The Journal of Steroid Biochemistry and Molecular Biology
Short communication7-Dehydrocholesterol reductase activity is independent of cytochrome P450 reductase
Highlights
► 7-Dehydrocholesterol reductase does not require cytochrome P450 reductase for activity. ► Cytochrome P450 reductase does not stimulate nor augment 7-dehydrocholesterol reductase activity. ► 7-Dehydrocholesterol reductase expression is elevated in microsomes from livers that lack cytochrome P450 reductase.
Introduction
7-Dehydrocholesterol reductase (DHCR7) is a 54 kDa microsomal enzyme that catalyzes the final step in cholesterol synthesis, the reduction of the sterol C(7-8) double bond, using NADPH as an electron source. Mutations in the DHCR7 gene that impair or inactivate the enzyme result in Smith-Lemli-Opitz Syndrome (SLOS), a disease characterized by developmental and neurological deficits due to cholesterol deficiency [1]. Human and rat DHCR7 cDNAs were cloned in 1998 and 1999, respectively [2], [3], and the enzymes were expressed in yeast and shown to be active in microsomal preparations. A subsequent study with rat liver microsomes [4] presented several lines of evidence that DHCR7 required NADPH-cytochrome P450 reductase (POR) for activity; this included a marked inhibition of DHCR7 activity in microsomes by addition of antibody to POR, as well as a reconstitution of DHCR7 activity by addition of purified POR to protease-treated microsomes and to partially purified DHCR7 preparations. Although NADPH-cytochrome P450 reductase is more widely recognized for its role in the reduction of cytochrome P450 in the oxidation of xenobiotics and steroids, POR is required for cholesterol synthesis, where it is the requisite electron donor to lanosterol demethylase (CYP51A1) [5] and the principal, but not exclusive, electron donor to squalene monooxygenase [6]. POR is ubiquitously expressed in mammalian cells and also in yeast.
DHCR7 has not yet been purified to homogeneity or expressed in a host lacking POR (e.g., Escherichia coli), so the requirement for POR for activity has not been unequivocally established. Moreover, the absence of a recognizable NADPH binding domain in this protein, or the closely related reductase, Δ14-sterol reductase (TM7SF2) has further clouded the issue. The recent availability of hepatic POR-null mice [7] provided an opportunity to examine the requirement for this electron transfer protein in mammalian microsomes that lack this reductase. Our results argue strongly that POR is not required for DHCR7 activity.
Section snippets
Materials and methods
7-Dehydrocholesterol reductase activity was measured by following the conversion of ergosterol to brassicasterol as described [8] in liver microsomes prepared from wild-type and hepatic POR-null mice [7]. Incubations (500 μl) contained 100 μg of microsomal protein and 60 μM ergosterol solubilized in 5% methyl-β-cyclodextrin (Sigma–Aldrich, St. Louis, MO) and were carried out for 60 min at 37 °C. Lipids were saponified (1 M ethanolic NaOH) for 1 h at 90 °C, extracted twice with hexane, derivatized, and
Results
As shown in Fig. 1, 7-dehydrocholesterol reductase activity, measured as the conversion of ergosterol to brassicasterol [8], was prominent in microsomes prepared from the livers of mice in which cytochrome P450 reductase expression is extinguished during maturation [7]. This activity was consistently 2–3-fold greater than that found in microsomes from the corresponding control (wild-type) mice when evaluated on the basis of microsomal protein concentration, time-course, or substrate
Discussion
The present results argue strongly against a role for cytochrome P450 reductase in 7-dehydrocholesterol reductase activity. The 2-fold increase in DHCR7 activity in liver microsomes from hepatic POR-null mice matched the 2-fold increase in immunoreactive DHCR7 protein, even while POR protein levels were reduced below detectable levels. This increase in DHCR7 activity and protein is consistent with the 1.3- to 1.9-fold increase in DHCR7 gene expression that results from the loss of POR
Acknowledgments
We thank Drs. Xinxin Ding and Jun Gu for providing the liver tissue used in these studies; generation and maintenance of this mouse line was supported in part by NIH Grant ES-07462. Ms. Lauren Smith carried out the initial studies, and Dr. Jack Goodman provided advice and guidance on the mass spectrometric analysis.
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