Activation of Acyl-Coenzyme A:Cholesterol Acyltransferase by Cholesterol or by Oxysterol in a Cell-free System

Cheng, Dong; Chang, Catherine C.Y.; Qu, Xun; Chang, Ta−Yuan

doi:10.1074/jbc.270.2.685

Cited by 161 publications

(142 citation statements)

References 46 publications

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“…Although the competitive binding of 25-and 27-HC against cholesterol to NPC1-NTD has been reported (14,15,23), the effect of oxysterols on NPC1-mediated cholesterol transport has not been verified, possibly because most of the assays (i.e. sterol regulatory element-binding protein cleavage and cholesterol esterification) used to analyze the function of NPC1 are indirectly measuring endoplasmic reticulum cholesterol level, and the readouts can be affected by oxysterols themselves (32,33). On the other hand, here we show that 25-and 27-HC block the endocytosis of NPC1L1 and inhibit the formation of NPC1L1-flotillin-cholesterol membrane microdomains (Fig.…”

Section: Discussionmentioning

confidence: 99%

The N-terminal Domain of NPC1L1 Protein Binds Cholesterol and Plays Essential Roles in Cholesterol Uptake

Zhang

et al. 2011

Journal of Biological Chemistry

100

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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.NPC1L1 is a multiple transmembrane protein that is essential for dietary cholesterol absorption and biliary cholesterol reabsorption (1-3). Our previous studies have revealed that NPC1L1 can move between the endocytic recycling compartment (ERC) 4 and plasma membrane (PM) (4). Cholesterol depletion induces the export of NPC1L1 from ERC to PM in a myosin Vb-rab11a-rab11-FIP2-dependent manner (5, 6). Reversely, cholesterol replenishment promotes the formation of cholesterol-enriched membrane microdomains containing NPC1L1 and flotillins, named NPC1L1-flotillin-cholesterol membrane microdomains (7). These microdomains are in turn internalized through clathrin-AP2-mediated endocytosis and, therefore, lead to the internalization of NPC1L1 and ensure the efficient cholesterol uptake (7). It's still a mystery how cholesterol is sensed to initiate the formation and endocytosis of NPC1L1-flotillin-cholesterol membrane microdomains. Niemann-Pick C (NPC) 1 is a homologue of NPC1L1 (8, 9). NPC1 and another protein, NPC2, are required for transporting the LDL-derived cholesterol out of late endosomes/lysosomes (10). Mutation of the npc1 or npc2 genes leads to the Niemann-Pick Type C disease, a fatal hereditary disorder characterized by accumulation of cholesterol in late endosomes/ lysosomes (9, 11). NPC1 and NPC1L1 show the similar protein topology. Both of them have 13 transmembrane helices containing a sterol-sensing domain, a large luminal N-terminal domain (NTD), and two large luminal loops (12, 13). Recently, it has been shown that the NTD of NPC1 directly binds cholesterol (14). The crystal structure of the cholesterol-bound NPC1-NTD reveals that the cholesterol molecule is embedded in the binding pocket with an ...

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Section: Discussionmentioning

confidence: 99%

The N-terminal Domain of NPC1L1 Protein Binds Cholesterol and Plays Essential Roles in Cholesterol Uptake

Zhang

et al. 2011

Journal of Biological Chemistry

100

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“…In insect cells, mouse and human ACAT1 and ACAT2 utilize a variety of oxysterols in addition to cholesterol as substrates (31,42). Ergosterol and plant sterols are poor substrates (31,43).…”

Section: Biochemical Activity and Regulationmentioning

confidence: 99%

The Enzymes of Neutral Lipid Synthesis

Buhman¹,

Chen²,

Farese³

2001

Journal of Biological Chemistry

150

115

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“…The membrane pellet was resuspended in 300 l buffer F, pH 7.5, without oleoyl-CoA. ACAT activity was assayed in vitro as described (25).…”

Section: Acat Assaymentioning

confidence: 99%

Modulation of Endosomal Cholesteryl Ester Metabolism by Membrane Cholesterol

Wang

Castoreno

Stockinger

et al. 2005

Journal of Biological Chemistry

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Cells acquire cholesterol in part by endocytosis of cholesteryl ester containing lipoproteins. In endosomes and lysosomes cholesteryl ester is hydrolyzed by acidic cholesteryl ester hydrolase producing cholesterol and fatty acids. Under certain pathological conditions, however, such as in atherosclerosis, excessive levels of cholesteryl ester accumulate in lysosomes for reasons that are poorly understood. Here, we have studied endosomal and lysosomal cholesteryl ester metabolism in cultured mouse macrophages and with cell-free extracts. We show that net hydrolysis of cholesteryl ester is coupled to the transfer of cholesterol to membranes. When membrane cholesterol levels are low, absorption of cholesterol effectively drives cholesteryl ester hydrolysis. When cholesterol levels in acceptor membranes approach saturation or when cholesterol export is blocked, cholesterol is re-esterified in endosomes. These results reveal a new facet of cellular cholesterol homeostasis and provide a potential explanation for cholesteryl ester accumulation in lysosomes of atherosclerotic cells.One of the major hallmarks of atherosclerosis is the progressive accumulation of intracellular and extracellular cholesterol (1-3). In early stages of atherosclerosis excess cholesterol is mainly deposited as cholesteryl fatty acyl esters ("cholesteryl ester"). As atherosclerosis progresses, both cholesteryl ester and cholesterol accumulate (4). Advanced lesions are characterized by the formation of extracellular lipid cores that consist of cholesteryl ester, crystals of cholesterol monohydrate, and debris (5-7). Intracellularly, cholesterol builds up as a component of membranes and as oily droplets of cholesteryl ester that form in the cytoplasm and inside lysosomes (4, 5). At the late stages of disease progression, lysosomes become the major site of lipid accumulation (4, 8).The mechanisms that are responsible for the formation of cholesteryl esters in atheromatous cells have only partly been resolved. It is clear that cholesteryl ester in cytoplasmic lipid droplets is produced by ER 1 -localized acyl-CoA:cholesterol acyltransferases (ACATs) that are allosterically activated by cholesterol (9, 10). The aberrant accumulation of cholesteryl ester in lysosomes, however, remains to be explained (8).Cholesterol in atherosclerotic lesions is predominantly derived from serum lipoproteins such as low density lipoprotein (LDL) (11). LDL consists of a hydrophobic core containing mainly cholesteryl ester and triglycerides surrounded by a lipid monolayer and apolipoprotein B100 (12). LDL and its atherogenic derivatives are recognized by cell surface receptors, internalized, and transferred to the endosomal system. Cholesteryl ester and triglycerides are then hydrolyzed by acidic cholesteryl ester hydrolase (aCEH), generating cholesterol and fatty acids (11, 13,14).It has previously been proposed that lysosomal cholesteryl ester accumulation in atheromatous cells might result from a deficiency of aCEH (5, 15). Most subsequent studies on aCEH activity in...

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Activation of Acyl-Coenzyme A:Cholesterol Acyltransferase by Cholesterol or by Oxysterol in a Cell-free System

Cited by 161 publications

References 46 publications

The N-terminal Domain of NPC1L1 Protein Binds Cholesterol and Plays Essential Roles in Cholesterol Uptake

The N-terminal Domain of NPC1L1 Protein Binds Cholesterol and Plays Essential Roles in Cholesterol Uptake

The Enzymes of Neutral Lipid Synthesis

Modulation of Endosomal Cholesteryl Ester Metabolism by Membrane Cholesterol

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