Dual metabolic pathway of 25‐hydroxyvitamin D<sub>3</sub> catalyzed by human CYP24

Sakaki, Toshiyuki; Sawada, Natsumi; Komai, Koichiro; Shiozawa, Shunichi; Yamada, Sachiko; Yamamoto, Keiko; Ohyama, Yoshihiko; Inouye, Kuniyo

doi:10.1046/j.1432-1327.2000.01680.x

Cited by 172 publications

(173 citation statements)

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“…25[OH]D and 1, 25[OH]D can also be converted into less active moieties by the 24-hydoxylase, cytochrome P450, family 24, subfamily A, polypeptide 1 (CYP24A1). The expression of CYP24A1 is induced by 1, 25[OH] 2 D itself, providing a robust mechanism for negative feedback [7]. Therefore, the relative expression of these hydroxylases can serve to tightly regulate 1, 25[OH]D levels and activity during an immune response.…”

Section: Introductionmentioning

confidence: 99%

Regulation of CYP27B1 and CYP24A1 hydroxylases limits cell‐autonomous activation of vitamin D in dendritic cells

et al. 2014

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Keywords: CYP27B1 r CYP24A1 r DCs r Macrophages r Vitamin D Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionThe capacity of vitamin D metabolites to modulate macrophage and DC function has been subject to extensive research interest in light of the association of vitamin D deficiency with a broad spectrum of immunological and infectious diseases. This gives Correspondence: Dr. Mahdad Noursadeghi e-mail: m.noursadeghi@ucl.ac.uk way to the potential for vitamin D supplementation [1][2][3][4] or ex vivo conditioning of DCs with vitamin D for cell therapy strategies [5]. Vitamin D is synthesized from 7-dehydrocholesterol in the skin following exposure to ultraviolet B radiation and thermal isomerisation, or replenished by dietary intake. It is 25-hydroxylated in the liver to form 25-hydroxyvitamin D (25[OH]D), which is in * These authors contributed equally to this work. Results 25[OH]D is not functionally activated during monocyte differentiation to immature DCsWe first compared the activation of 25[OH]D during monocyte differentiation into macrophages and immature DCs. Conventional cell culture media in this model are vitamin D deficient (Fig. 1A) (Fig. 1B). This was also associated with upregulation of the prototypic vitamin D-inducible gene cathelicidin (CAMP) in macrophages (Fig. 1C) (Fig. 1C). Importantly, upregulation of DC-SIGN during monocyte differentiation to DCs was not affected by the presence of 1,25[OH] 2 D (Fig. 1D) DCs show time-dependent increase in activation of 25[OH]D independent of CYP27B1 expression levelsWe confirmed previous observations [16] that expression of the vitamin D-activating hydroxylase CYP27B1 is significantly lower in DCs compared with MDMs at both transcript and protein levels ( Fig. 2A and B). In contrast, vitamin D receptor (VDR) expression was higher in DCs than MDMs ( Fig. 2A). Taken together, these data further support the hypothesis that 25[OH]D has no effect on DCs in the experiments described above, because of a failure to generate 1,25[OH] 2 D rather than an inability to respond to 1,25[OH] 2 D. We also confirmed that 24-h stimulation of DCs with LPS significantly increases transcript and protein levels of CYP27B1 ( Fig. 2B and C (Fig. 2D). However, there was no concomitant upregulation of CAMP (Fig. 2C) . Monocytes are typically differentiated to DCs over 4-7 days, therefore we considered the possibility that time in culture may confound comparisons between different studies. Interestingly, we did find 25[OH]D-dependent upregulation of CAMP gene expression in DCs that increased with time after at least 6 days in culture (Fig. 2E). This was not associated with any significant time-dependent increase in CYP27B1 expression ( DCs express truncated CYP27B1 transcriptsWe found that LPS stimulation was able to upregulate DC expression of CYP27B1 transcript to levels higher than that in MDMs ( Figs. 2A and C). However, the expression of CYP27B1 protein remains much lower in DCs compared with ...

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Section: Introductionmentioning

confidence: 99%

Regulation of CYP27B1 and CYP24A1 hydroxylases limits cell‐autonomous activation of vitamin D in dendritic cells

et al. 2014

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“…The characterization of CYP24A1 structure and function is complicated by observations that the enzyme displays both C 23 -and C 24 -hydroxylase activity that depends on which side-chain carbon (C 23 or C 24 ) is hydroxylated first. Several studies have demonstrated that speciesspecific CYP24A1 isoforms express different levels of C 23 -and C 24 -hydroxylase activities that result in side-chain lactone formation (C23 pathway) or side-chain cleavage and oxidation to a carboxylic acid (C24 pathway) [18,19] Human CYP24A1 possesses both C 23 -and C 24 -activity, while the rat form seems to catalyze the C 24 pathway exclusively. Although the structural basis of this altered specificity remains elusive, Hanamoto et al have recently demonstrated that the C 23 -hydroxylase activity of rat CYP24A1 could be bolstered by humanizing single residues (T416M, I500T) within the enzyme's putative active site [20] ( Figure 1).…”

Section: Introductionmentioning

confidence: 99%

Hybrid homology modeling and mutational analysis of cytochrome P450C24A1 (CYP24A1) of the Vitamin D pathway: Insights into substrate specificity and membrane bound structure–function

Annalora

Bobrovnikov-Marjon

Serda

et al. 2007

Archives of Biochemistry and Biophysics

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Cytochrome P450C24A1 (CYP24A1), a peripheral inner mitochondrial membrane hemoprotein and candidate oncogene, regulates the side-chain metabolism and biological function of vitamin D and many of its related analog drugs. Rational mutational analysis of rat CYP24A1 based on hybrid (2C5/ BM-3) homology modeling and affinity labeling studies clarified the role of key domains (Nterminus, A', A, and F-helices, β3a strand, & β5 hairpin) in substrate binding and catalysis. The scope of our study was limited by an inability to purify stable mutant enzyme targeting soluble domains (B', G, and I-helices) and suggested greater conformational flexibility among CYP24A1's membraneassociated domains. The most notable mutants developed by modeling were V391T and I500A, which displayed defective binding function and profound metabolic defects for 25-hydroxylated vitamin D 3 substrates similar to a non-functional F-helix mutant (F249T) that we previously reported. Val-391 (β3a strand) and Ile-500 (β5 hairpin) are modeled to interact with Phe-249 (F-helix) in a hydrophobic cluster that directs substrate binding events through interactions with the vitamin D cis-triene moiety. Prior affinity labeling studies identified an amino-terminal residue (Ser-57) as a putative active-site residue that interacts with the 3β-OH group of the vitamin D A-ring. Studies with 3-epi and 3-deoxy-1,25(OH) 2 D 3 analogs confirmed interactions between the 3β-OH group and Ser-57 effect substrate recognition and trafficking while establishing that the trans conformation of A-ring hydroxyl groups (1α & 3β) is obligate for high-affinity binding to rat CYP24A1. Our work suggests that CYP24A1's amphipathic nature allows for monotopic membrane insertion, whereby a pw2d-like substrate access channel is formed to shuttle secosteroid substrate from the membrane to the active-site. We hypothesize that CYP24A1 has evolved a unique amino-terminal membrane § Deceased

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“…Mutations affecting CYP24A1 function correlate with incidents of idiopathic infantile hypercalcemia or hypercalciuria or elevated serum 1,25(OH) 2 D3 in adults (11)(12)(13)(14). In humans, CYP24A1 mediates hydroxylation of the vitamin D side chain at either carbon-23 (C23) or carbon-24 (C24), then mediates all subsequent steps in each pathway to produce either the metabolite 1,25(OH) 2 D3-26,23-lactone or calcitroic acid from the C23 or C24 pathways, respectively (15)(16)(17). While calcitroic acid is excreted in urine, 1,25(OH) 2 D3-26, 23-lactone has been demonstrated to modulate vitamin D signaling by binding the vitamin D receptor (18,19).…”

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The cytochrome P450 24A1 interaction with adrenodoxin relies on multiple recognition sites that vary among species

Estrada

2018

Journal of Biological Chemistry

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Mitochondrial cytochromes P450 (P450s) are responsible for important metabolic reactions, including steps involved in steroid and vitamin D metabolism. The mitochondrial P450 24A1 (CYP24A1) is responsible for deactivation of the bioactive form of vitamin D, 1,25(OH) 2 D3. Its function relies on formation of a P450-redox partner complex with the ferredoxin and electron donor adrenodoxin (Adx). However, very little is known about how the Adx-CYP24A1 complex forms. In this study, we report the results of solution NMR in which we monitor isotopically labeled full-length Adx as it binds CYP24A1 in complex with the P450 inhibitor clotrimazole. The NMR titration data suggested a mode for P450-Adx interactions in which formation of the complex relies on contributions from multiple recognition sites on the Adx core domain, some of which have not previously been reported. To evaluate differences among CYP24A1-Adx complexes from different mammalian species and displaying distinct regioselectivity for 1,25(OH) 2 D3, all bound spectra were acquired in parallel for human (carbon-23 and -24 hydroxylase), rat (carbon-24 hydroxylase), and opossum (carbon-23 hydroxylase) CYP24A1 isoforms. Binding data from a series of single and double charge-neutralizing substitutions of Adx confirmed that species-specific CYP24A1 isoforms differ in binding to Adx, providing evidence that variations in redox partner interactions correlate with P450 regioselectivity. In summary, these findings reveal that CYP24A1-Adx interactions rely on several recognition sites and that variations in CYP24A1 isoforms modulate formation of the complex, thus providing insight into the variable and complex nature of mitochondrial P450-Adx interactions.Mitochondrial cytochromes P450 (P450) are responsible for a host of biological reactions, including steps necessary in steroid and vitamin D metabolism. Similar to microsomal P450s, their function requires the transfer of two electrons, delivered sequentially, in order to generate the active oxidizing species necessary for completion of one cycle of P450 catalysis (1). However, in contrast to microsomal P450 enzymes, for which reduction of P450 can be achieved by binding either a P450 oxidoreductase or, in some cases, the small heme protein cytochrome b 5 , mitochondrial enzymes rely entirely on formation of a transient complex with the soluble ferredoxin protein, Adrenodoxin (Adx). Adx folds into a compact structure consisting of three short α-helices and five anti-parallel β strands, with a [2Fe-2S] cluster coordinated near solvent accessible surfaces between helix-1 and helix-3 (2,3). Changes in the C-terminal region of bovine Adx have been shown to participate in mediating a monomer to dimer transition in response to reduction of the [2Fe-2S] cluster (2), thereby suggesting that dimers of Adx are functionally relevant.While the prevailing evidence suggests that complex formation between Adx with P450 relies on salt bridge interactions between the side chains of acidic residues on helix-3 (Asp-72 - Cytoch...

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Dual metabolic pathway of 25‐hydroxyvitamin D₃ catalyzed by human CYP24

Cited by 172 publications

References 26 publications

Regulation of CYP27B1 and CYP24A1 hydroxylases limits cell‐autonomous activation of vitamin D in dendritic cells

Regulation of CYP27B1 and CYP24A1 hydroxylases limits cell‐autonomous activation of vitamin D in dendritic cells

Hybrid homology modeling and mutational analysis of cytochrome P450C24A1 (CYP24A1) of the Vitamin D pathway: Insights into substrate specificity and membrane bound structure–function

The cytochrome P450 24A1 interaction with adrenodoxin relies on multiple recognition sites that vary among species

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Dual metabolic pathway of 25‐hydroxyvitamin D3 catalyzed by human CYP24

Cited by 172 publications

References 26 publications

Regulation of CYP27B1 and CYP24A1 hydroxylases limits cell‐autonomous activation of vitamin D in dendritic cells

Regulation of CYP27B1 and CYP24A1 hydroxylases limits cell‐autonomous activation of vitamin D in dendritic cells

Hybrid homology modeling and mutational analysis of cytochrome P450C24A1 (CYP24A1) of the Vitamin D pathway: Insights into substrate specificity and membrane bound structure–function

The cytochrome P450 24A1 interaction with adrenodoxin relies on multiple recognition sites that vary among species

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Dual metabolic pathway of 25‐hydroxyvitamin D₃ catalyzed by human CYP24