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  • PRT 4165 structure The Acat gene was identified by functiona

    2019-07-11

    The Acat1 gene was identified by functional complementation of a Chinese hamster ovary cell mutant lacking ACAT activity [12]. Unlike most other genes, human Acat1 is located in two different chromosomes, PRT 4165 structure 1 and 7, with each site containing a distinct promoter: chromosome 1 contains exons 1–16, and chromosome 7 contains the optional long exon Xa [13]. The majority of ACAT1 mRNAs is transcribed from exons 1–16; this mRNA translates into a 50-kDa size protein on SDS-PAGE [14]. In addition, the pre-mRNAs produced from exons 1–16 and the pre-mRNAs produced from the optional exon Xa participate in a trans-splicing event to produce an endogenous, chimeric 4.3kb ACAT1 mRNA. Remarkably, the endogenous 4.3kb mRNA then undergoes a second trans-splicing event with an exogenous transcript encoded by the antisense strand of Amp(r) (asAmp), which is present in common Amp(r)-plasmids, to produce a novel mRNA species. This novel mRNA species translates into a 56-kDa size protein [15]. The 56-kDa protein has less ACAT enzyme activity than that of the 50-kDa protein [16]. The chimeric 4.3kb ACAT1 mRNA found in human cannot be found in mouse. In mouse the Acat1 gene is located solely on chromosome 1, contains 17 exons [17], and produces a single protein of 48-kDa size protein on SDS-PAGE [18]. Human Acat2 is located in chromosome 12 and produces a single 46-kDa size protein on SDS-PAGE [19]. Meiner et al. [18,20] generated and characterized the Acat1 and Acat2 knockout mice. These mice have served as valuable tools in lipoprotein metabolism, atherosclerosis and neurodegenerative disease research. The recombinant 50-kDa human ACAT1 has been purified to homogeneity with full retention in enzymatic activity [21]. When assayed in reconstituted liposomes or in mixed micelles, the enzyme responds to cholesterol as its substrate in a sigmoidal manner. ACAT2 activity also responds to cholesterol in a sigmoidal manner. Additional kinetic analyses show that both ACAT1 and ACAT2 can use a variety of sterols as substrates, and their activities are significantly activated by a variety of sterols. Among the sterols tested (including oxysterols, plant sterols, yeast sterols, and several synthetic sterol analogs), cholesterol is the best substrate and the best activator [22]. Those sterols that act both as ACAT activators and as substrates all contain a 3β hydroxyl group at steroid ring A. The sterols that contain alterations at the iso-octyl side chain, such as the plant sterol sitosterol, are both poor substrates and poor activators of ACAT. In addition, epi-cholesterol, which contains the 3α hydroxyl, is not a substrate and is a very poor activator. Likewise, enantiomeric cholesterol (ent-cholesterol), which is the mirror image of cholesterol and possesses essentially the same biophysical properties of cholesterol [23], is also not a substrate and a very poor activator [24]. These studies implicate that both ACAT1 and ACAT2 are allosteric enzymes, and the structural feature of a given sterol as an ACAT substrate deviates from that as an activator. A shortcoming of these studies was that a sterol-like molecule that is only a substrate but not an activator was not found. In the absence of such molecule, in order to test the structural features of various sterols as activators, sitosterol was chosen as a substrate [24]; however, sitosterol might act as an activator. Thus, by using sitosterol as the substrate in the ACAT activation assay, certain essential structural feature of a given sterol to act as an activator might have been masked.
    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]. Using bovine adrenal homogenates, Mellon and Hochberg [28] demonstrated that PREG could form lipoidal derivatives in vitro. In the adrenals of dog, rat and guinea-pig, PREG esters have been reported to be up to 40% of the total adrenal PREG content, while in human adrenals, PREG esters comprise more than 3 times the amount of free PREG [29].