ABBS 2005,38(03): Human Acyl-CoA:cholesterol Acyltransferase (ACAT) and its Potential as a Target for Pharmaceutical Intervention against Atherosclerosis

Received:
January 9, 2006

Accepted:
February 7, 2006

*Corresponding
author: E-mail, [email protected]

Abstract        Acyl-CoA:cholesterol acyltransferase (ACAT)
catalyzes the formation of cholesteryl esters from cholesterol and long-chain
fatty-acyl-coenzyme A. At the single-cell level, ACAT serves as a regulator of
intracellular cholesterol homeostasis. In addition, ACAT supplies cholesteryl
esters for lipoprotein assembly in the liver and small intestine. Under
pathological conditions, the accumulation of cholesteryl esters produced by
ACAT in macrophages contributes to foam cell formation, a hallmark of the early
stage of atherosclerosis. Several reviews addressing various aspects of ACAT
and ACAT inhibitors are available [1–8]. This
review briefly outlines the current knowledge on the biochemical properties of
human ACATs, and then focuses on discussing the merit of ACAT as a drug target
for pharmaceutical interventions against atherosclerosis.

Key words        acyl-CoA:cholesterol acyltransferase;
cholesterol; cholesteryl ester; atherosclerosis

Acyl-CoA:cholesterol
acyltransferase 1

Two ACAT genes have been
identified in mammals: ACAT1 and ACAT2. The ACAT1 gene was first identified in
1993 by virtue of its ability to functionally complement the ACAT deficiency of
a Chinese hamster ovary (CHO) cell mutant [9]. Using ACAT1-specific antibodies,
Miyazaki et al. [10] examined human atherosclerotic lesions using
immunohistochemical staining. These results showed that in early lesions of the
human aorta, mononuclear cells expressed only limited ACAT1. In contrast, the
same ACAT1 antibodies stained much more intensely in fatty streak lesions,
particularly in areas that contained macrophages with foamy transformation (Fig.
1). This demonstrates that ACAT1 is a marker for the early development of
human atherosclerosis. To determine the cell types in this region, in the same
study Miyazaki et al. used Oil-Red-O to identify the lipid-rich region,
followed by double immunostaining (Fig. 2). The results demonstrated
that monocytes/macrophages were the major cellular component of the
ACAT1-expressing cells in atherosclerotic lesions [10]. These findings have
potential clinical significance because it is known that plaque ruptures
associated with acute myocardial infarction generally occur in the plaque’s
shoulder regions that are filled with lipid-rich, foamy macrophages [11,12].

Human ACAT1 (hACAT1)
expressed in CHO cells or in insect H5 cells has been purified to homogeneity
with retention of catalytic activity [13]. hACAT1 is a homotetrameric integral
membrane protein [14] and mainly resides in the endoplasmic reticulum [15,16].
Unlike many enzymes involved in cholesterol metabolism, ACAT1 is not regulated
by cholesterol at the transcriptional level. The main mode of sterol-specific
regulation of ACAT1 is at the post-translational level, involving allosteric
activation by its own substrate, cholesterol [13]. Kinetic analysis comparing
various oxysterols to cholesterol as a substrate and as an activator revealed
that ACAT1 might contain two types of sterol binding sites. The first site is
the activator site, which strongly prefers cholesterol to oxysterols or any
other sterol analogs examined. Activation of ACAT1 by cholesterol causes the
second site, the substrate-binding site, to accommodate a variety of sterols,
including cholesterol, oxysterols, plant sterols, and various other sterols as
the enzymatic substrate and facilitates enzyme catalysis [17]. The mode of
activation was further analyzed by using a specific cholesterol analog called
enantiomeric cholesterol. Enantiomeric cholesterol is the mirror image of
cholesterol. In membranes, it possesses identical biophysical properties as
cholesterol [18]. The results showed that activation of ACAT1 occurs with
cholesterol, but not with enantiomeric cholesterol [19]. These results indicate
that the activation mechanism mainly involves stereo-specific interaction,
rather than biophysical effects of cholesterol on phospholipid membranes.

In hepatocytes, ACAT1
can provide cholesteryl esters (CEs) for very low-density lipoprotein (VLDL)
assembly [20,21]. In macrophages and other cell types, ACAT1 is involved in
forming CEs as lipid droplets [10,16]. The VLDL assembly process occurs within
the lumens of the intracellular membrane compartment(s), while the lipid
droplets are formed in the cell cytoplasm. For ACAT1 to fulfill its dual roles,
it has been hypothesized that the active site of ACAT1 may be located within
the plane(s) of the ER membrane, such that the enzyme biosynthesizes CEs within
the ER membrane [22]. This arrangement would enable the CE formed to leave the
cytoplasmic leaflet of the membrane to form lipid droplets or to be recruited
to the lumen of the membrane by the protein microsomal lipid transfer protein
(MTP) for the VLDL assembly process. The recent evidence reported by Guo et
al. [23] demonstrating that ACAT1 contains 9 transmembrane domains, with
the active site H460 located within transmembrane domain #7, provided the
experimental support for this hypothesis.

ACAT2

In 1998 the second ACAT
gene, designated as ACAT2, was simultaneously identified in monkeys, mice and
humans [24–26]. ACAT2
shares high homology with ACAT1 near the C-terminus, but not near the N-terminus.
The kinetic properties of ACAT2 are quite similar to those of ACAT1 [19,21].
However, membrane topology study has shown that human ACAT2 contains only two
detectable transmembrane domains [27]. Its active site, H432 (the equivalent of
HMore recently, using
Western blot, histochemical staining, and RT-PCR analyses, Sakashita et al. demonstrated
that immature macrophages expressed only ACAT1, but fully differentiated
macrophages expressed both ACAT1 and ACAT2. In addition, ACAT2 is also present
in macrophages of human atherosclerotic lesions [30]. Thus, the tissue
distribution of human ACAT2 is not as restricted as previously believed. The
limited data available suggest that within the same cell, ACAT1 expression may
be constitutive, while that of ACAT2 may be inducible.

ACAT inhibitors

Starting in the One early concern in
developing ACAT inhibitors was adrenal toxicity. The cause for adrenal toxicity
is probably caused by a certain chemical structure, which is unrelated to the
ability of the compound to inhibit ACAT. Recent advances in pharmaceutical
research, this concern seems to have been eliminated [41]. Another major
concern for using the ACAT inhibitor is cellular toxicity induced by free
cholesterol loading. By adding ACAT inhibitors to lipid-laden mouse peritoneal
macrophages maintained in cell culture with no cholesterol acceptor present,
investigators observed the crystal formation of free cholesterol inside the
cells, which in turn induced cellular toxicity and leads to cell death [42,43].
The in vivo significance of these findings is unclear at present.
However, this finding does raise concern for using ACAT inhibitors for treating
atherosclerotic lesions under conditions where cellular sterol efflux becomes
severely hampered.

Coronary heart disease
(CHD) is one of the leading causes of morbidity and mortality in industrialized
countries. Hypercholesterolemia is one of the major risk factors for
progression of atherosclerosis. The development of HMG-CoA reductase
inhibitors, i.e., statins, has significantly reduced the mortality of CHD
patients by 20%–35%, saving
the lives of millions of people each year [44]. On the other hand, even with
the aid of statins, CHD-related mortality remains high. There is still a need
to identify other compounds that would complement the action of statins and
might further reduce CHD incidence and mortality. One of these potential novel
agents is the ACAT inhibitor. Different from the effect of statins, which work
mainly by reducing plasma cholesterol, the ACAT inhibitors may work by directly
reducing the size of the lipid-rich core in the atherosclerotic plaques, thus
stabilizing the lesion against plaque rupture.

Under normal physiological
conditions, ACAT1 is expressed more ubiquitously than the more restricted ACAT2
expression. Recent data raise the possibility that ACAT2 may be inducible in
different tissues under various pathological conditions [29,30]. Therefore,
before the roles of ACAT1 and ACAT 

Acknowledgements

We thank Ellen CHANG and
Kathy SAVAGE for critical reading of this article.

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