Acyl-CoA:cholesterol acyltransferases (ACATs/SOATs): Enzymes with multiple sterols as substrates and as activators

doi:10.1016/j.jsbmb.2014.09.008

The Journal of Steroid Biochemistry and Molecular Biology

Volume 151, July 2015, Pages 102-107

https://doi.org/10.1016/j.jsbmb.2014.09.008 Get rights and content

Highlights

•
ACATs esterify both sterols and certain steroids including pregnenolonone.
•
ACATs contain two different steroidal binding sites.
•
ACATs are drug targets to treat several human diseases.
•
The novelty of ACAT enzymology can be exploited to develop allosteric ACAT inhibitors.

Abstract

Cholesterol is essential to the growth and viability of cells. The metabolites of cholesterol include: steroids, oxysterols, and bile acids, all of which play important physiological functions. Cholesterol and its metabolites have been implicated in the pathogenesis of multiple human diseases, including: atherosclerosis, cancer, neurodegenerative diseases, and diabetes. Thus, understanding how cells maintain the homeostasis of cholesterol and its metabolites is an important area of study. Acyl-coenzyme A:cholesterol acyltransferases (ACATs, also abbreviated as SOATs) converts cholesterol to cholesteryl esters and play key roles in the regulation of cellular cholesterol homeostasis. ACATs are most unusual enzymes because (i) they metabolize diverse substrates including both sterols and certain steroids; (ii) they contain two different binding sites for steroidal molecules. In mammals, there are two ACAT genes that encode two different enzymes, ACAT1 and ACAT2. Both are allosteric enzymes that can be activated by a variety of sterols. In addition to cholesterol, other sterols that possess the 3-beta OH at C-3, including PREG, oxysterols (such as 24(S)-hydroxycholesterol and 27-hydroxycholesterol, etc.), and various plant sterols, could all be ACAT substrates. All sterols that possess the iso-octyl side chain including cholesterol, oxysterols, various plant sterols could all be activators of ACAT. PREG can only be an ACAT substrate because it lacks the iso-octyl side chain required to be an ACAT activator. The unnatural cholesterol analogs epi-cholesterol (with 3-alpha OH in steroid ring B) and ent-cholesterol (the mirror image of cholesterol) contain the iso-octyl side chain but do not have the 3-beta OH at C-3. Thus, they can only serve as activators and cannot serve as substrates. Thus, within the ACAT holoenzyme, there are site(s) that bind sterol as substrate and site(s) that bind sterol as activator; these sites are distinct from each other. These features form the basis to further pursue ACAT structure–function analysis, and can be explored to develop novel allosteric ACAT inhibitors for therapeutic purposes.

This article is part of a Special Issue entitled ‘Steroid/Sterol signaling’.

Section snippets

ACAT enzymes

Acyl-CoA:cholesterol acyltransferases (ACATs), also known as sterol O-acyltransferases (SOATs), play important roles in cellular cholesterol homeostasis and are drug targets for therapeutic intervention of several diseases including atherosclerosis (reviewed in [1]), Alzheimer’s disease [2], [3], [4], [5] and cancer [6]. In mammals, two genes that encode two different proteins exist: Acat1 and Acat2 [7]. Along with acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), ACAT1 and ACAT2 are founding

PREG esterification and ACAT

PREG is the obligatory precursor for all steroid hormones. Biosynthesis of PREG occurs in mitochondria, using cholesterol (CHOL) as the precursor [25,26]. Once produced, PREG can be converted by enzymes in the mitochondria and in the ER to various steroid hormones. In addition, PREG can be stored as fatty acyl esters. Steroid fatty acyl esters can provide a means to quickly provide a substrate pool in times of need. Lipoidal conjugates of PREG were first identified in the bovine adrenal [27].

Analysis of ACAT activators when PREG is used as the substrate

Rogers et al. [34] showed that, in the absence of cholesterol, PREG is a very poor substrate. However, the addition of cholesterol in the assay mixture increases the rate of PREG esterification by 100-fold. The result of the converse experiment showed that when CHOL was used as the substrate, PREG had minimal effect on CHOL esterification. These results show that PREG is a substrate but it cannot serve as an activator for ACAT1. Similar results were obtained when ACAT2 was used as the enzyme

ACAT and neurosteroid esterification

Neurosteroids including the steroids PREG, DHEA, progesterone and allopregnanolone etc., are synthesized de novo in the brain by neurons and astrocytes, or are produced in peripheral tissues but accumulate in the central nervous system [39,40]. Various neurosteroids possess many interesting biological activities. For example, neurosteroids can allosterically regulate gamma-aminobutyric acid receptors [41]. PREG sulfate, which contains a sulfate ester at the 3β-hydroxyl moiety, can potentiate

ACAT and oxysterol esterification

Cholesterol is the precursor for various oxidized sterols, such as: 7α-hydroxycholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxcholesterol, 25-hydroxycholesterol and 27-hydroxycholesterol [48]. 7α-hydroxycholesterol is the obligatory precursor for bile acid biosynthesis. 22(R)-hydroxycholesterol is the obligatory sterol intermediate in order to form PREG. 24(S)-hydroxycholesterol is the major oxysterol present in the brain, and plays important role in mediating the excretion of cholesterol

Future perspectives

ACATs are drug targets. Previously, most of the effort has been focused on targeting ACAT to treat atherosclerosis. Recent work demonstrated substantial benefits of inhibiting ACAT1 in mouse models of Alzheimer’s disease [2], [3], [4], [5]. An additional area of interest is the role ACAT in cancer: ACAT inhibition has been used to block carcinogenesis in vitro in a variety of cancer types including breast cancer [59], glioblastoma [60], and lymphocytic leukemia [61]. In addition, ACAT1 has been

Acknowledgements

We thank Joseph Granger and Bryan Neumann for careful reading of this manuscript. This work is supported by NIH grants AG37609 and HL 60306 to TYC and CCYC.

References (63)

B. Hutter-Paier et al.
The ACAT inhibitor CP-113,818 markedly reduces amyloid pathology in a mouse model of Alzheimer’s disease
Neuron
(2004)
S.R. Murphy et al.
Acat1 knockdown gene therapy decreases amyloid-beta in a mouse model of Alzheimer’s disease
Mol. Ther.
(2013)
S. Yue et al.
Cholesteryl ester accumulation induced by PTEN loss and PI3K/AKT activation underlies human prostate cancer aggressiveness
Cell Metab.
(2014)
K.F. Buhman et al.
Mammalian acyl-CoA:cholesterol acyltransferases
Biochim. Biophys. Acta
(2000)
C. Yu et al.
Molecular cloning and characterization of two isoforms of saccharomyces cerevisiae acyl-coA:sterol acyltransferase
J. Biol. Chem.
(1996)
C.C. Chang et al.
Molecular cloning and functional expression of human acyl-coenzyme A:cholesterol acyltransferase cDNA in mutant Chinese hamster ovary cells
J. Biol. Chem.
(1993)
B.L. Li et al.
Human acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) gene organization and evidence that the 4.3-kb ACAT-1 mRNA is produced from two different chromosomes
J. Biol. Chem.
(1999)
L. Yang et al.
Human acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) sequences located in two different chromosomes (7 and 1) are required to produce a novel ACAT1 isoenzyme with additional sequence at the N terminus
J. Biol. Chem.
(2004)
P.J. Uelmen et al.
Tissue-specific expression and cholesterol regulation of acylcoenzyme A:cholesterol acyltransferase (ACAT) in mice. Molecular cloning of mouse ACAT cDNA, chromosomal localization, and regulation of ACAT in vivo and in vitro
J. Biol. Chem.
(1995)
C.C. Chang et al.
Immunological quantitation and localization of ACAT-1 and ACAT-2 in human liver and small intestine
J. Biol. Chem.
(2000)

C.C. Chang et al.

Recombinant acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) purified to essential homogeneity utilizes cholesterol in mixed micelles or in vesicles in a highly cooperative manner

J. Biol. Chem.

(1998)

Y. Zhang et al.

Cholesterol is superior to 7-ketocholesterol or 7 alpha-hydroxycholesterol as an allosteric activator for acyl-coenzyme A:cholesterol acyltransferase 1

J. Biol. Chem.

(2003)

D.A. Mannock et al.

Effects of natural and enantiomeric cholesterol on the thermotropic phase behavior and structure of egg sphingomyelin bilayer membranes

Biophys. J.

(2003)

S. Mellon-Nussbaum et al.

Biosynthesis of lipoidal derivatives of pregnenolone and dehydroisoandrosterone by the adrenal

J. Biol. Chem.

(1980)

B. Lavallee et al.

Formation of pregnenolone- and dehydroepiandrosterone-fatty acid esters by lecithin–cholesterol acyltransferase in human plasma high density lipoproteins

Biochim. Biophys. Acta

(1996)

M. Damon et al.

GLC/MS identification of new pregnenolone metabolites in confluent embryonic rat fibroblast cultures

J. Steroid Biochem.

(1982)

M.A. Rogers et al.

Cellular pregnenolone esterification by acyl-CoA:cholesterol acyltransferase

J. Biol. Chem.

(2012)

Z.Y. Guo et al.

The active site His-460 of human acyl-coenzyme A:cholesterol acyltransferase 1 resides in a hitherto undisclosed transmembrane domain

J. Biol. Chem.

(2005)

C. Yu et al.

Human acyl-CoA:cholesterol acyltransferase-1 is a homotetrameric enzyme in intact cells and in vitro

J. Biol. Chem.

(1999)

E.E. Baulieu et al.

Neurosteroids: a new brain function?

J. Steroid Biochem. Mol. Biol.

(1990)

S.H. Mellon et al.

Neurosteroids: biochemistry and clinical significance

Trends Endocrinol. Metab.

(2002)

M. Takebayashi et al.

Differential regulation by pregnenolone sulfate of intracellular Ca²⁺ increase by amino acids in primary cultured rat cortical neurons

Neurochem. Int.

(1998)

N. Tagawa et al.

Strain differences of neurosteroid levels in mouse brain

Steroids

(2006)

D.W. Russell

Oxysterol biosynthetic enzymes

Biochim. Biophys. Acta

(2000)

L.L. Smith et al.

Biological activities of oxysterols

Free Radic. Biol. Med.

(1989)

S.E. Szedlacsek et al.

Esterification of oxysterols by human plasma lecithin–cholesterol acyltransferase

J. Biol. Chem.

(1995)

N.E. Freeman et al.

Acyl-coenzyme A:cholesterol acyltransferase promotes oxidized LDL/oxysterol-induced apoptosis in macrophages

J. Lipid Res.

(2005)

A.E. Rusinol et al.

A unique mitochondria-associated membrane fraction from rat liver has a high capacity for lipid synthesis and contains pre-Golgi secretory proteins including nascent lipoproteins

J. Biol. Chem.

(1994)

T.Y. Chang et al.

Acyl-coenzyme A:cholesterol acyltransferases

Am. J. Phys.

(2009)

E.Y. Bryleva et al.

ACAT1 gene ablation increases 24(S)-hydroxycholesterol content in the brain and ameliorates amyloid pathology in mice with AD

Proc. Natl. Acad. Sci. U. S. A.

(2010)

R. Bhattacharyya et al.

Palmitoylation of amyloid precursor protein regulates amyloidogenic processing in lipid rafts

J. Neurosci.

(2013)

Cited by (112)

27-Hydroxylation of oncosterone by CYP27A1 switches its activity from pro-tumor to anti-tumor
2023, Journal of Lipid Research
Oncosterone (6-oxo-cholestane-3β,5α-diol; OCDO) is an oncometabolite and a tumor promoter on estrogen receptor alpha-positive breast cancer (ER(+) BC) and triple-negative breast cancers (TN BC). OCDO is an oxysterol formed in three steps from cholesterol: 1) oxygen addition at the double bond to give α- or β- isomers of 5,6-epoxycholestanols (5,6-EC), 2) hydrolyses of the epoxide ring of 5,6-ECs to give cholestane-3β,5α,6β-triol (CT), and 3) oxidation of the C6 hydroxyl of CT to give OCDO. On the other hand, cholesterol can be hydroxylated by CYP27A1 at the ultimate methyl carbon of its side chain to give 27-hydroxycholesterol ((25R)-Cholest-5-ene-3beta,26-diol, 27HC), which is a tumor promoter for ER(+) BC. It is currently unknown whether OCDO and its precursors can be hydroxylated at position C27 by CYP27A1, as is the impact of such modification on the proliferation of ER(+) and TN BC cells. We investigated, herein, whether 27H-5,6-ECs ((25R)-5,6-epoxycholestan-3β,26-diol), 27H-CT ((25R)-cholestane-3β,5α,6β,26-tetrol) and 27H-OCDO ((25R)-cholestane-6-oxo-3β,5α,26-triol) exist as metabolites and can be produced by cells expressing CYP27A1. We report, for the first time, that these compounds exist as metabolites in humans. We give pharmacological and genetic evidence that CYP27A1 is responsible for their production. Importantly, we found that 27-hydroxy-OCDO (27H-OCDO) inhibits BC cell proliferation and blocks OCDO and 27-HC-induced proliferation in BC cells, showing that this metabolic conversion commutes the proliferative properties of OCDO into antiproliferative ones. These data suggest an unprecedented role of CYP27A1 in the control of breast carcinogenesis by inhibiting the tumor promoter activities of oncosterone and 27-HC.
Chemical synthesis and biochemical properties of cholestane-5α,6β-diol-3-sulfonate: A non-hydrolysable analogue of cholestane-5α,6β-diol-3β-sulfate
2023, Journal of Steroid Biochemistry and Molecular Biology
Cholestane-3β,5α,6β-triol (CT) is a primary metabolite of 5,6-epoxycholesterols (5,6-EC) that is catalyzed by the cholesterol-5,6-epoxide hydrolase (ChEH). CT is a well-known biomarker for Niemann-Pick disease type C (NP-C), a progressive inherited neurodegenerative disease. On the other hand, CT is known to be metabolized by the 11β-hydroxysteroid-dehydrogenase of type 2 (11β-HSD2) into a tumor promoter named oncosterone that stimulates the growth of breast cancer tumors. Sulfation is a major metabolic transformation leading to the production of sulfated oxysterols. The production of cholestane-5α,6β-diol-3β-O-sulfate (CDS) has been reported in breast cancer cells. However, no data related to CDS biological properties have been reported so far. These studies have been hampered because sulfate esters of sterols and steroids are rapidly hydrolyzed by steroid sulfatase to give free steroids and sterols. In order to get insight into the biological properties of CDS, we report herein the synthesis and the characterization of cholestane-5α,6β-diol-3β-sulfonate (CDSN), a non-hydrolysable analogue of CDS. We show that CDSN is a potent inhibitor of 11β-HSD2 that blocks oncosterone production on cell lysate. The inhibition of oncosterone biosynthesis of a whole cell assay was observed but results from the blockage by CDSN of the uptake of CT in MCF-7 cells. While CDSN inhibits MCF-7 cell proliferation, we found that it potentiates the cytotoxic activity of post-lanosterol cholesterol biosynthesis inhibitors such as tamoxifen and PBPE. This effect was associated with an increase of free sterols accumulation and the appearance of giant multilamellar bodies, a structural feature reminiscent of Type C Niemann-Pick disease cells and consistent with a possible inhibition by CDSN of NPC1. Altogether, our data showed that CDSN is biologically active and that it is a valuable tool to study the biological properties of CDS and more specifically its impact on immunity and viral infection.
Is reverse cholesterol transport regulated by active cholesterol?
2023, Journal of Lipid Research
This review considers the hypothesis that a small portion of plasma membrane cholesterol regulates reverse cholesterol transport in coordination with overall cellular homeostasis. It appears that almost all of the plasma membrane cholesterol is held in stoichiometric complexes with bilayer phospholipids. The minor fraction of cholesterol that exceeds the complexation capacity of the phospholipids is called active cholesterol. It has an elevated chemical activity and circulates among the organelles. It also moves down its chemical activity gradient to plasma HDL, facilitated by the activity of ABCA1, ABCG1, and SR-BI. ABCA1 initiates this process by perturbing the organization of the plasma membrane bilayer, thereby priming its phospholipids for translocation to apoA-I to form nascent HDL. The active excess sterol and that activated by ABCA1 itself follow the phospholipids to the nascent HDL. ABCG1 similarly rearranges the bilayer and sends additional active cholesterol to nascent HDL, while SR-BI simply facilitates the equilibration of the active sterol between plasma membranes and plasma proteins. Active cholesterol also flows downhill to cytoplasmic membranes where it serves both as a feedback signal to homeostatic ER proteins and as the substrate for the synthesis of mitochondrial 27-hydroxycholesterol (27HC). 27HC binds the LXR and promotes the expression of the aforementioned transport proteins. 27HC-LXR also activates ABCA1 by competitively displacing its inhibitor, unliganded LXR. § Considerable indirect evidence suggests that active cholesterol serves as both a substrate and a feedback signal for reverse cholesterol transport. Direct tests of this novel hypothesis are proposed.
A basic model for the association of ligands with membrane cholesterol: application to cytolysin binding
2023, Journal of Lipid Research
Almost all the cholesterol in cellular membranes is associated with phospholipids in simple stoichiometric complexes. This limits the binding of sterol ligands such as filipin and perfringolysin O (PFO) to a small fraction of the total. We offer a simple mathematical model that characterizes this complexity. It posits that the cholesterol accessible to ligands has two forms: active cholesterol, which is that not complexed with phospholipids; and extractable cholesterol, that which ligands can capture competitively from the phospholipid complexes. Simulations based on the model match published data for the association of PFO oligomers with liposomes, plasma membranes, and the isolated endoplasmic reticulum. The model shows how the binding of a probe greatly underestimates cholesterol abundance when its affinity for the sterol is so weak that it competes poorly with the membrane phospholipids. Two examples are the understaining of plasma membranes by filipin and the failure of domain D4 of PFO to label their cytoplasmic leaflets. Conversely, the exaggerated staining of endolysosomes suggests that their cholesterol, being uncomplexed, is readily available. The model is also applicable to the association of cholesterol with intrinsic membrane proteins. For example, it supports the hypothesis that the sharp threshold in the regulation of homeostatic endoplasmic reticulum proteins by cholesterol derives from the cooperativity of their binding to the sterol weakly held by the phospholipids. Thus, the model explicates the complexity inherent in the binding of ligands like PFO and filipin to the small accessible fraction of membrane cholesterol.
Identification of gene signatures and molecular mechanisms underlying the mutual exclusion between psoriasis and leprosy
2024, Scientific Reports
Cholesterol: A friend to viruses
2024, International Reviews of Immunology

View all citing articles on Scopus

View full text

ReviewAcyl-CoA:cholesterol acyltransferases (ACATs/SOATs): Enzymes with multiple sterols as substrates and as activators

Highlights

Abstract

Section snippets

ACAT enzymes

PREG esterification and ACAT

Analysis of ACAT activators when PREG is used as the substrate

ACAT and neurosteroid esterification

ACAT and oxysterol esterification

Future perspectives

Acknowledgements

Neuron

Mol. Ther.

Cell Metab.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biophys. J.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Steroid Biochem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Steroid Biochem. Mol. Biol.

Trends Endocrinol. Metab.

Neurochem. Int.

Steroids

Biochim. Biophys. Acta

Free Radic. Biol. Med.

J. Biol. Chem.

J. Lipid Res.

J. Biol. Chem.

Acyl-coenzyme A:cholesterol acyltransferases

Am. J. Phys.

ACAT1 gene ablation increases 24(S)-hydroxycholesterol content in the brain and ameliorates amyloid pathology in mice with AD

Proc. Natl. Acad. Sci. U. S. A.

Palmitoylation of amyloid precursor protein regulates amyloidogenic processing in lipid rafts

J. Neurosci.

Review
Acyl-CoA:cholesterol acyltransferases (ACATs/SOATs): Enzymes with multiple sterols as substrates and as activators