ABSTRACT
For HIV to become infectious, any new virion produced from an infected cell must undergo a maturation process that involves the assembly of viral polyproteins Gag and Gag-Pol at the membrane surface. The self-assembly of these viral proteins drives formation of a new viral particle as well as the activation of HIV protease, which is needed to cleave the polyproteins so that the final core structure of the virus will properly form. Molecules that interfere with HIV maturation will prevent any new virions from infecting additional cells. In this manuscript, we characterize the unique mechanism by which a mercaptobenzamide thioester small molecule (SAMT-247) interferes with HIV maturation via a series of selective acetylations at highly conserved cysteine and lysine residues in Gag and Gag-Pol polyproteins. The results provide the first insights into how acetylation can be utilized to perturb the process of HIV maturation and reveal a new strategy to limit the infectivity of HIV.
Subject(s)
Anti-HIV Agents/pharmacology , Benzamides/pharmacology , HIV/drug effects , Protein Unfolding/drug effects , Virus Assembly/drug effects , gag Gene Products, Human Immunodeficiency Virus/drug effects , Acetylation , Amino Acid Sequence , Cell Line , Cysteine/chemistry , Fusion Proteins, gag-pol/chemistry , Fusion Proteins, gag-pol/drug effects , Humans , Lysine/chemistry , gag Gene Products, Human Immunodeficiency Virus/chemistryABSTRACT
Short chain fatty acids (SCFAs) play a central role in health and disease. One function of these signaling molecules is to serve as precursors for short chain fatty acylation, a class of metabolically-derived posttranslational modifications (PTMs) that are established by lysine acetyltransferases (KATs) and lysine deacetylases (KDACs). Via this mechanism, short chain fatty acylation serves as an integrated reporter of metabolism as well as KAT and KDAC activity, and has the potential to illuminate the role of these processes in disease. However, few methods to study short chain fatty acylation exist. Here we report a bioorthogonal pro-metabolite strategy for profiling short chain fatty acylation in living cells. Inspired by the dietary component tributyrin, we synthesized a panel of ester-caged bioorthogonal short chain fatty acids. Cellular evaluation of these agents led to the discovery of an azido-ester that is metabolized to its cognate acyl-coenzyme A (CoA) and affords robust protein labeling profiles. We comprehensively characterize the metabolic dependence, toxicity, and histone deacetylase (HDAC) inhibitor sensitivity of these bioorthogonal pro-metabolites, and apply an optimized probe to identify novel candidate protein targets of short chain fatty acids in cells. Our studies showcase the utility of bioorthogonal pro-metabolites for unbiased profiling of cellular protein acylation, and suggest new approaches for studying the signaling functions of SCFAs in differentiation and disease.
ABSTRACT
Replication foci are generated by many viruses to concentrate and localize viral DNA synthesis to specific regions of the cell. Expression of the HPV16 E1 and E2 replication proteins in keratinocytes results in nuclear foci that recruit proteins associated with the host DNA damage response. We show that the Brd4 protein localizes to these foci and is essential for their formation. However, when E1 and E2 begin amplifying viral DNA, Brd4 is displaced from the foci and cellular factors associated with DNA synthesis and homologous recombination are recruited. Differentiated HPV-infected keratinocytes form similar nuclear foci that contain amplifying viral DNA. We compare the different foci and show that, while they have many characteristics in common, there is a switch between early Brd4-dependent foci and mature Brd4-independent replication foci. However, HPV genomes encoding mutated E2 proteins that are unable to bind Brd4 can replicate and amplify the viral genome. We propose that, while E1, E2 and Brd4 might bind host chromatin at early stages of infection, there is a temporal and functional switch at later stages and increased E1 and E2 levels promote viral DNA amplification, displacement of Brd4 and growth of a replication factory. The concomitant DNA damage response recruits proteins required for DNA synthesis and repair, which could then be utilized for viral DNA replication. Hence, while Brd4 can enhance replication by concentrating viral processes in specific regions of the host nucleus, this interaction is not absolutely essential for HPV replication.
Subject(s)
DNA Replication/physiology , DNA, Viral/biosynthesis , Genome, Viral/physiology , Human papillomavirus 16/physiology , Nuclear Proteins/metabolism , Papillomavirus Infections/metabolism , Transcription Factors/metabolism , Virus Replication/physiology , Cell Cycle Proteins , Cell Line , Chromatin/genetics , Chromatin/metabolism , Chromatin/virology , DNA, Viral/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , Nuclear Proteins/genetics , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , Papillomavirus Infections/genetics , Transcription Factors/geneticsABSTRACT
Cytochrome P450 2E1 (CYP2E1) is a key enzyme in the metabolic activation of many low molecular weight toxicants and also an important contributor to oxidative stress. A noninvasive method to monitor CYP2E1 activity in vivo would be of great value for studying the role of CYP2E1 in chemical-induced toxicities and stress-related diseases. In this study, a mass spectrometry-based metabolomic approach was used to identify a metabolite biomarker of CYP2E1 through comparing the urine metabolomes of wild-type (WT), Cyp2e1-null, and CYP2E1-humanized mice. Metabolomic analysis with multivariate models of urine metabolites revealed a clear separation of Cyp2e1-null mice from WT and CYP2E1-humanized mice in the multivariate models of urine metabolomes. Subsequently, 2-piperidone was identified as a urinary metabolite that inversely correlated to the CYP2E1 activity in the three mouse lines. Backcrossing of WT and Cyp2e1-null mice, together with targeted analysis of 2-piperidone in mouse serum, confirmed the genotype dependency of 2-piperidone. The accumulation of 2-piperidone in the Cyp2e1-null mice was mainly caused by the changes in the biosynthesis and degradation of 2-piperidone because compared with the WT mice, the conversion of cadaverine to 2-piperidone was higher, whereas the metabolism of 2-piperidone to 6-hydroxy-2-piperidone was lower in the Cyp2e1-null mice. Overall, untargeted metabolomic analysis identified a correlation between 2-piperidone concentrations in urine and the expression and activity of CYP2E1, thus providing a noninvasive metabolite biomarker that can be potentially used in to monitor CYP2E1 activity.
Subject(s)
Cytochrome P-450 CYP2E1/metabolism , Metabolomics/methods , Piperidones/urine , Animals , Biomarkers , Cadaverine/metabolism , Female , Mice , Phenotype , Tandem Mass SpectrometryABSTRACT
The pregnane X receptor (PXR) has been postulated to play a role in the metabolism of α-tocopherol owing to the up-regulation of hepatic cytochrome P450 (P450) 3A in human cell lines and murine models after α-tocopherol treatment. However, in vivo studies confirming the role of PXR in α-tocopherol metabolism in humans presents significant difficulties and has not been performed. PXR-humanized (hPXR), wild-type, and Pxr-null mouse models were used to determine whether α-tocopherol metabolism is influenced by species-specific differences in PXR function in vivo. No significant difference in the concentration of the major α-tocopherol metabolites was observed among the hPXR, wild-type, and Pxr-null mice through mass spectrometry-based metabolomics. Gene expression analysis revealed significantly increased expression of Cyp3a11 as well as several other P450s only in wild-type mice, suggesting species-specificity for α-tocopherol activation of PXR. Luciferase reporter assay confirmed activation of mouse PXR by α-tocopherol. Analysis of the Cyp2c family of genes revealed increased expression of Cyp2c29, Cyp2c37, and Cyp2c55 in wild-type, hPXR, and Pxr-null mice, which suggests PXR-independent induction of Cyp2c gene expression. This study revealed that α-tocopherol is a partial agonist of PXR and that PXR is necessary for Cyp3a induction by α-tocopherol. The implications of a novel role for α-tocopherol in Cyp2c gene regulation are also discussed.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Liver/drug effects , Receptors, Steroid/drug effects , Receptors, Steroid/metabolism , alpha-Tocopherol/pharmacology , Animals , Biomarkers/urine , Biotransformation , Chromatography, Liquid , Cytochrome P-450 CYP3A/metabolism , Cytochrome P-450 Enzyme System/genetics , Drug Partial Agonism , Gene Expression Regulation, Enzymologic/drug effects , Genes, Reporter , Hep G2 Cells , Humans , Isoenzymes , Liver/enzymology , Male , Mass Spectrometry , Metabolomics/methods , Mice , Mice, Knockout , Mice, Transgenic , Pregnane X Receptor , Receptors, Steroid/deficiency , Receptors, Steroid/genetics , Species Specificity , Time Factors , Transfection , alpha-Tocopherol/urineABSTRACT
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is among the most potent environmentally toxic compounds. Serum metabolomics identified azelaic acid monoesters as significantly increased metabolites after TCDD treatment, due to downregulation of hepatic carboxylesterase 3 (CES3, also known as triglyceride hydrolase) expression in an arylhydrocarbon receptor (AhR)-dependent manner in mice. The decreased CES3 expression was accomplished by TCDD-stimulated TGFß-SMAD3 and IL6-STAT3 signaling, but not by direct AhR signaling. Methionine- and choline-deficient (MCD) diet-treated mice also showed enhanced serum azelaic acid monoester levels after attenuation of hepatic CES3 expression, while db/db mice did not, thus suggesting an association with steatohepatitis. Forced expression of CES3 reversed serum azelaic acid monoester/azelaic acid ratios and hepatic TGFß mRNA levels in TCDD- and MCD diet-treated mice and ameliorated steatohepatitis induced by MCD diet. These results support the view that azelaic acid monoesters are possible indicators of TCDD exposure and steatohepatitis and suggest a link between CES3, TGFß, and steatohepatitis.
Subject(s)
Diet , Fatty Liver/metabolism , Metabolomics , Animals , Carboxylic Ester Hydrolases/metabolism , Cells, Cultured , Dicarboxylic Acids/blood , Dicarboxylic Acids/metabolism , Fatty Liver/chemically induced , Hepatocytes/cytology , Hepatocytes/metabolism , Inflammation/metabolism , Interleukin-6/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Obese , Polychlorinated Dibenzodioxins/toxicity , Receptors, Aryl Hydrocarbon/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction , Transforming Growth Factor beta/metabolismABSTRACT
Transforming growth factor-ß (TGFß) is activated as a result of liver injury, such as cholestasis. However, its influence on endogenous metabolism is not known. This study demonstrated that TGFß regulates hepatic phospholipid and bile acid homeostasis through MAD homolog 3 (SMAD3) activation as revealed by lithocholic acid-induced experimental intrahepatic cholestasis. Lithocholic acid (LCA) induced expression of TGFB1 and the receptors TGFBR1 and TGFBR2 in the liver. In addition, immunohistochemistry revealed higher TGFß expression around the portal vein after LCA exposure and diminished SMAD3 phosphorylation in hepatocytes from Smad3-null mice. Serum metabolomics indicated increased bile acids and decreased lysophosphatidylcholine (LPC) after LCA exposure. Interestingly, in Smad3-null mice, the metabolic alteration was attenuated. LCA-induced lysophosphatidylcholine acyltransferase 4 (LPCAT4) and organic solute transporter ß (OSTß) expression were markedly decreased in Smad3-null mice, whereas TGFß induced LPCAT4 and OSTß expression in primary mouse hepatocytes. In addition, introduction of SMAD3 enhanced the TGFß-induced LPCAT4 and OSTß expression in the human hepatocellular carcinoma cell line HepG2. In conclusion, considering that Smad3-null mice showed attenuated serum ALP activity, a diagnostic indicator of cholangiocyte injury, these results strongly support the view that TGFß-SMAD3 signaling mediates an alteration in phospholipid and bile acid metabolism following hepatic inflammation with the biliary injury.
Subject(s)
Bile Acids and Salts/metabolism , Liver/metabolism , Phospholipids/metabolism , Signal Transduction , Smad3 Protein/metabolism , Transforming Growth Factor beta/metabolism , Animals , Cells, Cultured , Hep G2 Cells , Hepatocytes/chemistry , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Lithocholic Acid , Liver/drug effects , Liver/injuries , Male , Mice , Mice, Inbred C57BL , Mice, NudeABSTRACT
BACKGROUND: Contradictory results from clinical trials that examined the role of vitamin E in chronic disease could be a consequence of interindividual variation, caused by factors such as xenobiotic use. Cometabolism of vitamin E with other pharmaceutical products could affect the bioavailability of the drug. Thus, it is necessary to understand fully the metabolic routes and biological endpoints of vitamin E. OBJECTIVE: The objective was to uncover novel metabolites and roles of vitamin E in humans and mouse models. DESIGN: Human volunteers (n = 10) were fed almonds for 7 d and then an α-tocopherol dietary supplement for 14 d. Urine and serum samples were collected before and after dosing. C57BL/6 mice (n = 10) were also fed α-tocopherol-deficient and -enriched diets for 14 d. Urine, serum, and feces were collected before and after dosing, and liver samples were collected after euthanization. Ultraperformance liquid chromatography electrospray ionization time-of-flight mass spectrometry and multivariate data analysis tools were used to analyze the samples. RESULTS: Three novel urinary metabolites of α-tocopherol were discovered in humans and mice: α-carboxyethylhydroxychroman (α-CEHC) glycine, α-CEHC glycine glucuronide, and α-CEHC taurine. Another urinary metabolite, α-CEHC glutamine, was discovered in mice after α-CEHC gavage. Increases in liver fatty acids and decreases in serum and liver cholesterol were observed in mice fed the α-tocopherol-enriched diet. CONCLUSION: Novel metabolites and metabolic pathways of vitamin E were identified by mass spectrometry-based metabolomics and will aid in understanding the disposition and roles of vitamin E in vivo.
Subject(s)
Chromans/metabolism , alpha-Tocopherol/metabolism , Adolescent , Adult , Amino Acids/chemistry , Amino Acids/urine , Animals , Cholesterol/blood , Cholesterol/metabolism , Chromans/administration & dosage , Chromans/chemistry , Chromans/urine , Chromatography, High Pressure Liquid , Female , Glucuronides/chemistry , Glucuronides/urine , Humans , Liver/metabolism , Male , Metabolomics/methods , Mice , Mice, Inbred C57BL , Molecular Structure , Species Specificity , Spectrometry, Mass, Electrospray Ionization , Taurine/analogs & derivatives , Taurine/chemistry , Taurine/urine , Vitamin E Deficiency/blood , Vitamin E Deficiency/metabolism , Vitamin E Deficiency/urine , Young Adult , alpha-Tocopherol/administration & dosageABSTRACT
Mass spectrometry-based metabolomics has previously demonstrated utility for identifying biomarkers of ionizing radiation exposure in cellular, mouse and rat in vivo radiation models. To provide a valuable link from small laboratory rodents to humans, γ-radiation-induced urinary biomarkers were investigated using a nonhuman primate total-body-irradiation model. Mass spectrometry-based metabolomics approaches were applied to determine whether biomarkers could be identified, as well as the previously discovered rodent biomarkers of γ radiation. Ultra-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry analysis was carried out on a time course of clean-catch urine samples collected from nonhuman primates (n = 6 per cohort) exposed to sham, 1.0, 3.5, 6.5 or 8.5 Gy doses of (60)Co γ ray (â¼0.55 Gy/min) ionizing radiation. By multivariate data analysis, 13 biomarkers of radiation were discovered: N-acetyltaurine, isethionic acid, taurine, xanthine, hypoxanthine, uric acid, creatine, creatinine, tyrosol sulfate, 3-hydroxytyrosol sulfate, tyramine sulfate, N-acetylserotonin sulfate, and adipic acid. N-Acetyltaurine, isethionic acid, and taurine had previously been identified in rats, and taurine and xanthine in mice after ionizing radiation exposure. Mass spectrometry-based metabolomics has thus successfully revealed and verified urinary biomarkers of ionizing radiation exposure in the nonhuman primate for the first time, which indicates possible mechanisms for ionizing radiation injury.
Subject(s)
Biomarkers/urine , Metabolomics , Radiation Injuries/urine , Spectrometry, Mass, Electrospray Ionization/methods , Animals , Area Under Curve , Dose-Response Relationship, Radiation , Female , Gamma Rays , Macaca mulatta , Male , Mice , RatsABSTRACT
Radiation metabolomics has aided in the identification of a number of biomarkers in cells and mice by ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) and in rats by gas chromatography-coupled mass spectrometry (GCMS). These markers have been shown to be both dose- and time-dependent. Here UPLC-ESI-QTOFMS was used to analyze rat urine samples taken from 12 rats over 7 days; they were either sham-irradiated or γ-irradiated with 3 Gy after 4 days of metabolic cage acclimatization. Using multivariate data analysis, nine urinary biomarkers of γ radiation in rats were identified, including a novel mammalian metabolite, N-acetyltaurine. These upregulated urinary biomarkers were confirmed through tandem mass spectrometry and comparisons with authentic standards. They include thymidine, 2'-deoxyuridine, 2'deoxyxanthosine, N(1)-acetylspermidine, N-acetylglucosamine/galactosamine-6-sulfate, N-acetyltaurine, N-hexanoylglycine, taurine and, tentatively, isethionic acid. Of these metabolites, 2'-deoxyuridine and thymidine were previously identified in the rat by GCMS (observed as uridine and thymine) and in the mouse by UPLC-ESI-QTOFMS. 2'Deoxyxanthosine, taurine and N-hexanoylglycine were also seen in the mouse by UPLC-ESI-QTOFMS. These are now unequivocal cross-species biomarkers for ionizing radiation exposure. Downregulated biomarkers were shown to be related to food deprivation and starvation mechanisms. The UPLC-ESI-QTOFMS approach has aided in the advance for finding common biomarkers of ionizing radiation exposure.
Subject(s)
Biomarkers/urine , Metabolome/radiation effects , Proteome/analysis , Proteome/metabolism , Whole-Body Irradiation/adverse effects , Animals , Gamma Rays , Male , Radiation Dosage , Rats , Rats, WistarABSTRACT
We modified a series of (N)-methanocarba nucleoside 5'-uronamides to contain dialkyne groups on an extended adenine C2 substituent, as synthetic intermediates leading to potent and selective A(3) adenosine receptor (AR) agonists. The proximal alkyne was intended to promote receptor recognition, and the distal alkyne reacted with azides to form triazole derivatives (click cycloaddition). Click chemistry was utilized to couple an octadiynyl A(3)AR agonist to azido-containing fluorescent, chemically reactive, biotinylated, and other moieties with retention of selective binding to the A(3)AR. A bifunctional thiol-reactive crosslinking reagent was introduced. The most potent and selective novel compound was a 1-adamantyl derivative (K(i) 6.5nM), although some of the click products had K(i) values in the range of 200-400nM. Other potent, selective derivatives (K(i) at A(3)AR innM) were intended as possible receptor affinity labels: 3-nitro-4-fluorophenyl (10.6), alpha-bromophenacyl (9.6), thiol-reactive isothiazolone (102), and arylisothiocyanate (37.5) derivatives. The maximal functional effects in inhibition of forskolin-stimulated cAMP were measured, indicating that this class of click adducts varied from partial to full A(3)AR agonist compared to other widely used agonists. Thus, this strategy provides a general chemical approach to linking potent and selective A(3)AR agonists to reporter groups of diverse structure and to carrier moieties.
Subject(s)
Adenosine A3 Receptor Agonists , Alkynes/pharmacology , Nucleosides/pharmacology , Alkynes/chemical synthesis , Alkynes/chemistry , Animals , CHO Cells , Cells, Cultured , Cricetinae , Cricetulus , Drug Design , Humans , Molecular Structure , Nucleosides/chemical synthesis , Nucleosides/chemistry , Receptor, Adenosine A3/metabolism , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Farnesoid X receptor (FXR) is a nuclear receptor that regulates genes involved in synthesis, metabolism, and transport of bile acids and thus plays a major role in maintaining bile acid homeostasis. In this study, metabolomic responses were investigated in urine of wild-type and Fxr-null mice fed cholic acid, an FXR ligand, using ultra-performance liquid chromatography (UPLC) coupled with electrospray time-of-flight mass spectrometry (TOFMS). Multivariate data analysis between wild-type and Fxr-null mice on a cholic acid diet revealed that the most increased ions were metabolites of p-cresol (4-methylphenol), corticosterone, and cholic acid in Fxr-null mice. The structural identities of the above metabolites were confirmed by chemical synthesis and by comparing retention time (RT) and/or tandem mass fragmentation patterns of the urinary metabolites with the authentic standards. Tauro-3alpha,6,7alpha,12alpha-tetrol (3alpha,6,7alpha,12alpha-tetrahydroxy-5beta-cholestan-26-oyltaurine), one of the most increased metabolites in Fxr-null mice on a CA diet, is a marker for efficient hydroxylation of toxic bile acids possibly through induction of Cyp3a11. A cholestatic model induced by lithocholic acid revealed that enhanced expression of Cyp3a11 is the major defense mechanism to detoxify cholestatic bile acids in Fxr-null mice. These results will be useful for identification of biomarkers for cholestasis and for determination of adaptive molecular mechanisms in cholestasis.
Subject(s)
Adaptation, Physiological , Bile Acids and Salts/pharmacology , Gene Deletion , Metabolic Networks and Pathways , Metabolomics , Receptors, Cytoplasmic and Nuclear/deficiency , Urinalysis , Animals , Bile Acids and Salts/metabolism , Cholic Acids/metabolism , Cholic Acids/pharmacology , Chromatography, High Pressure Liquid , Cresols/metabolism , Cresols/urine , Gene Expression Regulation/drug effects , Glucocorticoids/metabolism , Glucocorticoids/urine , Liver/drug effects , Liver/metabolism , Male , Mass Spectrometry , Mice , Multivariate Analysis , Phenotype , Receptors, Cytoplasmic and Nuclear/geneticsABSTRACT
Schistosomiasis is a chronic parasitic disease affecting hundreds of millions of individuals worldwide. Current treatment depends on a single agent, praziquantel, raising concerns of emergence of resistant parasites. Here, we continue our explorations of an oxadiazole-2-oxide class of compounds we recently identified as inhibitors of thioredoxin glutathione reductase (TGR), a selenocysteine-containing flavoenzyme required by the parasite to maintain proper cellular redox balance. Through systematic evaluation of the core molecular structure of this chemotype, we define the essential pharmacophore, establish a link between the nitric oxide donation and TGR inhibition, determine the selectivity for this chemotype versus related reductase enzymes, and present evidence that these agents can be modified to possess appropriate drug metabolism and pharmacokinetic properties. The mechanistic link between exogenous NO donation and parasite injury is expanded and better defined. The results of these studies verify the utility of oxadiazole-2-oxides as novel inhibitors of TGR and as efficacious antischistosomal agents.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Nitric Oxide/chemistry , Oxadiazoles/chemistry , Oxadiazoles/pharmacology , Schistosomiasis/drug therapy , Schistosomicides/chemistry , Schistosomicides/pharmacology , Animals , Biological Availability , Drug Discovery , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/therapeutic use , Humans , Inhibitory Concentration 50 , Models, Molecular , Multienzyme Complexes/antagonists & inhibitors , Multienzyme Complexes/chemistry , NADH, NADPH Oxidoreductases/antagonists & inhibitors , NADH, NADPH Oxidoreductases/chemistry , Oxadiazoles/pharmacokinetics , Oxadiazoles/therapeutic use , Protein Conformation , Rats , Schistosoma/drug effects , Schistosomiasis/enzymology , Schistosomicides/pharmacokinetics , Schistosomicides/therapeutic use , Solubility , Structure-Activity Relationship , Substrate SpecificityABSTRACT
Pregnane X receptor (PXR) is an important nuclear receptor xenosensor that regulates the expression of metabolic enzymes and transporters involved in the metabolism of xenobiotics and endobiotics. In this study, ultra-performance liquid chromatography (UPLC) coupled with electrospray time-of-flight mass spectrometry (TOFMS), revealed altered urinary metabolomes in both Pxr-null and wild-type mice treated with the mouse PXR activator pregnenolone 16alpha-carbonitrile (PCN). Multivariate data analysis revealed that PCN significantly attenuated the urinary vitamin E metabolite alpha-carboxyethyl hydroxychroman (CEHC) glucuronide together with a novel metabolite in wild-type but not Pxr-null mice. Deconjugation experiments with beta-glucuronidase and beta-glucosidase suggested that the novel urinary metabolite was gamma-CEHC beta-D-glucoside (Glc). The identity of gamma-CEHC Glc was confirmed by chemical synthesis and by comparing tandem mass fragmentation of the urinary metabolite with the authentic standard. The lower urinary CEHC was likely due to PXR-mediated repression of hepatic sterol carrier protein 2 involved in peroxisomal beta-oxidation of branched-chain fatty acids (BCFA). Using a combination of metabolomic analysis and a genetically modified mouse model, this study revealed that activation of PXR results in attenuated levels of the two vitamin E conjugates, and identification of a novel vitamin E metabolite, gamma-CEHC Glc. Activation of PXR results in attenuated levels of the two vitamin E conjugates that may be useful as biomarkers of PXR activation.
Subject(s)
Chromans/metabolism , Metabolomics , Propionates/metabolism , Receptors, Steroid/metabolism , Vitamin E/metabolism , Vitamins/metabolism , Animals , Chromans/chemistry , Gene Expression , Humans , Liver/metabolism , Male , Mass Spectrometry/methods , Mice , Mice, Knockout , Molecular Structure , Oxidation-Reduction , Pregnane X Receptor , Pregnenolone Carbonitrile/chemistry , Pregnenolone Carbonitrile/metabolism , Propionates/chemistry , Receptors, Steroid/genetics , Urine/chemistryABSTRACT
Ligand binding to the glucocorticoid receptor (GR) results in receptor binding to glucocorticoid response elements (GREs) and the formation of transcriptional regulatory complexes. Equally important, these complexes are continuously disassembled, with active processes driving GR off GREs. We found that co-chaperone p23-dependent disruption of GR-driven transcription depended on the ligand binding domain (LBD). Next, we examined the importance of the LBD and of ligand dissociation in GR-GRE dissociation in living cells. We showed in fluorescence recovery after photobleaching studies that dissociation of GR from GREs is faster in the absence of the LBD. Furthermore, GR interaction with a target promoter revealed ligand-specific exchange rates. However, using covalently binding ligands, we demonstrated that ligand dissociation is not required for receptor dissociation from GREs. Overall, these studies showed that activities impinging on the LBD regulate GR exchange with GREs but that the dissociation of GR from GREs is independent from ligand dissociation.
Subject(s)
Molecular Chaperones/metabolism , Receptors, Glucocorticoid/metabolism , Transcriptional Activation , Animals , Cell Line, Tumor , Cloning, Molecular , Fluorescence Recovery After Photobleaching , Humans , Ligands , Mice , Molecular Chaperones/genetics , Mutation , Promoter Regions, Genetic , Protein Binding , Protein Structure, Tertiary , Rats , Receptors, Glucocorticoid/genetics , Response ElementsABSTRACT
To investigate the determinants of promoter-specific gene regulation by the glucocorticoid receptor (GR), we compared the composition and function of regulatory complexes at two NFkappaB-responsive genes that are differentially regulated by GR. Transcription of the IL-8 and IkappaBalpha genes is stimulated by TNFalpha in A549 cells, but GR selectively represses IL-8 mRNA synthesis by inhibiting Ser2 phosphorylation of the RNA polymerase II (pol II) C-terminal domain (CTD). The proximal kappaB elements at these genes differ in sequence by a single base pair, and both recruited RelA and p50. Surprisingly, GR was recruited to both of these elements, despite the fact that GR failed to repress the IkappaBalpha promoter. Rather, the regulatory complexes formed at IL-8 and IkappaBalpha were distinguished by differential recruitment of the Ser2 CTD kinase, P-TEFb. Disruption of P-TEFb function by the Cdk-inhibitor, DRB, or by small interfering RNA selectively blocked TNFalpha stimulation of IL-8 mRNA production. GR competed with P-TEFb recruitment to the IL-8 promoter. Strikingly, IL-8 mRNA synthesis was repressed by GR at a post-initiation step, demonstrating that promoter proximal regulatory sequences assemble complexes that impact early and late stages of mRNA synthesis. Thus, GR accomplishes selective repression by targeting promoter-specific components of NFkappaB regulatory complexes.
Subject(s)
Gene Expression Regulation , Interleukin-8/genetics , NF-kappa B/metabolism , Positive Transcriptional Elongation Factor B/metabolism , Promoter Regions, Genetic/genetics , Receptors, Glucocorticoid/metabolism , Chromatin Immunoprecipitation , Dichlororibofuranosylbenzimidazole/pharmacology , Glutathione Transferase/metabolism , Humans , I-kappa B Proteins/genetics , Models, Biological , NF-KappaB Inhibitor alpha , Oligonucleotides , Phosphorylation , Positive Transcriptional Elongation Factor B/antagonists & inhibitors , RNA Polymerase II/antagonists & inhibitors , RNA Polymerase II/metabolism , RNA, Small Interfering/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic/drug effects , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Members of the mammalian p160 family, such as GRIP1, are known as glucocorticoid receptor (GR) coactivators; at certain glucocorticoid response elements (GREs), however, GRIP1 acts as a GR corepressor. We characterized functional interactions of GR and GRIP1 in a repression complex where GR tethers to DNA-bound activator protein-1 (AP-1), as at the human collagenase-3 gene, and tested whether the identified interactions were similar or different at other response elements. At the AP-1 tethering GRE, we mapped the GRIP1 corepressor activity to a domain distinct from the two known GRIP1 activation domains; it exhibited intrinsic GR-independent repression potential when recruited to DNA via Gal4 DNA-binding domain. Interestingly, neither the domain nor the activity was detected in the other two p160 family members, SRC1 and RAC3. The same GRIP1 corepression domain was required for GR-mediated repression at the nuclear factor-kappaB (NF-kappaB) tethering GRE of the human IL-8 gene. In contrast, at the osteocalcin gene GRE, where GR represses transcription by binding to a DNA site overlapping the TATA box, both GRIP1 and SRC1 corepressed, and the GRIP1-specific repression domain was dispensable. Thus, in a single cell type, GR and GRIP1 conferred one mode of activation and two modes of repression by selectively engaging distinct surfaces of GRIP1 in a response element-specific manner.