyword(s):

Electron Microscopy ◽

Capsid Proteins ◽

Cryo Electron Microscopy ◽

Bk Polyomavirus ◽

Structural Insights

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Development and Physiological Regulation of Intestinal Lipid Absorption. III. Intestinal transporters and cholesterol absorption

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00061.2008 ◽

2008 ◽

Vol 294 (4) ◽

pp. G839-G843 ◽

Cited By ~ 49

Author(s):

David Y. Hui ◽

Eric D. Labonté ◽

Philip N. Howles

Keyword(s):

Basolateral Membrane ◽

Scavenger Receptor ◽

Cholesterol Absorption ◽

Lipid Absorption ◽

Intestinal Lumen ◽

Intestinal Cholesterol Absorption ◽

Physiological Regulation ◽

A Cell ◽

Intestinal Transporters ◽

Niemann Pick

Intestinal cholesterol absorption is modulated by transport proteins in enterocytes. Cholesterol uptake from intestinal lumen requires several proteins on apical brush-border membranes, including Niemann-Pick C1-like 1 (NPC1L1), scavenger receptor B-I, and CD36, whereas two ATP-binding cassette half transporters, ABCG5 and ABCG8, on apical membranes work together for cholesterol efflux back to the intestinal lumen to limit cholesterol absorption. NPC1L1 is essential for cholesterol absorption, but its function as a cell surface transporter or an intracellular cholesterol transport protein needs clarification. Another ATP transporter, ABCA1, is present in the basolateral membrane to mediate HDL secretion from enterocytes.

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Structures of dimeric human NPC1L1 provide insight into mechanisms for cholesterol absorption

Science Advances ◽

10.1126/sciadv.abh3997 ◽

2021 ◽

Vol 7 (34) ◽

pp. eabh3997

Author(s):

Tao Long ◽

Yang Liu ◽

Yu Qin ◽

Russell A. DeBose-Boyd ◽

Xiaochun Li

Keyword(s):

Low Density Lipoprotein ◽

Transmembrane Helix ◽

Dietary Cholesterol ◽

Density Lipoprotein ◽

Cholesterol Absorption ◽

Oligomeric State ◽

Cryo Electron Microscopy ◽

Niemann Pick ◽

Circulating Levels ◽

Insight Into

Polytopic Niemann-Pick C1-like 1 (NPC1L1) plays a major role in intestinal absorption of biliary cholesterol, vitamin E (VE), and vitamin K (VK). The drug ezetimibe inhibits NPC1L1-mediated absorption of cholesterol, lowering of circulating levels of low-density lipoprotein cholesterol. Here, we report cryo–electron microscopy structures of human NPC1L1 (hNPC1L1) bound to either cholesterol or a lipid resembling VE. These findings, together with functional assays, reveal that the same intramolecular channel in hNPC1L1 mediates transport of VE and cholesterol. hNPC1L1 exists primarily as a homodimer; dimerization is mediated by aromatic residues within a region of transmembrane helix 2 that exhibits a horizonal orientation in the membrane. Mutation of tryptophan-347 lies in this region disrupts dimerization and the resultant monomeric NPC1L1 exhibits reduced efficiency of cholesterol uptake. These findings identify the oligomeric state of hNPC1L1 as a target for therapies that inhibit uptake of dietary cholesterol and reduce the incidence of cardiovascular disease.

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Pravastatin Modulate Niemann-Pick C1-Like 1 and ATP-Binding Cassette G5 and G8 to Influence Intestinal Cholesterol Absorption

Journal of Pharmacy & Pharmaceutical Sciences ◽

10.18433/j3m029 ◽

2015 ◽

Vol 18 (5) ◽

pp. 765 ◽

Cited By ~ 2

Author(s):

Atsushi Kawase ◽

Seiji Hata ◽

Mai Takagi ◽

Masahiro Iwaki

Keyword(s):

Mrna Expression ◽

Hepg2 Cells ◽

Regulatory Element ◽

Cholesterol Absorption ◽

Atp Binding ◽

Mrna Levels ◽

Rt Pcr ◽

Intestinal Cholesterol Absorption ◽

Niemann Pick ◽

Atp Binding Cassette

Purpose. Niemann-Pick C1-like 1 (NPC1L1), ATP-binding cassette (ABC)G5, and ABCG8 mediate intestinal cholesterol absorption. It is unclear whether pravastatin (PR) or ezetimibe (EZ) affect expression of these transporters. We examined the effects of PR and EZ on NPC1L1, ABCG5, and ABCG8 expression in human hepatoma HepG2 cells and the murine small intestine. We also assessed expression of the transcription factors liver X receptor (LXR)a, LXRb and sterol regulatory element-binding protein. Methods. Transporter mRNA levels were determined in murine small intestines 6 and 24 h after oral PR and EZ administration by real-time reverse-transcriptase polymerase chain reaction (RT-PCR). In PR- and EZ-treated HepG2 cells, transporter and transcription factor mRNA and protein levels were examined by RT-PCR and western blot, respectively. Results. Significant decreases in NPC1L1, ABCG5, and ABCG8 mRNA expression were observed in the duodenum, but not jejunum and ileum, of mice 24 h after treatment with PR, but not EZ. In HepG2 cells, PR but not EZ treatment for 24 h also significantly decreased NPC1L1 protein and ABCG5, and ABCG8 mRNA expression, while increasing LXRa mRNA levels. Conclusion. PR but not EZ treatment reduced duodenal cholesterol transporter expression in mice. PR-induced increases in LXRa mRNA levels may be involved in attenuation of NPC1L1 expression, subsequently decreasing intestinal cholesterol absorption. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

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