Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids
ReviewOxysterol biosynthetic enzymes
Introduction
According to a recent exhaustive review [1], the biosynthesis of oxysterols was first documented in 1956 by Frederickson and Ono, who incubated radiolabeled cholesterol with a subcellular fraction enriched in mitochondria and showed that both 25-hydroxycholesterol and 27-hydroxycholesterol1 were formed. The enzymatic synthesis of a third oxysterol, 24-hydroxycholesterol2, was reported later [2]. Oxysterols remained a biochemical curiosity until it was shown in 1974 that they were potent inhibitors of sterol synthesis in cultured cells [3], [4]. This demonstration suggested a regulatory role for oxysterols in lipid metabolism and it has since served as a stimulus to explore their physiological actions.
Research during the ensuing 26 years revealed that oxysterols participate in several different aspects of lipid metabolism (Fig. 1). They are regulators of gene expression, substrates for bile acid synthesis, and mediators of sterol transport. As regulatory molecules, they inhibit the production of transcription factors required for the expression of genes in the cholesterol supply pathways [5], and they are ligands that activate members of the nuclear hormone receptor gene family [6], [7], [8]. Oxysterols are inactivated by conversion into bile acids, and in some instances the essential need for bile acids can be met solely by the metabolism of oxysterols [9]. They also may be substrates for steroid hormone synthesis [10]. Tissues such as the lung and brain secrete measurable amounts of oxysterols into the circulation, which are then transported to the liver and converted into bile acids [11], [12]. This secretion represents a form of reverse cholesterol transport [13], a mechanism that peripheral tissues use to return cholesterol to the liver and thus to maintain homeostasis.
Individual oxysterols engage in multiple roles in lipid metabolism (Fig. 2). For example, 24-hydroxycholesterol is a ligand for the nuclear hormone receptor, liver X receptor (LXR) [6], [8], is metabolized into bile acids by a dedicated pathway [14], and is a secreted product of cholesterol metabolism in the central nervous system [11]. 25-Hydroxycholesterol potently suppresses the production of mature sterol regulatory element binding proteins (SREBP, see below), which are transcription factors that activate genes in the cholesterol supply pathways [5], [15]. This oxysterol is also a substrate for bile acid synthesis [16]. 27-Hydroxycholesterol is the most abundant oxysterol in the tissues and plasma of the mouse [17] and is active as a regulatory molecule [3], [4] and bile acid substrate [18], and in reverse cholesterol transport [12] (Fig. 2).
Three different cholesterol hydroxylase enzymes synthesize these oxysterols (Fig. 3). Cholesterol 24-hydroxylase produces 24-hydroxycholesterol, and to a lesser extent 25-hydroxycholesterol [19]. Cholesterol 25-hydroxylase produces 25-hydroxycholesterol [15], whereas sterol 27-hydroxylase synthesizes 27-hydroxycholesterol and to a lesser extent 24- and 25-hydroxycholesterol [20]. Although each enzyme resides in the membrane compartment of the cell and utilizes NADPH as a cofactor to catalyze oxysterol biosynthesis, they differ substantially in sequence, tissue distribution and subcellular localization.
Section snippets
Sterol 27-hydroxylase
This enzyme was the first cholesterol hydroxylase to be isolated. Kjell Wikvall developed a purification scheme for sterol 27-hydroxylase that began with detergent-solubilized protein from rabbit liver mitochondria [21]. His colleagues established the sequences of several peptides produced from the purified enzyme and these sequences were used to design oligonucleotide probes that identified the encoding cDNA [22]. Analysis of the sequence of the cloned cDNA reveals sterol 27-hydroxylase to be
Isolation of cDNAs encoding cholesterol 24-hydroxylase and cholesterol 25-hydroxylase
The cholesterol 24-hydroxylase and 25-hydroxylase enzymes are present in trace amounts within mammalian tissues and are far less abundant than sterol 27-hydroxylase. Their isolation by classical protein purification methods was predicted to require large investments in starting material and cold room time. To overcome these challenges, an expression cloning strategy was developed to isolate their cDNAs (Fig. 4). The first step consisted of the construction of a cDNA library in an expression
Cholesterol 24-hydroxylase
Conceptual translation of cholesterol 24-hydroxylase cDNAs isolated from the mouse and the human revealed highly conserved proteins of 500 amino acids (Table 1). The encoded enzymes are microsomal members of the cytochrome P-450 family whose sequences are 93% identical [19]. The official name for cholesterol 24-hydroxylase is CYP46 [36].
Cholesterol 24-hydroxylase is predominantly expressed in the brain as judged by protein and RNA blotting studies [19]. This tissue distribution is unique among P
Cholesterol 25-hydroxylase
The 25-hydroxylase enzymes from mouse and human are small, hydrophobic proteins of approx. 300 amino acids [15] (Table 1). Unlike sterol 27-hydroxylase and cholesterol 24-hydroxylase, 25-hydroxylase is not a cytochrome P-450 but rather belongs to a smaller family of non-heme iron containing proteins [46]. The iron is present as Fe-O-Fe or Fe-OH-Fe and is coordinated with clusters of conserved histidine residues in these proteins. Members of this family include bacterial enzymes such as alkane
Structural relationships between cholesterol hydroxylases
Cholesterol is found throughout the plant and animal kingdoms [53], which raises the question of how widespread oxysterol biosynthetic enzymes are. Cloning studies to date show that each of the cholesterol hydroxylases discussed above is present in mice and humans, and biochemical data indicate that the 7α-hydroxylation of 25-hydroxycholesterol occurs in many different vertebrate species [16], including birds, and a homologue of the CYP39A1 oxysterol 7α-hydroxylase that acts on
Future directions
There are many questions that remain to be answered concerning the synthesis and biology of oxysterols. For example, what is the relative importance of oxysterols as substrates for bile acid synthesis versus regulators of transcription versus mediators of reverse cholesterol transport? Are there additional hydroxylases with tissue-specific expression patterns? Do defects in the synthesis of 24- or 25-hydroxycholesterol underlie human genetic disease? Are the cholesterol hydroxylases targets for
Acknowledgements
The experimental work discussed in this review was carried out by Stefan Andersson, James J. Cali, Daphne L. Davis, Jia Li-Hawkins, Erik G. Lund and Margrit Schwarz, and was supported by grant HL-20948 from the US National Institutes of Health. I thank David J. Mangelsdorf and Joyce J. Repa for critical reading of the manuscript.
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