D. Cheng et al., ACTIVATION OF ACYL-COENZYME A-CHOLESTEROL ACYLTRANSFERASE BY CHOLESTEROL OR BY OXYSTEROL IN A CELL-FREE SYSTEM, The Journal of biological chemistry, 270(2), 1995, pp. 685-695
Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an intracellular
enzyme that catalyzes the conjugation of long chain fatty acid and ch
olesterol to form cholesteryl esters. It is an integral membrane prote
in located in the endoplasmic reticulum. Experiments performed in inta
ct mammalian cells have shown that the rate of cholesteryl ester synth
esis in intact cells, as well as the ACAT activity from cell extracts,
are greatly activated by the addition of low density lipoprotein (LDL
) or oxygenated sterols such as 25-hydroxycholesterol to the growth me
dium, However, the molecular mechanism(s) by which sterol(s) stimulate
the ACAT activity remains to be elucidated. Recently, our laboratory
reported the expression cloning of human ACAT cDNA (Chang, C. C. Y., H
uh, H. Y., Cadigan, K. M., and Chang, T. Y. 1993) J. Biol. Chem. 268,
20747-20755). In the current study, we report the expression of human
ACAT cDNA in insect Sf9 cells, Uninfected Sf9 cells do not express det
ectable ACAT-like activity. Infecting these cells with recombinant vir
us containing ACAT cDNA caused these cells to express high levels of A
CAT protein and high levels of ACAT activity when assayed in vitro. Th
e catalytic properties of ACAT expressed in these cells were found to
be similar to those found in human tissue culture cells, The combinati
on of high level of ACAT protein expression and the low level of cellu
lar cholesterol content in the infected cells have provided us a novel
opportunity to establish a simple cell-free system, whereby stimulati
on of ACAT by sterols can be readily demonstrated, Using this system,
we have shown that cholesterol itself can serve as an ACAT activator i
n vitro, in addition to its role as an ACAT substrate. The current wor
k provides the experimental basis to hypothesize that, inside mammalia
n cells, cholesterol itself may serve as a physiological regulator of
ACAT.