Cytochrome P450 metabolism of the post-lanosterol intermediates explains enigmas of cholesterol synthesis

Abstract

Cholesterol synthesis is among the oldest metabolic pathways, consisting of the Bloch and Kandutch-Russell branches. Following lanosterol, sterols of both branches are proposed to be dedicated to cholesterol. We challenge this dogma by mathematical modeling and with experimental evidence. It was not possible to explain the sterol profile of testis in cAMP responsive element modulator tau (Crem τ) knockout mice with mathematical models based on textbook pathways of cholesterol synthesis. Our model differs in the inclusion of virtual sterol metabolizing enzymes branching from the pathway. We tested the hypothesis that enzymes from the cytochrome P450 (CYP) superfamily can participate in the catalysis of non-classical reactions. We show that CYP enzymes can metabolize multiple sterols in vitro, establishing novel branching points of cholesterol synthesis. In conclusion, sterols of cholesterol synthesis can be oxidized further to metabolites not dedicated to production of cholesterol. Additionally, CYP7A1, CYP11A1, CYP27A1, and CYP46A1 are parts of a broader cholesterol synthesis network.

Figure 1. Expression levels of cholesterogenic genes.

Expression is represented as fold change of each individual mouse compared to the wild-type (WT) average for each gene separately. Triangles (blue) and circles (red) present WT and Crem^−/− animals, respectively. Lines represent average expression for each gene according to the genotype. Asterisks present p-values: *** for p < 0.001, ** for p < 0.01, * for p < 0.05. Gene abbreviations are according to Unigene (see Abbreviations in Supplementary information).

Figure 2. Cholesterol synthesis sterol concentrations.

Boxplots represent log₁₀(μg/g testes wet weight) measurements of the sterols lanosterol, T-MAS (testis meiosis-activating sterol), lathosterol, 7-DHC (7-dehydrocholesterol), desmosterol, and cholesterol for each genotype (WT, wild-type; KO, Crem^−/−) with associated p-values.

Figure 3. The cholesterol synthesis pathway, combined with a mathematical model.

The underlined genes (gray) and sterol intermediates (black) were measured in the present study. Enzyme (gene) abbreviations are according to Unigene (see Abbreviations in Supplementary information). E1 and E2 present multiple enzymes combined, while E3, E4, E5 and E6 present minimal requirements for mathematical model illustration of the sterol intermediate levels measured. Numbers (%) show initial division of metabolic flux at branching points, while numbers in squared brackets show final model division values. DHL, 24,25-dihydrolanosterol; FF-MAS, follicular-fluid meiosis-activating sterol; T-MAS, testis meiosis-activating sterol; 7-DHC, 7-dehydrocholesterol.

Figure 4. Optimization of enzyme levels (Model: Experiment seven).

Optimization results for enzyme levels (black) compared to experimental mRNA levels (gray) for experiment seven of the simulation. E3–E6 represent the virtual enzymes. mRNA levels are set to value 1.00.

Figure 5. LC-MS profiles of desmosterol incubation with different CYP enzymes.

(1) CYP7A1, (2) CYP11A1, (3) CYP27A1, and (4) CYP46A1. An APCI⁺ ionization mode was used, and m/z 383 (for hydroxy and epoxy products, with loss of H₂O to give m/z 383 [MH-18]⁺) was monitored in each case except CYP11A1 where it was monitored at m/z 299 (for pregnenolone, with loss of H₂O to give m/z 299 [MH-18]⁺). A, assay without enzyme. B, assay with enzyme.

Figure 6. Identification of sterol products.

(1) ¹H NMR spectra of (A) desmosterol and (B) purified product obtained from reaction of CYP7A1 and desmosterol. (2) GC-MS fragmentation of the product (TMS ether derivative) obtained from reaction of lathosterol with CYP27A1. (3) ¹H NMR spectra of (A) lathosterol and (B) purified product obtained from reaction of CYP27A1 with lathosterol. (4). GC-MS fragmentation of the product (TMS ether derivative) obtained from reaction of lathosterol with CYP46A1. The fragment m/z 131 is indicative of loss of the elements of C(CH₃)₂OSi(CH₃)₃, consistent with hydroxylation at C25 (parts 2,4A). The fragment m/z 145 (addition of one methylene) is indicative of loss of the elements of (CH₃)₂CH₂COSi(CH₃)₃, consistent with hydroxylation at C24 (part 4B). These fragmentation patterns (base peaks) were matched with standard 24- and 25-hydroxycholesterol (data not shown) with the assumption that a change in the position of double bond in “B” ring of sterols would not have any effect on the α-cleavage of an TMS isopropyl ether of the side chain, which is far from the “B” ring of the sterol.

Figure 7. Interplay between cholesterol biosynthesis and RORγ pathway.

Cholesterol biosynthesis intermediates downstream of lanosterol have recently been proposed to act as endogenous RORγ ligands. Analysis of the transformation of several cholesterol intermediates with four CYP enzymes revealed potential novel metabolites. These compounds have increased polarity due to the addition of a keto or a hydroxyl group to either the main sterol ring or to the side chain. Although they have yet to be characterized with regards to their RORγ activating potential, the results of Santori et al. indicate that the addition of hydroxyl groups to positions 7, 24, 25, and 27 do not abolish RORγ specific activity.