Evidence of Allosteric Coupling between Substrate Binding and Adx Recognition in the Vitamin D Carbon-24 Hydroxylase CYP24A1.
Ontology highlight
ABSTRACT:
SUBMITTER: Kumar A
PROVIDER: S-EPMC7233526 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Calcitriol, a regulator of calcium homeostasis with antitumor properties, is degraded by the product of the CYP24A1 gene, which is downregulated in human prostate cancer by unknown mechanisms. We found that CYP24A1 expression is inversely correlated with promoter DNA methylation in prostate cancer cell lines. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (DAC) activates CYP24A1 expression in prostate cancer cells. In vitro methylation of the CYP24A1 promoter represses its promoter activity. Furthermore, inhibition of histone deacetylases by trichostatin A (TSA) enhances the expression of CYP24A1 in prostate cancer cells. Quantitative chromatin immunoprecipitation-PCR (ChIP-qPCR) reveals that specific histone modifications are associated with the CYP24A1 promoter region. Treatment with TSA increases H3K9ac and H3K4me2 and simultaneously decreases H3K9me2 at the CYP24A1 promoter. ChIP-qPCR assay reveals that treatment with DAC and TSA increases the recruitment of vitamin D receptor to the CYP24A1 promoter. Reverse transcriptase-PCR analysis of paired human prostate samples revealed that CYP24A1 expression is downregulated in prostate malignant lesions compared with adjacent histologically benign lesions. Bisulfite pyrosequencing shows that CYP24A1 gene is hypermethylated in malignant lesions compared with matched benign lesions. Our findings indicate that repression of CYP24A1 gene expression in human prostate cancer cells is mediated in part by promoter DNA methylation and repressive histone modifications.
| S-EPMC2928678 | biostudies-literature
Project description:ContextIdiopathic infantile hypercalcemia (IIH) is a disorder the genetic etiology and physiological basis of which are not well understood.ObjectiveThe objective of the study was to describe the underlying physiology and genetic cause of hypercalcemia in an infant with severe IIH and to extend these genetic findings into an additional cohort of children with IIH.DesignThis was an inpatient study of a single patient with consanguineous parents at an academic medical center with follow-up in a specialty clinic cohort.PatientsThe patient population was one patient with severe IIH for gene discovery and physiological testing and 27 patients with idiopathic infantile hypercalcemia in the replication cohort.InterventionsInterventions included a calcium isotopic absorption study as well as homozygosity mapping and whole-exome sequencing in a single patient followed up by gene sequencing in replication cohort.Main outcome measureFractional absorption of calcium and genetic variants causing hypercalcemia were measured.ResultsIntestinal calcium absorption was extremely elevated (?90%). A rare homozygous deletion in the CYP24A1 gene was found, leading to the loss of a single highly conserved amino acid. In vivo functional studies confirmed decreased 24-hydroxylase activity because the subject had undetectable levels of 24,25-dihydroxyvitamin D. No coding variants in CYP24A1 were found in the 27 additional patients with IIH.ConclusionsOur study confirms that CYP24A1 plays a causal role in some but not all cases of IIH via markedly increased intestinal absorption of calcium, suggesting that genetic diagnosis could be helpful in a subset of IIH patients. This case demonstrates the power of an unbiased, genome-wide approach accompanied by informative physiological studies to provide new insights into human biology.
| S-EPMC3275367 | biostudies-literature
Project description:Epigenetic alterations occur in tumor-associated vessels in the tumor microenvironment. Methylation of the CYP24A1 gene promoter differs in endothelial cells isolated from tumors and non-tumor microenvironments in mice. The epigenetic makeup of endothelial cells of human tumor-associated vasculature is unknown due to difficulty of isolating endothelial cells populations from a heterogeneous tissue microenvironment. To ascertain CYP24A1 promoter methylation in tumor-associated endothelium, we utilized laser microdissection guided by CD31 immunohistochemistry to procure endothelial cells from human prostate tumor specimens. Prostate tissues were obtained following robotic radical prostatectomy from men with clinically localized prostate cancer. Adjacent histologically benign prostate tissues were used to compare endothelium from benign versus tumor microenvironments. Sodium bisulfite sequencing of CYP24A1 promoter region showed that the average CYP24A1 promoter methylation in the endothelium was 20% from the tumor microenvironment compared with 8.2% in the benign microenvironment (p< 0.05). A 2-fold to 17-fold increase in CYP24A1 promoter methylation was observed in the prostate tumor endothelium compared with the matched benign prostate endothelium in four patient samples, while CYP24A1 remained unchanged in two patient sample. In addition, there is no correlation of the level of CYP24A1 promoter methylation in prostate tumor-associated endothelium with that of epithelium/stroma. This study demonstrates that the CYP24A1 promoter is methylated in tumor-associated endothelium, indicating that epigenetic alterations in CYP24A1 may play a role in determining the phenotype of tumor-associated vasculature in the prostate tumor microenvironment.
| S-EPMC3219083 | biostudies-other
Project description:Elevated serum vitamin D with hypercalciuria can result in nephrocalcinosis and nephrolithiasis. This study evaluated the cause of excess 1,25-dihydroxycholecalciferol (1?,25(OH)2D3) in the development of those disorders in two individuals.Two patients with elevated vitamin D levels and nephrocalcinosis or nephrolithiasis were investigated at the National Institutes of Health (NIH) Clinical Center and the NIH Undiagnosed Diseases Program, by measuring calcium, phosphate, and vitamin D metabolites, and by performing CYP24A1 mutation analysis.Both patients exhibited hypercalciuria, hypercalcemia, low parathyroid hormone, elevated vitamin D (1?,25(OH)2D3), normal 25-OHD3, decreased 24,25(OH)2D, and undetectable activity of 1,25(OH)2D-24-hydroxylase (CYP24A1), the enzyme that inactivates 1?,25(OH)2D3. Both patients had bi-allelic mutations in CYP24A1 leading to loss of function of this enzyme. On the basis of dbSNP data, the frequency of predicted deleterious bi-allelic CYP24A1 variants in the general population is estimated to be as high as 4%-20%.The results of this study show that 1,25(OH)2D-24-hydroxylase deficiency due to bi-allelic mutations in CYP24A1 causes elevated serum vitamin D, hypercalciuria, nephrocalcinosis, and renal stones.
| S-EPMC3613951 | biostudies-literature
Project description:Studies of 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24A1) have demonstrated that it is a bifunctional enzyme capable of the 24-hydroxylation of 1alpha,25-(OH)(2)D(3), leading to the excretory form, calcitroic acid, and 23-hydroxylation, culminating in 1alpha,25-(OH)(2)D(3)-26,23-lactone. The degree to which CYP24A1 performs either 23- or 24-hydroxylation is species-dependent. In this paper, we show that the human enzyme that predominantly 24-hydroxylates its substrate differs from the opossum enzyme that 23-hydroxylates it at only a limited number of amino acid residues. Mutagenesis of the human form at a single substrate-binding residue (A326G) dramatically changes the regioselectivity of the enzyme from a 24-hydroxylase to a 23-hydroxylase, whereas other modifications have no effect. Ala-326 is located in the I-helix, close to the terminus of the docked 25-hydroxylated side chain in a CYP24A1 homology model, a result that we interpret indicates that substitution of a glycine at 326 provides extra space for the side chain of the substrate to move deeper into the pocket and place it in a optimal stereochemical position for 23-hydroxylation. We discuss the physiological ramifications of these results for species possessing the A326G substitution, as well as implications for optimal vitamin D analog design.
| S-EPMC1937525 | biostudies-literature
Project description:CYP24A1 is a mitochondrial cytochrome P450 (CYP) that catabolizes 1?,25-dihydroxyvitamin D(3) (1?,25-(OH)(2)D(3)) to different products: calcitroic acid or 1?,25-(OH)(2)D(3)-26,23-lactone via multistep pathways commencing with C24 and C23 hydroxylation, respectively. Despite the ability of CYP24A1 to catabolize a wide range of 25-hydroxylated analogs including 25-hydroxyvitamin D(3), the enzyme is unable to metabolize the synthetic prodrug, 1?-hydroxyvitamin D(3) (1?-OH-D(3)), presumably because it lacks a C25-hydroxyl. In the current study we show that a single V391L amino acid substitution in the ?3a-strand of human CYP24A1 converts this enzyme from a catabolic 1?,25-(OH)(2)D(3)-24-hydroxylase into an anabolic 1?-OH-D(3)-25-hydroxylase, thereby forming the hormone, 1?,25-(OH)(2)D(3). Furthermore, because the mutant enzyme retains its basal ability to catabolize 1?,25-(OH)(2)D(3) via C24 hydroxylation, it can also make calcitroic acid. Previous work has shown that an A326G mutation is responsible for the regioselectivity differences observed between human (primarily C24-hydroxylating) and opossum (C23-hydroxylating) CYP24A1. When the V391L and A326G mutations were combined (V391L/A326G), the mutant enzyme continued to form 1?,25-(OH)(2)D(3) from 1?-OH-D(3), but this initial product was diverted via the C23 hydroxylation pathway into the 26,23-lactone. The relative position of Val-391 in the ?3a-strand of a homology model and the crystal structure of rat CYP24A1 is consistent with hydrophobic contact of Val-391 and the substrate side chain near C21. We interpret that the substrate specificity of V391L-modified human CYP24A1 toward 1?-OH-D(3) is enabled by an altered contact with the substrate side chain that optimally positions C25 of the 1?-OH-D(3) above the heme for hydroxylation.
| S-EPMC3190681 | biostudies-literature
Project description:?-Butyrobetaine hydroxylase (BBOX) is a non-heme Fe(II) - and 2-oxoglutarate-dependent oxygenase that catalyzes the stereoselective hydroxylation of an unactivated C-H bond of ?-butyrobetaine (?BB) in the final step of carnitine biosynthesis. BBOX contains an aromatic cage for the recognition of the positively charged trimethylammonium group of the ?BB substrate. Enzyme binding and kinetic analyses on substrate analogues with P and As substituting for N in the trimethylammonium group show that the analogues are good BBOX substrates, which follow the efficiency trend N(+) >P(+) >As(+). The results reveal that an uncharged carbon analogue of ?BB is not a BBOX substrate, thus highlighting the importance of the energetically favorable cation-? interactions in productive substrate recognition.
| S-EPMC4736438 | biostudies-literature
Project description:Multiple sclerosis (MS) is a common disease of the central nervous system and a major cause of disability amongst young adults. Genome-wide association studies have identified many novel susceptibility loci including rs2248359. We hypothesized that genotypes of this locus could increase the risk of MS by regulating expression of neighboring gene, CYP24A1 which encodes the enzyme responsible for initiating degradation of 1,25-dihydroxyvitamin D3.We investigated this hypothesis using paired gene expression and genotyping data from three independent datasets of neurologically healthy adults of European descent. The UK Brain Expression Consortium (UKBEC) consists of post-mortem samples across 10 brain regions originating from 134 individuals (1231 samples total). The North American Brain Expression Consortium (NABEC) consists of cerebellum and frontal cortex samples from 304 individuals (605 samples total). The brain dataset from Heinzen and colleagues consists of prefrontal cortex samples from 93 individuals. Additionally, we used gene network analysis to analyze UKBEC expression data to understand CYP24A1 function in human brain.The risk allele, rs2248359-C, is strongly associated with increased expression of CYP24A1 in frontal cortex (p-value=1.45×10-13), but not white matter. This association was replicated using data from NABEC (p-value=7.2×10-6) and Heinzen and colleagues (p-value=1.2×10-4). Network analysis shows a significant enrichment of terms related to immune response in eight out of the 10 brain regions.The known MS risk allele rs2248359-C increases CYP24A1 expression in human brain providing a genetic link between MS and vitamin D metabolism, and predicting that the physiologically active form of vitamin D3 is protective. Vitamin D3's involvement in MS may relate to its immunomodulatory functions in human brain.Medical Research Council UK; King Faisal Specialist Hospital and Research Centre, Saudi Arabia; Intramural Research Program of the National Institute on Aging, National Institutes of Health, USA.
| S-EPMC4278441 | biostudies-literature
Project description:The specificity of SCF ubiquitin ligase-mediated protein degradation is determined by F-box proteins. We identified a biplanar dicarboxylic acid compound, called SCF-I2, as an inhibitor of substrate recognition by the yeast F-box protein Cdc4 using a fluorescence polarization screen to monitor the displacement of a fluorescein-labeled phosphodegron peptide. SCF-I2 inhibits the binding and ubiquitination of full-length phosphorylated substrates by SCF(Cdc4). A co-crystal structure reveals that SCF-I2 inserts itself between the beta-strands of blades 5 and 6 of the WD40 propeller domain of Cdc4 at a site that is 25 A away from the substrate binding site. Long-range transmission of SCF-I2 interactions distorts the substrate binding pocket and impedes recognition of key determinants in the Cdc4 phosphodegron. Mutation of the SCF-I2 binding site abrogates its inhibitory effect and explains specificity in the allosteric inhibition mechanism. Mammalian WD40 domain proteins may exhibit similar allosteric responsiveness and hence represent an extensive class of druggable target.
| S-EPMC4445864 | biostudies-literature
Project description:Escherichia coli chaperonin, GroEL, helps proteins fold under nonpermissive conditions. During the reaction cycle, GroEL undergoes allosteric transitions in response to binding of a substrate protein (SP), ATP, and the cochaperonin GroES. Using coarse-grained representations of the GroEL and GroES structures, we explore the link between allosteric transitions and the folding of a model SP, a de novo-designed four-helix bundle protein, with low spontaneous yield. The ensemble of GroEL-bound SP is less structured than the bulk misfolded structures. Upon binding, which kinetically occurs in two stages, the SP loses not only native tertiary contacts but also experiences a decrease in helical content. During multivalent binding and the subsequent ATP-driven transition of GroEL the SP undergoes force-induced stretching. Upon encapsulation, which occurs upon GroES binding, the SP finds itself in a "hydrophilic" cavity in which it can reach the folded conformation. Surprisingly, we find that the yield of the native state in the expanded GroEL cavity is relatively small even after it remains in it for twice the spontaneous folding time. Thus, in accord with the iterative annealing mechanism, multiple rounds of binding, partial unfolding, and release of the SP are required to enhance the yield of the folded SP.
| S-EPMC1885583 | biostudies-literature