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Irina A Pikuleva - One of the best experts on this subject based on the ideXlab platform.
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Cholesterol Hydroxylating Cytochrome P450 46A1: From Mechanisms of Action to Clinical Applications
Frontiers Media SA, 2021Co-Authors: Irina A Pikuleva, Nathalie CartierAbstract:Cholesterol, an essential component of the brain, and its local metabolism are involved in many neurodegenerative diseases. The blood-brain barrier is impermeable to cholesterol; hence, cholesterol homeostasis in the central nervous system represents a balance between in situ biosynthesis and elimination. Cytochrome P450 46A1 (CYP46A1), a central nervous system-specific enzyme, converts cholesterol to 24-hydroxycholesterol, which can freely cross the blood-brain barrier and be degraded in the liver. By the dual action of initiating cholesterol efflux and activating the cholesterol synthesis pathway, CYP46A1 is the key enzyme that ensures brain cholesterol turnover. In humans and mouse models, CYP46A1 activity is altered in Alzheimer’s and Huntington’s diseases, spinocerebellar ataxias, glioblastoma, and autism spectrum disorders. In mouse models, modulations of CYP46A1 activity mitigate the manifestations of Alzheimer’s, Huntington’s, Nieman-Pick type C, and Machao-Joseph (spinocerebellar ataxia type 3) diseases as well as amyotrophic lateral sclerosis, epilepsy, glioblastoma, and prion infection. Animal studies revealed that the CYP46A1 activity effects are not limited to cholesterol maintenance but also involve critical cellular pathways, like gene transcription, endocytosis, misfolded protein clearance, vesicular transport, and synaptic transmission. How CYP46A1 can exert central control of such essential brain functions is a pressing question under investigation. The potential therapeutic role of CYP46A1, demonstrated in numerous models of brain disorders, is currently being evaluated in early clinical trials. This review summarizes the past 70 years of research that has led to the identification of CYP46A1 and brain cholesterol homeostasis as powerful therapeutic targets for severe pathologies of the CNS.
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N,N-Dimethyl-3β-hydroxycholenamide Reduces Retinal Cholesterol via Partial Inhibition of Retinal Cholesterol Biosynthesis Rather Than its Liver X Receptor Transcriptional Activity
Frontiers Media S.A., 2018Co-Authors: Nicole El-darzi, Natalia Mast, Artem Astafev, Aicha Saadane, Morrie Lam, Irina A PikulevaAbstract:N,N-dimethyl-3β-hydroxycholenamide (DMHCA) is an experimental pharmaceutical and a steroidal liver X receptor (LXR) agonist, which does not induce undesired hepatic lipogenesis. Herein, DMHCA was evaluated for its retinal effects on normal C57BL/6J and Cyp27a1−/−CYP46A1−/− mice; the latter having higher retinal total and esterified cholesterol in addition to retinal vascular abnormalities. Different doses and two formulations were used for DMHCA delivery either via drinking water (C57BL/6J mice) or by oral gavage (Cyp27a1−/−CYP46A1−/− mice). The duration of treatment was 1 week for C57BL/6J mice and 2 or 4 weeks for Cyp27a1−/−CYP46A1−/− mice. In both genotypes, the higher DMHCA doses (37–80 mg/kg of body weight/day) neither increased serum triglycerides nor serum cholesterol but altered the levels of retinal sterols. Total retinal cholesterol was decreased in the DMHCA-treated mice, mainly due to a decrease in retinal unesterified cholesterol. In addition, retinal levels of cholesterol precursors lanosterol, zymosterol, desmosterol, and lathosterol were changed in Cyp27a1−/−CYP46A1−/− mice. In both genotypes, DMHCA effect on retinal expression of the LXR target genes was only moderate and gender-specific. Collectively, the data obtained provide evidence for a decrease in retinal cholesterol as a result of DMHCA acting in the retina as an enzyme inhibitor of cholesterol biosynthesis rather than a LXR transcriptional activator. Specifically, DMHCA appears to partially inhibit the cholesterol biosynthetic enzyme Δ24-dehydrocholesterol reductase rather than upregulate the expression of LXR target genes involved in reverse cholesterol transport. The identified DMHCA dosages, formulations, and routes of delivery as well as the observed effects on the retina should be considered in future studies using DMHCA as a potential therapeutic for age-related macular degeneration and diabetic retinopathy
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Cytochrome P450 27A1 Deficiency and Regional Differences in Brain Sterol Metabolism Cause Preferential Cholestanol Accumulation in the Cerebellum
Journal of Biological Chemistry, 2017Co-Authors: Natalia Mast, Kyle W. Anderson, Illarion V Turko, Curtis Tatsuoka, Yong Li, Irina A PikulevaAbstract:Abstract Cytochrome P450 27A1 (CYP27A1 or sterol 27-hydroxylase) is a ubiquitous, multifunctional enzyme catalyzing regio- and stereo-specific 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, whose lack likely 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 27a1 deficiency and regional differences in brain sterol metabolism cause preferential cholestanol accumulation in the cerebellum
Journal of Biological Chemistry, 2017Co-Authors: Natalia Mast, Illarion V Turko, Curtis Tatsuoka, Kyle Anderson, Joseph Lin, Irina A PikulevaAbstract: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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Sterol quantifications in the brain of CYP46A1-/- mice and wild type controls.
2017Co-Authors: Natalia Mast, Kyle W. Anderson, Ingemar Bjorkhem, Joseph B. Lin, Irina A PikulevaAbstract:Data for cholesterol, lathosterol, desmosterol, 24-hydroxycholesterol, and 27-hydroxycholesterol were taken from [49]. The data represent the mean ± SD of three measurements in individual (n = 3) 3–5 month old mice. Statistical significance was assessed two-way ANOVA followed by pairwise comparisons made using the Bonferroni correction. Significant comparisons of free sterols are denoted by the following color code: pink asterisks, significant changes between CYP46A1-/- females versus wild type females; blue asterisks, significant changes between CYP46A1-/- males versus wild type males; gray asterisks, significant changes between female and male mice of the same strain; and black asterisks, significant changes between the genotypes when data were collapsed across genders. *, P≤0.05; **, P≤0.01; ***, P≤0.001; and ND, not detectable (the limit of detection was 1 pmol/mg protein).
Natalia Mast - One of the best experts on this subject based on the ideXlab platform.
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synthesis and pharmacokinetic study of a 11c labeled cholesterol 24 hydroxylase inhibitor using in loop 11c co2 fixation method
Bioorganic & Medicinal Chemistry Letters, 2020Co-Authors: Zhen Chen, Natalia Mast, Xiaoyun Deng, Tuo Shao, Hualong Fu, Qingzhen Yu, Jiahui Chen, Jian Rong, Yihan Shao, Lee JosephsonAbstract:Abstract Cholesterol 24-hydroxylase, also known as CYP46A1 (EC 1.14.13.98), is a monooxygenase and a member of the cytochrome P450 family. CYP46A1 is specifically expressed in the brain where it controls cholesterol elimination by producing 24S-hydroxylcholesterol (24-HC) as the major metabolite. Modulation of CYP46A1 activity may affect Aβ deposition and p-tau accumulation by changing 24-HC formation, which thereafter serves as potential therapeutic pathway for Alzheimer’s disease. In this work, we showcase the efficient synthesis and preliminary pharmacokinetic evaluation of a novel cholesterol 24-hydroxylase inhibitor 1 for use in positron emission tomography.
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N,N-Dimethyl-3β-hydroxycholenamide Reduces Retinal Cholesterol via Partial Inhibition of Retinal Cholesterol Biosynthesis Rather Than its Liver X Receptor Transcriptional Activity
Frontiers Media S.A., 2018Co-Authors: Nicole El-darzi, Natalia Mast, Artem Astafev, Aicha Saadane, Morrie Lam, Irina A PikulevaAbstract:N,N-dimethyl-3β-hydroxycholenamide (DMHCA) is an experimental pharmaceutical and a steroidal liver X receptor (LXR) agonist, which does not induce undesired hepatic lipogenesis. Herein, DMHCA was evaluated for its retinal effects on normal C57BL/6J and Cyp27a1−/−CYP46A1−/− mice; the latter having higher retinal total and esterified cholesterol in addition to retinal vascular abnormalities. Different doses and two formulations were used for DMHCA delivery either via drinking water (C57BL/6J mice) or by oral gavage (Cyp27a1−/−CYP46A1−/− mice). The duration of treatment was 1 week for C57BL/6J mice and 2 or 4 weeks for Cyp27a1−/−CYP46A1−/− mice. In both genotypes, the higher DMHCA doses (37–80 mg/kg of body weight/day) neither increased serum triglycerides nor serum cholesterol but altered the levels of retinal sterols. Total retinal cholesterol was decreased in the DMHCA-treated mice, mainly due to a decrease in retinal unesterified cholesterol. In addition, retinal levels of cholesterol precursors lanosterol, zymosterol, desmosterol, and lathosterol were changed in Cyp27a1−/−CYP46A1−/− mice. In both genotypes, DMHCA effect on retinal expression of the LXR target genes was only moderate and gender-specific. Collectively, the data obtained provide evidence for a decrease in retinal cholesterol as a result of DMHCA acting in the retina as an enzyme inhibitor of cholesterol biosynthesis rather than a LXR transcriptional activator. Specifically, DMHCA appears to partially inhibit the cholesterol biosynthetic enzyme Δ24-dehydrocholesterol reductase rather than upregulate the expression of LXR target genes involved in reverse cholesterol transport. The identified DMHCA dosages, formulations, and routes of delivery as well as the observed effects on the retina should be considered in future studies using DMHCA as a potential therapeutic for age-related macular degeneration and diabetic retinopathy
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Cytochrome P450 27A1 Deficiency and Regional Differences in Brain Sterol Metabolism Cause Preferential Cholestanol Accumulation in the Cerebellum
Journal of Biological Chemistry, 2017Co-Authors: Natalia Mast, Kyle W. Anderson, Illarion V Turko, Curtis Tatsuoka, Yong Li, Irina A PikulevaAbstract:Abstract Cytochrome P450 27A1 (CYP27A1 or sterol 27-hydroxylase) is a ubiquitous, multifunctional enzyme catalyzing regio- and stereo-specific 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, whose lack likely 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 27a1 deficiency and regional differences in brain sterol metabolism cause preferential cholestanol accumulation in the cerebellum
Journal of Biological Chemistry, 2017Co-Authors: Natalia Mast, Illarion V Turko, Curtis Tatsuoka, Kyle Anderson, Joseph Lin, Irina A PikulevaAbstract: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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Sterol quantifications in the brain of CYP46A1-/- mice and wild type controls.
2017Co-Authors: Natalia Mast, Kyle W. Anderson, Ingemar Bjorkhem, Joseph B. Lin, Irina A PikulevaAbstract:Data for cholesterol, lathosterol, desmosterol, 24-hydroxycholesterol, and 27-hydroxycholesterol were taken from [49]. The data represent the mean ± SD of three measurements in individual (n = 3) 3–5 month old mice. Statistical significance was assessed two-way ANOVA followed by pairwise comparisons made using the Bonferroni correction. Significant comparisons of free sterols are denoted by the following color code: pink asterisks, significant changes between CYP46A1-/- females versus wild type females; blue asterisks, significant changes between CYP46A1-/- males versus wild type males; gray asterisks, significant changes between female and male mice of the same strain; and black asterisks, significant changes between the genotypes when data were collapsed across genders. *, P≤0.05; **, P≤0.01; ***, P≤0.001; and ND, not detectable (the limit of detection was 1 pmol/mg protein).
Nina Isoherranen - One of the best experts on this subject based on the ideXlab platform.
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the retinoic acid hydroxylase cyp26a1 has minor effects on postnatal vitamin a homeostasis but is required for exogenous atra clearance
Journal of Biological Chemistry, 2019Co-Authors: Guo Zhong, Jessica M Snyder, Cathryn A Hogarth, Weize Huang, Traci B Topping, Jeffrey T Lafrance, Laura Palau, Lindsay C Czuba, Gabriel Ghiaur, Nina IsoherranenAbstract:The all-trans-retinoic acid (atRA) hydroxylase Cyp26a1 is essential for embryonic development and may play a key role in regulating atRA clearance also in adults. We hypothesized that loss of Cyp26a1 activity via inducible knockout in juvenile or adult mice would result in decreased atRA clearance and increased tissue atRA concentrations and atRA-related adverse effects. To test these hypotheses, Cyp26a1 was knocked out in juvenile and adult male and female Cyp26a1 floxed mice using standard Cre–Lox technology and tamoxifen injections. Biochemical and histological methods were used to study the effects of global Cyp26a1 knockout. The Cyp26a1 knockout did not result in consistent histopathological changes in any major organs. Cyp26a1−/− mice gained weight normally and exhibited no adverse phenotypes for up to 1 year after loss of Cyp26a1 expression. Similarly, atRA concentrations were not increased in the liver, testes, spleen, or serum of these mice, and the Cyp26a1 knockout did not cause compensatory induction of lecithin:retinol acetyltransferase (Lrat) or retinol dehydrogenase 11 (Rdh11) mRNA or a decrease in aldehyde dehydrogenase 1a1 (Aldh1a1) mRNA in the liver compared with tamoxifen-treated controls. However, the Cyp26a1−/− mice showed increased bone marrow cellularity and decreased frequency of erythroid progenitor cells in the bone marrow consistent with a retinoid-induced myeloid skewing of hematopoiesis. In addition, the Cyp26a1 knockout decreased clearance of exogenous atRA by 70% and increased atRA half-life 6-fold. These findings demonstrate that despite lacking a major impact on endogenous atRA signaling, Cyp26a1 critically contributes as a barrier for exogenous atRA exposure.
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identification of tazarotenic acid as the first xenobiotic substrate of human retinoic acid hydroxylase cyp26a1 and cyp26b1
Journal of Pharmacology and Experimental Therapeutics, 2016Co-Authors: Robert S Foti, Alex Zelter, Nina Isoherranen, Leslie J Dickmann, Brian Buttrick, Philippe Diaz, Dominique DouguetAbstract:Cytochrome P450 (CYP) 26A1 and 26B1 are heme-containing enzymes responsible for metabolizing all-trans retinoic acid (at-RA). No crystal structures have been solved, and therefore homology models that provide structural information are extremely valuable for the development of inhibitors of cytochrome P450 family 26 (CYP26). The objectives of this study were to use homology models of CYP26A1 and CYP26B1 to characterize substrate binding characteristics, to compare structural aspects of their active sites, and to support the role of CYP26 in the metabolism of xenobiotics. Each model was verified by dockingat-RA in the active site and comparing the results to known metabolic profiles ofat-RA. The models were then used to predict the metabolic sites of tazarotenic acid with results verified by in vitro metabolite identification experiments. The CYP26A1 and CYP26B1 homology models predicted that the benzothiopyranyl moiety of tazarotenic acid would be oriented toward the heme of each enzyme and suggested that tazarotenic acid would be a substrate of CYP26A1 and CYP26B1. Metabolite identification experiments indicated that CYP26A1 and CYP26B1 oxidatively metabolized tazarotenic acid on the predicted moiety, with in vitro rates of metabolite formation by CYP26A1 and CYP26B1 being the highest across a panel of enzymes. Molecular analysis of the active sites estimated the active-site volumes of CYP26A1 and CYP26B1 to be 918 A(3)and 977 A(3), respectively. Overall, the homology models presented herein describe the enzyme characteristics leading to the metabolism of tazarotenic acid by CYP26A1 and CYP26B1 and support a potential role for the CYP26 enzymes in the metabolism of xenobiotics.
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induction of cyp26a1 by metabolites of retinoic acid evidence that cyp26a1 is an important enzyme in the elimination of active retinoids
Molecular Pharmacology, 2015Co-Authors: Ariel R Topletz, Wendel L Nelson, Robert S Foti, Sasmita Tripathy, Jakob A Shimshoni, Nina IsoherranenAbstract:All-trans-retinoic acid (atRA), the active metabolite of vitamin A, induces gene transcription via binding to nuclear retinoic acid receptors (RARs). The primary hydroxylated metabolites formed from atRA by CYP26A1, and the subsequent metabolite 4-oxo-atRA, bind to RARs and potentially have biologic activity. Hence, CYP26A1, the main atRA hydroxylase, may function either to deplete bioactive retinoids or to form active metabolites. This study aimed to determine the role of CYP26A1 in modulating RAR activation via formation and elimination of active retinoids. After treatment of HepG2 cells with atRA, (4S)-OH-atRA, (4R)-OH-atRA, 4-oxo-atRA, and 18-OH-atRA, mRNAs of CYP26A1 and RARβ were increased 300- to 3000-fold, with 4-oxo-atRA and atRA being the most potent inducers. However, >60% of the 4-OH-atRA enantiomers were converted to 4-oxo-atRA in the first 12 hours of treatment, suggesting that the activity of the 4-OH-atRA was due to 4-oxo-atRA. In human hepatocytes, atRA, 4-OH-atRA, and 4-oxo-atRA induced CYP26A1 and 4-oxo-atRA formation was observed from 4-OH-atRA. In HepG2 cells, 4-oxo-atRA formation was observed even in the absence of CYP26A1 activity and this formation was not inhibited by ketoconazole. In human liver microsomes, 4-oxo-atRA formation was supported by NAD+, suggesting that 4-oxo-atRA formation is mediated by a microsomal alcohol dehydrogenase. Although 4-oxo-atRA was not formed by CYP26A1, it was depleted by CYP26A1 (Km = 63 nM and intrinsic clearance = 90 μl/min per pmol). Similarly, CYP26A1 depleted 18-OH-atRA and the 4-OH-atRA enantiomers. These data support the role of CYP26A1 to clear bioactive retinoids, and suggest that the enzyme forming active 4-oxo-atRA may be important in modulating retinoid action.
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comparison of the function and expression of cyp26a1 and cyp26b1 the two retinoic acid hydroxylases
Biochemical Pharmacology, 2012Co-Authors: Ariel R Topletz, Jayne E Thatcher, Alex Zelter, Justin D Lutz, Suzanne Tay, Wendel L Nelson, Nina IsoherranenAbstract:Abstract All-trans -retinoic acid ( at RA) is an important signaling molecule in all chordates. The cytochrome P450 enzymes CYP26 are believed to partially regulate cellular concentrations of at RA via oxidative metabolism and hence affect retinoid homeostasis and signaling. CYP26A1 and CYP26B1 are at RA hydroxylases that catalyze formation of similar metabolites in cell systems. However, they have only 40% sequence similarity suggesting differences between the two enzymes. The aim of this study was to determine whether CYP26A1 and CYP26B1 have similar catalytic activity, form different metabolites from at RA and are expressed in different tissues in adults. The mRNA expression of CYP26A1 and CYP26B1 correlated between human tissues except for human cerebellum in which CYP26B1 was the predominant CYP26 and liver in which CYP26A1 dominated. Quantification of CYP26A1 and CYP26B1 protein in human tissues was in agreement with the mRNA expression and showed correlation between the two isoforms. Qualitatively, recombinant CYP26A1 and CYP26B1 formed the same primary and sequential metabolites from at RA. Quantitatively, CYP26B1 had a lower K m (19 nM) and V max (0.8 pmol/min/pmol) than CYP26A1 ( K m = 50 nM and V max = 10 pmol/min/pmol) for formation of 4-OH-RA. The major at RA metabolites 4-OH-RA, 18-OH-RA and 4-oxo-RA were all substrates of CYP26A1 and CYP26B1, and CYP26A1 had a 2–10-fold higher catalytic activity towards all substrates tested. This study shows that CYP26A1 and CYP26B1 are qualitatively similar RA hydroxylases with overlapping expression profiles. CYP26A1 has higher catalytic activity than CYP26B1 and seems to be responsible for metabolism of at RA in tissues that function as a barrier for at RA exposure.
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substrate specificity and ligand interactions of cyp26a1 the human liver retinoic acid hydroxylase
Molecular Pharmacology, 2011Co-Authors: Jayne E Thatcher, Wendel L Nelson, Brian Buttrick, Jakob A Shimshoni, Scott A Shaffer, David R Goodlett, Nina IsoherranenAbstract:All-trans-retinoic acid (atRA) is the active metabolite of vitamin A. atRA is also used as a drug, and synthetic atRA analogs and inhibitors of retinoic acid (RA) metabolism have been developed. The hepatic clearance of atRA is mediated primarily by CYP26A1, but design of CYP26A1 inhibitors is hindered by lack of information on CYP26A1 structure and structure-activity relationships of its ligands. The aim of this study was to identify the primary metabolites of atRA formed by CYP26A1 and to characterize the ligand selectivity and ligand interactions of CYP26A1. On the basis of high-resolution tandem mass spectrometry data, four metabolites formed from atRA by CYP26A1 were identified as 4-OH-RA, 4-oxo-RA, 16-OH-RA and 18-OH-RA. 9-cis-RA and 13-cis-RA were also substrates of CYP26A1. Forty-two compounds with diverse structural properties were tested for CYP26A1 inhibition using 9-cis-RA as a probe, and IC50 values for 10 inhibitors were determined. The imidazole- and triazole-containing inhibitors [S-(R*,R*)]-N-[4-[2-(dimethylamino)-1-(1H-imidazole-1-yl)propyl]-phenyl]2-benzothiazolamine (R116010) and (R)-N-[4-[2-ethyl-1-(1H-1,2,4-triazol-1-yl)butyl]phenyl]-2-benzothiazolamine (R115866) were the most potent inhibitors of CYP26A1 with IC50 values of 4.3 and 5.1 nM, respectively. Liarozole and ketoconazole were significantly less potent with IC50 values of 2100 and 550 nM, respectively. The retinoic acid receptor (RAR) γ agonist CD1530 was as potent an inhibitor of CYP26A1 as ketoconazole with an IC50 of 530 nM, whereas the RARα and RARβ agonists tested did not significantly inhibit CYP26A1. The pan-RAR agonist 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid and the peroxisome proliferator-activated receptor ligands rosiglitazone and pioglitazone inhibited CYP26A1 with IC50 values of 3.7, 4.2, and 8.6 μM, respectively. These data demonstrate that CYP26A1 has high ligand selectivity but accepts structurally related nuclear receptor agonists as inhibitors.
Jing-pian Peng - One of the best experts on this subject based on the ideXlab platform.
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regulation of cyp26a1 on th17 cells in mouse peri implantation
Journal of Cellular and Molecular Medicine, 2014Co-Authors: Haiyan Liu, Ying Yang, Hong-fei Xia, Huhe Chao, Zhenkun Liu, Zhihui Song, Jing-pian PengAbstract:Cytochrome P450 26A1 (cyp26a1) is expressed in the mouse uterus during peri-implantation. The repression of this protein is closely associated with a reduction in implantation sites, suggesting a specific role for cyp26a1 in pregnancy and prompting questions concerning how a metabolic enzyme can generate this distinct outcome. To explore the effective downstream targets of cyp26a1 and confirm if its role in peri-implantation depends on its metabolic substrate RA (retinoic acid), we characterized the changes in the peripheral blood, spleen and uterine implantation sites using the cyp26a1 gene vaccine constructed before. Flow cytometry results showed a significant increase in CD4+RORγt+ Th17 cells in both the peripheral blood and spleen in the experimental group. The expression of RORγt and IL-17 presented the Th17 cells reduction in uterus followed by the suppression of cyp26a1 expression. For greater certainty, cyp26a1 antibody blocking model and RNA interference model were constructed to determine the precise target immune cell group. High performance liquid chromatography results showed a significant increase in uterine at-RA followed by the immunization of cyp26a1 gene vaccine. Both the ascertain by measuring RARα protein levels in peri-implantation uterus after gene vaccine immunization and researches using the specific agonist and antagonist against RARα suggested that RARα may be the main RA receptor for signal transduction. These results provided more evidence for the signal messenger role of RA in cyp26a1 regulation from the other side. Here, we showed that the cyp26a1-regulated Th17 cells are dependent on at-RA signalling, which is delivered through RARα in mouse peri-implantation.
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the antitumor immunopreventive effects of a dna vaccine against cyp26a1 on mouse breast carcinoma
Vaccine, 2011Co-Authors: Rongchun Wang, Ying Yang, Zhenkun Liu, Wen Chen, Jing-pian PengAbstract:Abstract Background CYP26a1, which functioned mainly as a retinoic acid (RA) catabolic enzyme, has been shown to be oncogenic and to support cell survival in many breast carcinoma cells. Objectives The purpose of the study was to investigate the antitumor effect of a DNA vaccine targeting CYP26a1 on breast tumors development in mice which highly express CYP26a1 and to further clarify its potential mechanism. Methods After three times immunization of the DNA vaccine, the BALB/c mice were inoculated with the engineered 4T1 breast cancer cells expressing CYP26a1. Primary tumors were measured every 4 days after tumor cell inoculation. The primary tumors were surgically removed and weighted after 30 days of inoculation. The anti-CYP26a1 antibody titer of the antiserum was measured by an enzyme-linked immunosorbent assay (ELISA). The effect of the vaccine on apoptosis of the primary tumor was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Apoptosis-related proteins in primary tumor were detected by Western blotting. The expression of the Th1 and Th2 type cytokines was detected by RT-PCR. Results The vaccine could elicit the production of anti-CYP26a1 antibody and significantly inhibit the growth of the primary tumor compared to the control groups ( p Conclusion Our study shows that the vaccine targeting CYP26a1 significantly inhibits the primary tumor growth and progression by activating the apoptosis pathway and by eliciting both humoral and cellular immune responses.
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retinoic acid metabolizing enzyme cyp26a1 is implicated in rat embryo implantation
Human Reproduction, 2010Co-Authors: Hong-fei Xia, Ying Yang, Jing Sun, Jing-pian PengAbstract:background: The retinoic acid metabolizing enzyme Cyp26a1 plays a pivotal role in vertebrate embryo development. Cyp26a1 was characterized previously as a differentially expressed gene in peri-implantation rat uteri via suppressive subtracted hybridization analysis. However, the role of Cyp26a1 in rat embryo implantation remained elusive. methods: The expression of Cyp26a1 in the uteri of early pregnancy, pseudopregnancy and artificial decidualization was detected by northern blotting, real time-PCR, in situ hybridization, western blotting and immunofluorescent staining. The effect of Cyp26a1 on apoptosis of endometrial stromal cells (ESCs) isolated from rat uteri was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and Hoechst staining. Apoptosis-related proteins in ESCs were detected by western blotting. results: Cyp26a1 showed distinctive expression patterns in embryos and uteri during the peri-implantation period, with a remarkable increase (P , 0.01 versus Days 4–5) in mRNA and protein in the implantation phase (Days 5.5–6.5 of pregnancy). CYP26A1 was specifically localized in glandular epithelium, luminal epithelium and decidua basalis. The level of CYP26A1 protein was significantly increased in uteri of artificial decidualization (P , 0.01 versus control). Forced Cyp26a1 overexpression significantly reduced the sensitivity of ESCs to etoposideinduced apoptosis, with reductions in p53 (P , 0.01) and Fas (P , 0.05) proteins versus control, while in contrast, FasL (P , 0.01) and proliferating cell nuclear antigen (P , 0.05) proteins increased. conclusions: Cyp26a1 is spatiotemporally expressed in the uterus during embryo implantation and decidualization. Overexpression of Cyp26a1 attenuates the process of uterine stromal cell apoptosis, probably via down-regulating the expression of p53 and FasL.
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retinoic acid metabolizing enzyme cytochrome p450 26a1 cyp26a1 is essential for implantation functional study of its role in early pregnancy
Journal of Cellular Physiology, 2010Co-Authors: Bingchen Han, Ying Yang, Hong-fei Xia, Jing Sun, Jing-pian PengAbstract:Vitamin A (VA) is required for normal fetal development and successful pregnancy. Excessive VA intake during pregnancy may lead to adverse maternal and fetal effects. Cytochrome P450 26A1 (cyp26a1), a retinoic acid (RA)-metabolizing enzyme, is involved in VA metabolism. It has been shown that cyp26a1 is expressed in female reproductive tract, especially in uterus. In order to investigate the role of cyp26a1 during pregnancy, we constructed a recombinant plasmid DNA vaccine encoding cyp26a1 protein and immunized mice with the plasmid. Compared to control groups, the pregnancy rate of the cyp26a1 plasmid-immunized mice were significantly decreased (P < 0.01). Further results showed that both cyp26a1 mRNA and protein were specifically induced in the uterus during implantation period and localized in the uterine luminal epithelium. Importantly, the number of implantation sites was also significantly reduced (P < 0.05) after the uterine injection of cyp26a1-specific antisense oligos or anti-cyp26a1 antibody on day 3 of pregnancy. Accordingly, the expression of RA-related cellular retinoic acid binding protein 1 and tissue transglutaminase was markedly increased (P < 0.05) in the uterine luminal epithelium after intrauterine injection treatments. These data demonstrate that uterine cyp26a1 activity is important for the maintenance of pregnancy, especially during the process of blastocyst implantation. J. Cell. Physiol. 223: 471–479, 2010. © 2010 Wiley-Liss, Inc.
Illarion V Turko - One of the best experts on this subject based on the ideXlab platform.
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Cytochrome P450 27A1 Deficiency and Regional Differences in Brain Sterol Metabolism Cause Preferential Cholestanol Accumulation in the Cerebellum
Journal of Biological Chemistry, 2017Co-Authors: Natalia Mast, Kyle W. Anderson, Illarion V Turko, Curtis Tatsuoka, Yong Li, Irina A PikulevaAbstract:Abstract Cytochrome P450 27A1 (CYP27A1 or sterol 27-hydroxylase) is a ubiquitous, multifunctional enzyme catalyzing regio- and stereo-specific 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, whose lack likely 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 27a1 deficiency and regional differences in brain sterol metabolism cause preferential cholestanol accumulation in the cerebellum
Journal of Biological Chemistry, 2017Co-Authors: Natalia Mast, Illarion V Turko, Curtis Tatsuoka, Kyle Anderson, Joseph Lin, Irina A PikulevaAbstract: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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Mapping of the Allosteric Site in Cholesterol Hydroxylase CYP46A1 for Efavirenz, a Drug That Stimulates Enzyme Activity
Journal of Biological Chemistry, 2016Co-Authors: Kyle W. Anderson, Natalia Mast, Jeffrey W. Hudgens, Illarion V Turko, Irina A PikulevaAbstract: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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quantification of cholesterol metabolizing p450s cyp27a1 and CYP46A1 in neural tissues reveals a lack of enzyme product correlations in human retina but not human brain
Journal of Proteome Research, 2011Co-Authors: Wei Li Liao, Natalia Mast, Illarion V Turko, Gun Young Heo, Nathan G Dodder, Rachel E Reem, Suber S Huang, Pier Luigi Dipatre, Irina A PikulevaAbstract:Cytochrome P450 enzymes (CYP or P450) 46A1 and 27A1 play important roles in cholesterol elimination from the brain and retina, respectively, yet they have not been quantified in human organs because of their low abundance and association with membrane. On the basis of our previous development of a multiple reaction monitoring (MRM) workflow for measurements of low-abundance membrane proteins, we quantified CYP46A1 and CYP27A1 in human brain and retina samples from four donors. These enzymes were quantified in the total membrane pellet, a fraction of the whole tissue homogenate, using 15N-labled recombinant P450s as internal standards. The average P450 concentrations/mg of total tissue protein were 345 fmol of CYP46A1 and 110 fmol of CYP27A1 in the temporal lobe, and 60 fmol of CYP46A1 and 490 fmol of CYP27A1 in the retina. The corresponding P450 metabolites were then measured in the same tissue samples and compared to the P450 enzyme concentrations. Investigation of the enzyme−product relationships and an...
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combined use of mass spectrometry and heterologous expression for identification of membrane interacting peptides in cytochrome p450 46a1 and nadph cytochrome p450 oxidoreductase
Archives of Biochemistry and Biophysics, 2009Co-Authors: Natalia Mast, Irina A Pikuleva, Wei Li Liao, Illarion V TurkoAbstract:Cytochrome P450 46A1 (CYP46A1) and NADPH-cytochrome P450 oxidoreductase (CPR) are the components of the brain microsomal mixed-function monooxygenase system that catalyzes the conversion of cholesterol to 24-hydroxycholesterol. Both CYP46A1 and CPR are monotopic membrane proteins that are anchored to the endoplasmic reticulum via the N-terminal transmembrane domain. The exact mode of peripheral association of CYP46A1 and CPR with the membrane is unknown. Therefore, we studied their membrane topology by using an approach in which solution-exposed portion of heterologously expressed membrane-bound CYP46A1 or CPR was removed by digestion with either trypsin or chymotrypsin followed by extraction of the residual peptides and their identification by mass spectrometry. The identified putative membrane-interacting peptides were mapped onto available crystal structures of CYP46A1 and CPR and the proteins were positioned in the membrane considering spatial location of the missed cleavage sites located within these peptide as well as the flanking residues whose cleavage produced these peptides. Experiments were then carried out to validate the inference from our studies that the substrate, cholesterol, enters CYP46A1 from the membrane. As for CPR, its putative membrane topology indicates that the Q153R and R316W missense mutations found in patients with disordered steroidogenesis are located within the membrane-associated regions. This information may provide insight in the deleterious nature of these mutations.
