Expression of human cytochrome P450 46A1 in Escherichia coli: effects of N- and C-terminal modifications

doi:10.1016/j.abb.2004.05.012

Archives of Biochemistry and Biophysics

Volume 428, Issue 1, 1 August 2004, Pages 99-108

https://doi.org/10.1016/j.abb.2004.05.012 Get rights and content

Abstract

Heterologous expression in Escherichia coli, subcellular distribution, solubility, and catalytic and substrate-binding properties of four truncated cytochromes P450 46A1 were investigated in the present study. All four lacked the N-terminal transmembrane region (residues 3–27), and, in addition, Δ46A1H had a 4× His-tag fused to the C-terminus; HΔ46A1 had the N-terminal 4× His-tag; HΔ46A1Δ had a 4× His-tag at the N-terminus and did not contain a proline-rich region at the C-terminus (residues 494–499); and Δ46A1Δ lacked the C-terminal proline-rich region. The truncated enzymes were expressed at 390–650 nmol/L culture levels, distributed at about a 1:1 ratio between the membrane fraction and the cytosol in low ionic strength buffer, and were predominantly monomers in detergent-free buffer. They had moderately decreased catalytic efficiencies for either cholesterol or 24S-hydroxycholesterol or both, whereas their substrate-binding constants were either unchanged or decreased 2-fold. The two forms, Δ46A1Δ and HΔ46A1Δ, both lacking the C-terminal proline-rich region seem to be good candidates for future crystallographic studies because they contain only 0.3–0.8% of high molecular weight aggregates and their catalytic efficiencies are decreased no more than 2.3-fold.

Section snippets

Construction and expression of truncated P450s

Four truncated forms were generated (Fig. 1) using templates and primers shown in Table 1. A cDNA for CYP46A1 was obtained from Dr. D. Russell, University of Texas Southwestern Medical Center, and used to prepare an expression construct for a full-length CYP46A1 in the pCW vector [10]. This construct served as a polymerase chain reaction (PCR) template to engineer Δ46A1H. The forward 5^′-primer was designed to introduce an NdeI restriction site and GCT as the second codon, and to delete codons

Expression of truncated P450s in E. coli

According to a hydropathy plot analysis (Fig. 1A) and transmembrane region prediction algorithm (http://www.ch.embnet.org/software/TMPRED_form.html), a stretch of hydrophobic residues 3–23 of CYP46A1 spans the membrane. Initially we generated an enzyme that lacked this putative transmembrane region Δ(3–24) and had the following N-terminal sequence: MetAlaArgAlaArgSerArgTyr. Because no P450 spectrum was detected in the E. coli cells expressing this construct, we increased the extent of

Discussion

Four forms of CYP46A1 with different modifications at the N- and C-termini were engineered and characterized to select the most suitable enzyme for subsequent biophysical studies. All four P450s were the truncated variants, and their E. coli expression levels were higher than that of the full-length CYP46A1 (Fig. 1). From the three parameters tested, deletion of the N-terminal membrane anchor, deletion of the C-terminal proline-rich region, and position of a His-tag (either the N-terminal or

Acknowledgements

Oligonucleotide synthesis and DNA sequencing were carried out by the recombinant DNA Laboratory and the Protein Chemistry Laboratory, respectively, at the University of Texas Medical Branch.

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Until recently, genetic manipulations were the only means whereby CYP46A1 activity was increased in vivo. We were successful with a different approach based on our studies of the biochemical and biophysical properties of CYP46A1 (Mast et al., 2003, 2004, 2009, 2010, 2012, 2013a,b; Shafaati et al., 2010; White et al., 2008). We discovered that CYP46A1 is an allosteric enzyme and that the administration of the anti-HIV medication efavirenz (EFV) to normal (C57BL/6J) mice activates CYP46A1 and cerebral cholesterol turnover in mouse brain (Anderson et al., 2016; Mast et al., 2014).
Cytochrome P450 46A1 (CYP46A1 or cholesterol 24-hydroxylase) controls cholesterol elimination from the brain and plays a role in higher order brain functions. Genetically enhanced CYP46A1 expression in mouse models of Alzheimer's disease mitigates the manifestations of this disease. We enhanced CYP46A1 activity pharmacologically by treating 5XFAD mice, a model of rapid amyloidogenesis, with a low dose of the anti-HIV medication efavirenz. Efavirenz was administered from 1 to 9 months of age, and mice were evaluated at specific time points. At one month of age, cholesterol homeostasis was already disturbed in the brain of 5XFAD mice. Nevertheless, efavirenz activated CYP46A1 and mouse cerebral cholesterol turnover during the first four months of administration. This treatment time also reduced amyloid burden and microglia activation in the cortex and subiculum of 5XFAD mice as well as protein levels of amyloid precursor protein and the expression of several genes involved in inflammatory response. However, mouse short-term memory and long-term spatial memory were impaired, whereas learning in the context-dependent fear test was improved. Additional four months of drug administration (a total of eight months of treatment) improved long-term spatial memory in the treated as compared to the untreated mice, further decreased amyloid-β content in 5XFAD brain, and also decreased the mortality rate among male mice. We propose a mechanistic model unifying the observed efavirenz effects. We suggest that CYP46A1 activation by efavirenz could be a new anti-Alzheimer's disease treatment and a tool to study and identify normal and pathological brain processes affected by cholesterol maintenance.
In vitro cytochrome P450 46A1 (CYP46A1) activation by neuroactive compounds
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Cytochrome P450 46A1 (CYP46A1, cholesterol 24-hydroxylase) is the enzyme responsible for the majority of cholesterol elimination from the brain. Previously, we found that the anti-HIV drug efavirenz (EFV) can pharmacologically activate CYP46A1 in mice. Herein, we investigated whether CYP46A1 could also be activated by endogenous compounds, including major neurotransmitters. In vitro experiments with purified recombinant CYP46A1 indicated that CYP46A1 is activated by l-glutamate (l-Glu), l-aspartate, γ-aminobutyric acid, and acetylcholine, with l-Glu eliciting the highest increase (3-fold) in CYP46A1-mediated cholesterol 24-hydroxylation. We also found that l-Glu and other activating neurotransmitters bind to the same site on the CYP46A1 surface, which differs from the EFV-binding site. The other principal differences between EFV and l-Glu in CYP46A1 activation include an apparent lack of l-Glu binding to the P450 active site and different pathways of signal transduction from the allosteric site to the active site. EFV and l-Glu similarly increased the CYP46A1 k_cat, the rate of the “fast” phase of the enzyme reduction by the redox partner NADPH–cytochrome P450 oxidoreductase, and the amount of P450 reduced. Spectral titrations with cholesterol, in the presence of EFV or l-Glu, suggest that water displacement from the heme iron can be affected in activator-bound CYP46A1. Moreover, EFV and l-Glu synergistically activated CYP46A1. Collectively, our in vitro data, along with those from previous cell culture and in vivo studies by others, suggest that l-Glu-induced CYP46A1 activation is of physiological relevance.
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Cytochrome P450 27A1 (CYP27A1 or sterol 27-hydroxylase) is a ubiquitous, multifunctional enzyme catalyzing regio- and stereospecific hydroxylation of different sterols. In humans, complete CYP27A1 deficiency leads to cerebrotendinous xanthomatosis or nodule formation in tendons and brain (preferentially in the cerebellum) rich in cholesterol and cholestanol, the 5α-saturated analog of cholesterol. In Cyp27a1^−/− mice, xanthomas are not formed, despite a significant cholestanol increase in the brain and cerebellum. The mechanism behind cholestanol production has been clarified, yet little is known about its metabolism, except that CYP27A1 might metabolize cholestanol. It also is unclear why CYP27A1 deficiency results in preferential cholestanol accumulation in the cerebellum. We hypothesized that cholestanol might be metabolized by CYP46A1, the principal cholesterol 24-hydroxylase in the brain. We quantified sterols along with CYP27A1 and CYP46A1 in mouse models (Cyp27a1^−/−, Cyp46a1^−/−, Cyp27a1^−/−Cyp46a1^−/−, and two wild type strains) and human brain specimens. In vitro experiments with purified P450s were conducted as well. We demonstrate that CYP46A1 is involved in cholestanol removal from the brain and that several factors contribute to the preferential increase in cholestanol in the cerebellum arising from CYP27A1 deficiency. These factors include (i) low cerebellar abundance of CYP46A1 and high cerebellar abundance of CYP27A1, the lack of which probably selectively increases the cerebellar cholestanol production; (ii) spatial separation in the cerebellum of cholesterol/cholestanol-metabolizing P450s from a pool of metabolically available cholestanol; and (iii) weak cerebellar regulation of cholesterol biosynthesis. We identified a new physiological role of CYP46A1, an important brain enzyme and cytochrome P450 that could be activated pharmacologically.
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Cytochrome P450 46A1 (CYP46A1) is a microsomal enzyme and cholesterol 24-hydroxylase that controls cholesterol elimination from the brain. This P450 is also a potential target for Alzheimer disease because it can be activated pharmacologically by some marketed drugs, as exemplified by efavirenz, the anti-HIV medication. Previously, we suggested that pharmaceuticals activate CYP46A1 allosterically through binding to a site on the cytosolic protein surface, which is different from the enzyme active site facing the membrane. Here we identified this allosteric site for efavirenz on CYP46A1 by using a combination of hydrogen-deuterium exchange coupled to MS, computational modeling, site-directed mutagenesis, and analysis of the CYP46A1 crystal structure. We also mapped the binding region for the CYP46A1 redox partner oxidoreductase and found that the allosteric and redox partner binding sites share a common border. On the basis of the data obtained, we propose the mechanism of CYP46A1 allostery and the pathway for the signal transmission from the P450 allosteric site to the active site.
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Cytochrome P450 46A1 (CYP46A1) is a brain-specific cholesterol 24-hydroxylase responsible for the majority of cholesterol elimination from the brain. Genetically increased CYP46A1 expression in mice leads to improved cognition and decreases manifestations of Alzheimer disease. We found that four pharmaceuticals (efavirenz (EFV), acetaminophen, mirtazapine, and galantamine) prescribed for indications unrelated to cholesterol maintenance increased CYP46A1 activity in vitro. We then evaluated the anti-HIV medication EFV for the mode of interaction with CYP46A1 and the effect on mice. We propose a model for CYP46A1 activation by EFV and show that EFV enhanced CYP46A1 activity and cerebral cholesterol turnover in animals with no effect on the levels of brain cholesterol. The doses of EFV administered to mice and required for the stimulation of their cerebral cholesterol turnover are a hundred times lower than those prescribed to HIV patients. At such small doses, EFV may be devoid of adverse effects elicited by high drug concentrations. CYP46A1 could be a novel therapeutic target and a tool to further investigate the physiological and medical significance of cerebral cholesterol turnover.

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^☆: This work was supported in part by United States Public Health Service Grant No. GM62882 (to I.A.P.), Center Grant ES06676, Howard Hughes Medical Institute Grant No. 53000266, and Swedish Medical Research Council (to I.B.).

¹: Present address: Banyu Pharmaceutical, 3 Okubo, Tsukuba, Ibaraki 300-2611, Japan.

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Expression of human cytochrome P450 46A1 in Escherichia coli: effects of N- and C-terminal modifications☆

Abstract

Section snippets

Construction and expression of truncated P450s

Expression of truncated P450s in E. coli

Discussion

Acknowledgements

J. Lipid Res.

Biochim. Biophys. Acta

Neurosci. Lett.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

Mol. Cell

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

J. Biol. Chem.

Proc. Natl. Acad. Sci. USA