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  • Lavallee et al reported that PREG could be esterified


    Lavallee et al. [30] reported that PREG could be esterified by lecithin:cholesterol acyltransferase (LCAT). LCAT uses cholesterol and phosphatidyl choline present in the newly formed high-density lipoprotein (HDL) as substrates and convert them into cholesteryl esters and lysophosphatidyl choline. The LCAT enzyme is found in the blood. At the cellular level, Damon and Chavis [31] demonstrated that by adding labeled PREG to embryonic rat fibroblast they could obtain lipoidal PREG esters. Pahuja and Hochberg [32] used an in vitro assay and demonstrated the conversion of radiolabeled dehydroepiandrosterone (DHEA; a metabolite of PREG), estrogen, or corticosterone to their fatty acyl esters, when rat hepatic microsomes were used as the source of enzyme(s). However, the addition of a specific ACAT inhibitor to this in vitro assay did not reduce esterification of the steroids tested, suggesting that enzyme(s) other than ACAT [33] carried out the steroid esterification. More recently, based on ACAT sterol substrate specificity analysis, Rogers et al. [34] speculated that PREG, which Oseltamivir possesses the same classical A, B, C, D steroid ring and 3β-hydroxyl moiety but with a much shorter side chain attached to ring D, might be a substrate for ACAT. They tested this possibility by using recombinant ACAT1 or recombinant ACAT2 as the enzyme source, and showed that both Oseltamivir could catalyze the conversion of PREG to PREG esters. In addition, they added radio-labeled PREG to several mouse and human cell types and showed that it was efficiently converted to PREG esters; this esterification process could be inhibited by 80–90% when specific ACAT inhibitors were added at appropriate concentration. These results demonstrate that both ACAT1 and ACAT2 can convert PREG to PREG esters in vitro and in intact cells. DHEA was found to be a much inferior ACAT substrate to PREG. Rogers et al. next employed the Acat1 KO mouse, the Acat2 KO mouse, and the Lcat KO mouse to evaluate the roles of ACAT1, ACAT2, and LCAT in contributing to the total adrenal PREG ester levels in vivo. The result showed that neither Acat1 knockout nor Acat2 knockout caused significant reduction in levels of PREG fatty acyl ester, while the loss of Lcat resulted in a partial reduction of PREG fatty acyl ester in the adrenal. These results suggest that in the adrenal glands, multiple enzymes may be involved in the generation of PREG fatty acyl esters, with LCAT playing a larger role and with ACAT1/ACAT2 playing auxiliary roles. Interestingly, in the human adrenal H295R cell line, Ferraz-de-Souza et al. [35] reported a decreased ACAT1 mRNA expression when steroidogenic factor-1 is overexpressed, and increased ACAT1 mRNA expression when steroidogenic factor-1 is knocked down. Steroidogenic factor-1 is a master regulator of adrenal steroidogenesis and controls the expression of steroid acute regulatory protein (STAR) and CYP11a1 enzyme at the transcriptional level. This result suggests that ACAT1 may be involved in negatively regulating steroidogenesis in human adrenal cells.
    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 source. The purified ACAT1 is an intrinsically fluorescent protein; using this property, Chang et al. [9] devised a direct binding assay between purified ACAT1 and various ligands. Using this assay, Rogers et al. showed that ACAT1 directly binds to PREG with a Kd=0.6μM, which is 58-fold lower than the concentration for half maximal CHOL binding (35μM). This result showed that PREG binds to ACAT1 with strong affinity. Rogers et al. next used PREG as the substrate to probe the structural specificity of sterols that could serve as the activator. The results showed that essentially all sterols tested, including the unnatural cholesterol analogs epicholesterol, ent-cholesterol etc., could activate PREG esterification to a certain degree, with cholesterol providing the maximal activation. Based on these data, Rogers et al. proposes that ACAT1 contains two types of binding sites: a substrate site and an activator site (Fig. 1). ACAT1 has 9 transmembrane domains [36] and resides at the ER. The ACAT1 enzyme exists as a homotetramer [37], but it may act as a dimer of dimer [38]. Within each dimer, it may contain two identical sterol substrate sites (designated as site S), and one or two sterol activator site(s) (designated as site A). Site S preferentially binds PREG; it can also bind a variety of sterols including CHOL, plant sterols, and oxysterols. The binding between site S and the steroid is mainly stereospecific and does not involve extensive interaction with phospholipid in membrane. Site S does not bind ent-CHOL, or epi-CHOL. Site A prefers to bind CHOL but cannot bind PREG; it can also bind a variety of other sterols such as plant sterols, oxysterols, yeast sterol, epi-cholesterol, and ent-CHOL etc. The binding between site A and the steroid involves the ability of the sterol molecule to interact biophysically with phospholipid in membrane, in addition to the stereospecific structure of the sterol. When only PREG is present, PREG binds site S, but it fails to trigger appropriate conformational changes, and the enzyme can only catalyze PREG esterification at a very low rate. When PREG and CHOL (or other sterol analogs) are both present, binding of CHOL at site A causes conformational changes, enabling the enzyme to increase the rate of esterification reaction much more efficiently. It is unlikely that ACATs would be able to recognize steroids with a hydroxyl-moiety located at C-7, or at C11, or at C17 beta as substrates; however, these possibilities have not been formally tested.