An effective vaccine against influenza A virus based on the matrix protein 2 (M2).

The ever-increasing antigenic diversity of the hemagglutinin (HA) of influenza A virus (IAV) poses a significant challenge for effective vaccine development. Notably, the matrix protein 2 (M2) is a highly conserved 97 amino acid long transmembrane tetrameric protein present in the envelope of IAV. More than 99 % of IAV strains circulating in American swine herds share the identical pandemic (pdm) isoform of M2, making it an ideal target antigen for a vaccine that could elicit broadly protective immunity.

Optical annealing of peroxo-ferric intermediates in CYP17A1 and product formation.

Human cytochrome P450 CYP17A1 catalyzes the hydroxylation of pregnenolone and progesterone at the C17 position, with subsequent C17-C20 bond scission, to form dehydroepiandrosterone and androstenedione respectively. The first hydroxylation reaction is faster in HO than in DO, while the second carbon‑carbon bond scission event demonstrates an inverse solvent isotope effect, which is more pronounced for 17-hydroxy pregnenolone. In order to better understand the cause of this difference, we compared the optical absorption spectra of oxygenated CYP17A1 with the four substrates (pregnenolone, progesterone, 17-hydroxy pregnenolone and 17-hydroxy progesterone) in both HO and DO.

Midazolam as a Probe for Heterotropic Drug-Drug Interactions Mediated by CYP3A4.

Human cytochrome P450 CYP3A4 is involved in the processing of more than 35% of current pharmaceuticals and therefore is responsible for multiple drug-drug interactions (DDI). In order to develop a method for the detection and prediction of the possible involvement of new drug candidates in CYP3A4-mediated DDI, we evaluated the application of midazolam (MDZ) as a probe substrate. MDZ is hydroxylated by CYP3A4 in two positions: 1-hydroxy MDZ formed at lower substrate concentrations, and up to 35% of 4-hydroxy MDZ at high concentrations.

Midazolam as a Probe for Drug-Drug Interactions Mediated by CYP3A4: Homotropic Allosteric Mechanism of Site-Specific Hydroxylation.

We developed an efficient and sensitive probe for drug-drug interactions mediated by human CYP3A4 by using midazolam (MDZ) as a probe substrate. Using global analysis of four parameters over several experimental data sets, we demonstrate that the first MDZ molecule (MDZ1) binds with high affinity at the productive site near the heme iron and gives only hydroxylation at the 1 position (1OH). The second midazolam molecule (MDZ2) binds at an allosteric site at the membrane surface and perturbs the position and mobility of MDZ1 such that the minor hydroxylation product at the 4 position (4OH) is formed in a 1:2 ratio (35%).

Dark, Ultra-Dark and Ultra-Bright Nanodiscs for membrane protein investigations.

We describe the construction, expression and purification of three new membrane scaffold proteins (MSP) for use in assembling Nanodiscs. These new MSPs have a variety of luminescent properties for use in combination with several analytical methods. "Dark" MSP has no tryptophan residues, "Ultra-Dark" replaces both tryptophan and tyrosine with non-fluorescent side chains, and "Ultra-Bright" adds additional tryptophans to the parent membrane scaffold protein to provide a dramatic increase in native tryptophan fluorescence.

P450 CYP17A1 Variant with a Disordered Proton Shuttle Assembly Retains Peroxo-Mediated Lyase Efficiency.

Human cytochrome P450 CYP17A1 first catalyzes hydroxylation at the C17 position of either pregnenolone (PREG) or progesterone (PROG), and a subsequent C -C bond scission to produce dehydroepiandrosterone (DHEA) or androstenedione (AD). In the T306A mutant, replacement of the Threonine 306 alcohol functionality, essential for efficient proton delivery in the hydroxylase reaction, has only a small effect on the lyase activity. In this work, resonance Raman spectroscopy is employed to provide crucial structural insight, confirming that this mutant, with its disordered proton shuttle, fails to generate essential hydroxylase pathway intermediates, accounting for the loss in hydroxylase efficiency.

Biotransformation of the Mycotoxin Enniatin B1 by CYP P450 3A4 and Potential for Drug-Drug Interactions.

Enniatins (ENNs) are fungal secondary metabolites that frequently occur in grain in temperate climates. Their toxic potency is connected to their ionophoric character and lipophilicity. The biotransformation of ENNs predominantly takes place via cytochrome P450 3A (CYP 3A)-dependent oxidation reactions.

Allosteric Interactions in Human Cytochrome P450 CYP3A4: The Role of Phenylalanine 213.

The role of Phe213 in the allosteric mechanism of human cytochrome P450 CYP3A4 was studied using a combination of progesterone (PGS) and carbamazepine (CBZ) as probe substrates. We expressed, purified, and incorporated into POPC Nanodiscs three mutants, F213A, F213S, and F213Y, and compared them with wild-type (WT) CYP3A4 by monitoring spectral titration, the rate of NADPH oxidation, and steady-state product turnover rates with pure substrates and substrate mixtures. All mutants demonstrated higher activity with CBZ, lower activity with PGS, and a reduced level of activation of CBZ epoxidation by PGS, which was most pronounced in the F213A mutant.

Drug-Drug Interactions between Atorvastatin and Dronedarone Mediated by Monomeric CYP3A4.

Heterotropic interactions between atorvastatin (ARVS) and dronedarone (DND) have been deciphered using global analysis of the results of binding and turnover experiments for pure drugs and their mixtures. The in vivo presence of atorvastatin lactone (ARVL) was explicitly taken into account by using pure ARVL in analogous experiments. Both ARVL and ARVS inhibit DND binding and metabolism, while a significantly higher affinity of CYP3A4 for ARVL makes the latter the main modulator of activity (effector) in this system.

Heme Binding Biguanides Target Cytochrome P450-Dependent Cancer Cell Mitochondria.

The mechanisms by which cancer cell-intrinsic CYP monooxygenases promote tumor progression are largely unknown. CYP3A4 was unexpectedly associated with breast cancer mitochondria and synthesized arachidonic acid (AA)-derived epoxyeicosatrienoic acids (EETs), which promoted the electron transport chain/respiration and inhibited AMPKα. CYP3A4 knockdown activated AMPKα, promoted autophagy, and prevented mammary tumor formation.

The use of isomeric testosterone dimers to explore allosteric effects in substrate binding to cytochrome P450 CYP3A4.

Cytochrome P450 CYP3A4 is the main drug-metabolizing enzyme in the human liver, being responsible for oxidation of 50% of all pharmaceuticals metabolized by human P450 enzymes. Possessing a large substrate binding pocket, it can simultaneously bind several substrate molecules and often exhibits a complex pattern of drug-drug interactions. In order to better understand structural and functional aspects of binding of multiple substrate molecules to CYP3A4 we used resonance Raman and UV-VIS spectroscopy to document the effects of binding of synthetic testosterone dimers of different configurations, cis-TST2 and trans-TST2.

Incorporation of charged residues in the CYP2J2 F-G loop disrupts CYP2J2-lipid bilayer interactions.

CYP2J2 epoxygenase is an extrahepatic, membrane bound cytochrome P450 (CYP) that is primarily found in the heart and mediates endogenous fatty acid metabolism. CYP2J2 interacts with membranes through an N-terminal anchor and various non-contiguous hydrophobic residues. The molecular details of the motifs that mediate membrane interactions are complex and not fully understood.

Active site proton delivery and the lyase activity of human CYP17A1.

Cytochrome P450 CYP17A1 catalyzes a series of reactions that lie at the intersection of corticoid and androgen biosynthesis and thus occupies an essential role in steroid hormone metabolism. This multifunctional enzyme catalyzes the 17α-hydroxylation of Δ4- and Δ5-steroids progesterone and pregnenolone to form the corresponding 17α-hydroxy products through its hydroxylase activity, and a subsequent 17,20-carbon-carbon scission of pregnene-side chain produce the androgens androstenedione (AD) and dehydroepiandrosterone (DHEA). While the former hydroxylation reaction is believed to proceed through a conventional "Compound I" rebound mechanism, it has been suggested that the latter carbon cleavage is initiated by an iron-peroxy intermediate.

Kinetic solvent isotope effect in human P450 CYP17A1-mediated androgen formation: evidence for a reactive peroxoanion intermediate.

Human steroid hormone biosynthesis is the result of a complex series of chemical transformations operating on cholesterol, with key steps mediated by members of the cytochrome P450 superfamily. In the formation of the male hormone dehydroepiandrosterone, pregnenolone is first hydroxylated by P450 CYP17A1 at the 17-carbon, followed a second round of catalysis by the same enzyme that cleaves the C17-C20 bond, releasing acetic acid and the 17-keto product. In order to explore the mechanism of this C-C "lyase" activity, we investigated the kinetic isotope effect on the steady-state turnover of Nanodisc-incorporated CYP17A1.

Nanodiscs as a therapeutic delivery agent: inhibition of respiratory syncytial virus infection in the lung.

There is increasing interest in the application of nanotechnology to solve the difficult problem of therapeutic administration of pharmaceuticals. Nanodiscs, composed of a stable discoidal lipid bilayer encircled by an amphipathic membrane scaffold protein that is an engineered variant of the human Apo A-I constituent of high-density lipoproteins, have been a successful platform for providing a controlled lipid composition in particles that are especially useful for investigating membrane protein structure and function. In this communication, we demonstrate that nanodiscs are effective in suppressing respiratory syncytial viral (RSV) infection both in vitro and in vivo when self-assembled with the minor pulmonary surfactant phospholipid palmitoyloleoylphosphatidylglycerol (POPG).

Oxidase uncoupling in heme monooxygenases: human cytochrome P450 CYP3A4 in Nanodiscs.

The normal reaction mechanism of cytochrome P450 operates by utilizing two reducing equivalents to reduce atmospheric dioxygen, producing one molecule of water and an oxygenated product in an overall stoichiometry of 2 electrons:1 dioxygen:1 product. However, three alternate unproductive pathways exist where the intermediate iron-oxygen states in the catalytic cycle can yield reduced oxygen products without substrate metabolism. The first involves release of superoxide from the oxygenated intermediate while the second occurs after input of the second reducing equivalent.

Defining CYP3A4 structural responses to substrate binding. Raman spectroscopic studies of a nanodisc-incorporated mammalian cytochrome P450.

Resonance Raman (RR) spectroscopy is used to help define active site structural responses of nanodisc-incorporated CYP3A4 to the binding of three substrates: bromocriptine (BC), erythromycin (ERY), and testosterone (TST). We demonstrate that nanodisc-incorporated assemblies reveal much more well-defined active site RR spectroscopic responses as compared to those normally obtained with the conventional, detergent-stabilized, sampling strategies. While ERY and BC are known to bind to CYP3A4 with a 1:1 stoichiometry, only the BC induces a substantial conversion from low- to high-spin state, as clearly manifested in the RR spectra acquired herein.

Photoelectrochemical complexes for solar energy conversion that chemically and autonomously regenerate.

Naturally occurring photosynthetic systems use elaborate pathways of self-repair to limit the impact of photo-damage. Here, we demonstrate a complex consisting of two recombinant proteins, phospholipids and a carbon nanotube that mimics this process. The components self-assemble into a configuration in which an array of lipid bilayers aggregate on the surface of the carbon nanotube, creating a platform for the attachment of light-converting proteins.

Engineering extended membrane scaffold proteins for self-assembly of soluble nanoscale lipid bilayers.

High-density lipoproteins (HDLs) play an important role in human health through the metabolism and trafficking of cholesterol as well as providing the feedstocks for steroid hormone biosynthesis. These particles contain proteins, primarily Apo-AI and phospholipid and progress through various structural forms including 'lipid-poor', 'discoidal' and 'spherical' entities as cholesterol esters and lipid are incorporated. The discoidal form of HDL is stabilized in solution by two encircling belts of Apo-AI.

Functional reconstitution of monomeric CYP3A4 with multiple cytochrome P450 reductase molecules in Nanodiscs.

Traditional reconstitution of membrane cytochromes P450 monooxygenase system requires efficient solubilization of both P450 heme enzymes and redox partner NADPH dependent reductase, CPR, either in mixed micellar solution or by incorporation in liposomes. Here we describe a simple alternative approach to assembly of soluble complexes of monomeric human hepatic cytochrome P450 CYP3A4 with CPR by co-incorporation into nanoscale POPC bilayer Nanodiscs. Stable and fully functional complexes with different CPR:CYP3A4 stoichiometric ratios are formed within several minutes after addition of the full-length CPR to the solution of CYP3A4 preassembled into POPC Nanodiscs at 37 degrees C.

The critical iron-oxygen intermediate in human aromatase.

Aromatase (CYP19) is the target of several therapeutics used for breast cancer treatment and catalyzes the three-step conversion of androgens to estrogens, with an unusual C-C cleavage reaction in the third step. To better understand the CYP19 reaction, the oxy-ferrous complex of CYP19 with androstenedione substrate was cryotrapped, characterized by UV-vis spectroscopy, and cryoreduced to generate the next reaction cycle intermediate. EPR analysis revealed that the initial intermediate observed following cryoreduction is the unprotonated g(1)=2.

The ferrous-oxy complex of human aromatase.

In this communication, we document the self-assembly of heterologously expressed truncated human aromatase (CYP19) into nanometer scale phospholipids bilayers (Nanodiscs). The resulting P450 CYP19 preparation is stable and can tightly associate with the substrate androstenedione to form a nearly complete high-spin ferric protein. Ferrous CYP19 in Nanodiscs was mixed anaerobically in a rapid-scan stopped-flow with atmospheric dioxygen and the formation of the ferrous-oxy complex observed.