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The nutrient metal iron plays a key role in the survival of microorganisms. The iron-regulated surface determinant (Isd) system scavenges heme-iron from the human host, enabling acquisition of iron in iron-deplete conditions in Staphylococcus aureus during infection. The cell surface receptors IsdB and IsdH bind hemoproteins and transfer heme to IsdA, the final surface protein before heme-iron is transported through the peptidoglycan. To define the human B-cell response to IsdA, we isolated human monoclonal antibodies (mAbs) specific to the surface Isd proteins and determined their mechanism of action. We describe the first isolation of fully human IsdA and IsdH mAbs, as well as cross-reactive Isd mAbs. Two of the identified IsdA mAbs worked in a murine septic model of infection to reduce bacterial burden during staphylococcal infection. Their protection was a result of both heme-blocking and Fc-mediated effector functions, underscoring the importance of targeting S. aureus using diverse mechanisms.
© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: firstname.lastname@example.org.
RATIONALE - The most frequently occurring phthalate, di(2-ethylhexyl) phthalate (DEHP), causes adverse effects on glucose homeostasis and insulin sensitivity in several cell models and epidemiological studies. However, thus far, there is no information available on the molecular interaction of phthalates and one of the key regulators of the metabolism, the peroxisome proliferator-activated receptor gamma (PPARγ). Since the endogenous ligand of PPARγ, 15-deoxy-delta-12,14-prostaglandin J (15Δ-PGJ ), features structural similarity to DEHP and its main metabolites produced in human hepatic metabolism, mono(2-ethylhexyl) phthalate (MEHP) and mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), we tested the hypothesis of direct interactions between PPARγ and DEHP or its transformation products.
METHODS - Hydrogen/deuterium exchange mass spectrometry (HDX-MS) and docking were conducted to obtain structural insights into the interactions and surface plasmon resonance (SPR) analysis to reveal information about binding levels. To confirm the activation of PPARγ upon ligand binding on the cellular level, the GeneBLAzer® bioassay was performed.
RESULTS - HDX-MS and SPR analyses demonstrated that the metabolites MEHP and MEOHP, but not DEHP itself, bind to the ligand binding pocket of PPARγ. This binding leads to typical activation-associated conformational changes, as observed with its endogenous ligand 15Δ-PGJ . Furthermore, the reporter gene assay confirmed productive interaction. DEHP was inactive up to a concentration of 14 μM, while the metabolites MEHP and MEOHP were active at low micromolar concentrations.
CONCLUSIONS - In summary, this study gives structural insights into the direct interaction of PPARγ with MEHP and MEOHP and shows that the DEHP transformation products may modulate the lipid metabolism through PPARγ pathways.
© 2018 John Wiley & Sons, Ltd.
Cytochrome P450 (P450, CYP) research provides many opportunities for the application of kinetic isotope effect (KIE) strategies. P450s collectively catalyze oxidations of more substrates than any other group of enzymes, and CH bond cleavage is a major feature in a large fraction of these reactions. The presence of a significant primary deuterium KIE is evidence that hydrogen abstraction is at least partially rate-limiting in the reactions, and this appears to be the case in many P450 reactions. The first report of a KIE in (P450-linked) drug metabolism appeared in 1961 (for morphine N-demethylation), and in a number of cases, it has been possible to modulate the in vivo metabolism or toxicity of chemicals by deuterium substitution. A number of efforts are in progress to utilize deuterium substitution to alter the metabolism of drugs in an advantageous manner.
© 2017 Elsevier Inc. All rights reserved.
Uncovering mechanisms of antibody-mediated neutralization for viral infections requires epitope and paratope mapping in the context of whole viral particle interactions with the antibody in solution. In this study, we use amide hydrogen/deuterium exchange mass spectrometry to describe the interface of a dengue virus-neutralizing antibody, 2D22, with its target epitope. 2D22 binds specifically to DENV2, a serotype showing strain-specific structural expansion at human host physiological temperatures of 37°C. Our results identify the heavy chain of 2D22 to be the primary determinant for binding DENV2. Temperature-mediated expansion alters the mode of interaction of 2D22 binding. Importantly, 2D22 interferes with the viral expansion process and offers a basis for its neutralization mechanism. The relative magnitude of deuterium exchange protection upon antibody binding across the various epitope loci allows a deconstruction of the antibody-viral interface in host-specific environments and offers a robust approach for targeted antibody engineering.
Copyright © 2017 Elsevier Ltd. All rights reserved.
Cytochrome P450 46A1 (CYP46A1, cholesterol 24-hydroxylase) is the enzyme responsible for the majority of cholesterol elimination from the brain. Previously, we found that the anti-HIV drug efavirenz (EFV) can pharmacologically activate CYP46A1 in mice. Herein, we investigated whether CYP46A1 could also be activated by endogenous compounds, including major neurotransmitters. experiments with purified recombinant CYP46A1 indicated that CYP46A1 is activated by l-glutamate (l-Glu), l-aspartate, γ-aminobutyric acid, and acetylcholine, with l-Glu eliciting the highest increase (3-fold) in CYP46A1-mediated cholesterol 24-hydroxylation. We also found that l-Glu and other activating neurotransmitters bind to the same site on the CYP46A1 surface, which differs from the EFV-binding site. The other principal differences between EFV and l-Glu in CYP46A1 activation include an apparent lack of l-Glu binding to the P450 active site and different pathways of signal transduction from the allosteric site to the active site. EFV and l-Glu similarly increased the CYP46A1 , the rate of the "fast" phase of the enzyme reduction by the redox partner NADPH-cytochrome P450 oxidoreductase, and the amount of P450 reduced. Spectral titrations with cholesterol, in the presence of EFV or l-Glu, suggest that water displacement from the heme iron can be affected in activator-bound CYP46A1. Moreover, EFV and l-Glu synergistically activated CYP46A1. Collectively, our data, along with those from previous cell culture and studies by others, suggest that l-Glu-induced CYP46A1 activation is of physiological relevance.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Cytochrome P450 (P450, CYP) 21A2 is the major steroid 21-hydroxylase, converting progesterone to 11-deoxycorticosterone and 17α-hydroxyprogesterone (17α-OH-progesterone) to 11-deoxycortisol. More than 100 variants give rise to congenital adrenal hyperplasia (CAH). We previously reported a structure of WT human P450 21A2 with bound progesterone and now present a structure bound to the other substrate (17α-OH-progesterone). We found that the 17α-OH-progesterone- and progesterone-bound complex structures are highly similar, with only some minor differences in surface loop regions. Twelve P450 21A2 variants associated with either salt-wasting or nonclassical forms of CAH were expressed, purified, and analyzed. The catalytic activities of these 12 variants ranged from 0.00009% to 30% of WT P450 21A2 and the extent of heme incorporation from 10% to 95% of the WT. Substrate dissociation constants () for four variants were 37-13,000-fold higher than for WT P450 21A2. Cytochrome , which augments several P450 activities, inhibited P450 21A2 activity. Similar to the WT enzyme, high noncompetitive intermolecular kinetic deuterium isotope effects (≥ 5.5) were observed for all six P450 21A2 variants examined for 21-hydroxylation of 21--progesterone, indicating that C-H bond breaking is a rate-limiting step over a 10-fold range of catalytic efficiency. Using UV-visible and CD spectroscopy, we found that P450 21A2 thermal stability assessed in bacterial cells and with purified enzymes differed among salt-wasting- and nonclassical-associated variants, but these differences did not correlate with catalytic activity. Our in-depth investigation of CAH-associated P450 21A2 variants reveals critical insight into the effects of disease-causing mutations on this important enzyme.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Mouse models designed to examine hepatic metabolism are critical to diabetes and obesity research. Thus, a microscale method to quantitatively assess hepatic glucose and intermediary metabolism in conscious, unrestrained mice was developed. [(13)C3]propionate, [(2)H2]water, and [6,6-(2)H2]glucose isotopes were delivered intravenously in short- (9 h) and long-term-fasted (19 h) C57BL/6J mice. GC-MS and mass isotopomer distribution (MID) analysis were performed on three 40-μl arterial plasma glucose samples obtained during the euglycemic isotopic steady state. Model-based regression of hepatic glucose and citric acid cycle (CAC)-related fluxes was performed using a comprehensive isotopomer model to track carbon and hydrogen atom transitions through the network and thereby simulate the MIDs of measured fragment ions. Glucose-6-phosphate production from glycogen diminished, and endogenous glucose production was exclusively gluconeogenic with prolonged fasting. Gluconeogenic flux from phosphoenolpyruvate (PEP) remained stable, whereas that from glycerol modestly increased from short- to long-term fasting. CAC flux [i.e., citrate synthase (VCS)] was reduced with long-term fasting. Interestingly, anaplerosis and cataplerosis increased with fast duration; accordingly, pyruvate carboxylation and the conversion of oxaloacetate to PEP were severalfold higher than VCS in long-term fasted mice. This method utilizes state-of-the-art in vivo methodology and comprehensive isotopomer modeling to quantify hepatic glucose and intermediary fluxes during physiological stress in mice. The small plasma requirements permit serial sampling without stress and the affirmation of steady-state glucose kinetics. Furthermore, the approach can accommodate a broad range of modeling assumptions, isotope tracers, and measurement inputs without the need to introduce ad hoc mathematical approximations.
Copyright © 2015 the American Physiological Society.
Cytochrome P450 (P450) 21A2 is the major steroid 21-hydroxylase, and deficiency of this enzyme is involved in ∼95% of cases of human congenital adrenal hyperplasia, a disorder of adrenal steroidogenesis. A structure of the bovine enzyme that we published previously (Zhao, B., Lei, L., Kagawa, N., Sundaramoorthy, M., Banerjee, S., Nagy, L. D., Guengerich, F. P., and Waterman, M. R. (2012) Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants. J. Biol. Chem. 287, 10613-10622), containing two molecules of the substrate 17α-hydroxyprogesterone, has been used as a template for understanding genetic deficiencies. We have now obtained a crystal structure of human P450 21A2 in complex with progesterone, a substrate in adrenal 21-hydroxylation. Substrate binding and release were fast for human P450 21A2 with both substrates, and pre-steady-state kinetics showed a partial burst but only with progesterone as substrate and not 17α-hydroxyprogesterone. High intermolecular non-competitive kinetic deuterium isotope effects on both kcat and kcat/Km, from 5 to 11, were observed with both substrates, indicative of rate-limiting C-H bond cleavage and suggesting that the juxtaposition of the C21 carbon in the active site is critical for efficient oxidation. The estimated rate of binding of the substrate progesterone (kon 2.4 × 10(7) M(-1) s(-1)) is only ∼2-fold greater than the catalytic efficiency (kcat/Km = 1.3 × 10(7) M(-1) s(-1)) with this substrate, suggesting that the rate of substrate binding may also be partially rate-limiting. The structure of the human P450 21A2-substrate complex provides direct insight into mechanistic effects of genetic variants.
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Cytochrome P450 (P450) 4A11 is the only functionally active subfamily 4A P450 in humans. P450 4A11 catalyzes mainly ω-hydroxylation of fatty acids in liver and kidney; this process is not a major degradative pathway, but at least one product, 20-hydroxyeicosatetraenoic acid, has important signaling properties. We studied catalysis by P450 4A11 and the issue of rate-limiting steps using lauric acid ω-hydroxylation, a prototypic substrate for this enzyme. Some individual reaction steps were studied using pre-steady-state kinetic approaches. Substrate and product binding and release were much faster than overall rates of catalysis. Reduction of ferric P450 4A11 (to ferrous) was rapid and not rate-limiting. Deuterium kinetic isotope effect (KIE) experiments yielded low but reproducible values (1.2-2) for 12-hydroxylation with 12-(2)H-substituted lauric acid. However, considerable "metabolic switching" to 11-hydroxylation was observed with [12-(2)H3]lauric acid. Analysis of switching results [Jones, J. P., et al. (1986) J. Am. Chem. Soc. 108, 7074-7078] and the use of tritium KIE analysis with [12-(3)H]lauric acid [Northrop, D. B. (1987) Methods Enzymol. 87, 607-625] both indicated a high intrinsic KIE (>10). Cytochrome b5 (b5) stimulated steady-state lauric acid ω-hydroxylation ∼2-fold; the apoprotein was ineffective, indicating that electron transfer is involved in the b5 enhancement. The rate of b5 reoxidation was increased in the presence of ferrous P450 mixed with O2. Collectively, the results indicate that both the transfer of an electron to the ferrous·O2 complex and C-H bond-breaking limit the rate of P450 4A11 ω-oxidation.
Reported here are (i) a new synthetic approach for preparation of (ii) a new compound class, of -OH, for example, an -OH group is replaced with acetyl protecting group, protected 1,2-dehydrocholine analogs and (iii) a new synthetic route for betaine aldehyde. The CC bond of 1,2-dehydrocholine moiety can be used for molecular addition of parahydrogen producing -OH protected hyperpolarized choline by parahydrogen-induced polarization (PHIP). The reported synthetic approach allows for incorporation of (15) N and deuterium labels, which are necessary for preparation of highly polarized PHIP contrast agents. Isotope labeling with (15) N and/or deuterium was conducted. Hyperpolarized (15) N-choline enabled by the reported synthetic approach can be potentially used as an imaging biomarker of cancer similar to choline positron emission tomography tracers.
Copyright © 2013 John Wiley & Sons, Ltd.