The 2.0 A crystal structure of the ERalpha ligand-binding domain complexed with lasofoxifene

Lasofoxifene is a new and potent selective estrogen receptor modulator (SERM). The structural basis of its interaction with the estrogen receptor has been investigated by crystallographic analysis of its complex with the ligand-binding domain of estrogen receptor alpha at a resolution of 2.0 A. As with other SERMs, lasofoxifene diverts the receptor from its agonist-bound conformation by displacing the C-terminal AF-2 helix into the site at which the LXXLL motif of coactivator proteins would otherwise be able to bind. Lasofoxifene achieves this effect by occupying the space normally filled by residue Leu 540, as well as by modulating the conformation of residues of helix 11 (His 524, Leu 525). A well-defined salt bridge between lasofoxifene and Asp 351 suggests that charge neutralization in this region of the receptor may explain the some of the antiestrogenic effects of lasofoxifene. The results suggest general features of ERalpha/SERM recognition, and add a new dimension to efforts to rationalize differences between the biological activity profiles exhibited by these important pharmacological agents.     

Employment of a noninvasive magnetic method for evaluation of gastrointestinal transit in rats

ABSTRACT: AC Biosusceptometry (ACB) was previously employed towards recording gastrointestinal motility. Our data show a reliable and successful evaluation of gastrointestinal transit of liquid and solid meals in rats, considering the methods scarcity and number of experiments needed to endorsement of drugs and medicinal plants. ACB permits real time and simultaneous experiments using the same animal, preserving the physiological conditions employing both meals with simplicity and accuracy.     

Pseudomonas cepacia 2,2-dialkylglycine decarboxylase. Sequence and expression in Escherichia coli of structural and repressor genes

A 3969-base pair PstI-PstI fragment of Pseudomonas cepacia DNA containing the gene for the pyridoxal 5'-phosphate dependent 2,2-dialkylglycine decarboxylase (pyruvate) (EC 4.1.1.64) was cloned in Escherichia coli. The insert was sequenced by the dideoxy method using nested deletions from both ends, revealing a central 1302-base pair region that codes for the decarboxylase subunit. The recombinant enzyme was expressed in E. coli, purified to homogeneity, and sequenced at the amino terminus. Also, a cofactor-labeled active site peptide was sequenced. The carboxyl terminus of the deduced amino acid sequence is homologous with the carboxyl terminus of mammalian ornithine aminotransferase; the active site sequence is similar to the active site sequences of several other aminotransferases. No homologies with known decarboxylase sequences could be found. Expression of the decarboxylase gene is negatively controlled by a 687-nucleotide sequence upstream of and diverging from the structural gene. Expression is induced by S-isovaline, 2-methylalanine, and D-2-aminobutanoic acid, but not by glycine, D- or L-alanine, L-2-aminobutanoic acid, R-isovaline, or other alkyl amino acids.     

Structural insights into the catalytic mechanism of cyclophilin A

Cyclophilins constitute a ubiquitous protein family whose functions include protein folding, transport and signaling. They possess both sequence-specific binding and proline cis-trans isomerase activities, as exemplified by the interaction between cyclophilin A (CypA) and the HIV-1 CA protein. Here, we report crystal structures of CypA in complex with HIV-1 CA protein variants that bind preferentially with the substrate proline residue in either the cis or the trans conformation. Cis- and trans-Pro substrates are accommodated within the enzyme active site by rearrangement of their N-terminal residues and with minimal distortions in the path of the main chain. CypA Arg55 guanidinium group probably facilitates catalysis by anchoring the substrate proline oxygen and stabilizing sp3 hybridization of the proline nitrogen in the transition state.     

Deconstruction of activity-dependent covalent modification of heme in human neutrophil myeloperoxidase by multistage mass spectrometry (MS(4))

Myeloperoxidase (MPO) is known to be inactivated and covalently modified by treatment with hydrogen peroxide and agents similar to 3-(2-ethoxypropyl)-2-thioxo-2,3-dihydro-1H-purin-6(9H)-one (1), a 254.08 Da derivative of 2-thioxanthine. Peptide mapping by liquid chromatography and mass spectrometry detected modification by 1 in a labile peptide-heme-peptide fragment of the enzyme, accompanied by a mass increase of 252.08 Da. The loss of two hydrogen atoms was consistent with mechanism-based oxidative coupling. Multistage mass spectrometry (MS(4)) of the modified fragment in an ion trap/Orbitrap spectrometer demonstrated that 1 was coupled directly to heme. Use of a 10 amu window delivered the full isotopic envelope of each precursor ion to collision-induced dissociation, preserving definitive isotopic profiles for iron-containing fragments through successive steps of multistage mass spectrometry. Iron isotope signatures and accurate mass measurements supported the structural assignments. Crystallographic analysis confirmed linkage between the methyl substituent of the heme pyrrole D ring and the sulfur atom of 1. The final orientation of 1 perpendicular to the plane of the heme ring suggested a mechanism consisting of two consecutive one-electron oxidations of 1 by MPO. Multistage mass spectrometry using stage-specific collision energies permits stepwise deconstruction of modifications of heme enzymes containing covalent links between the heme group and the polypeptide chain.     

Structure-guided inhibitor design for human acetyl-coenzyme A carboxylase by interspecies active site conversion

Inhibition of acetyl-CoA carboxylases (ACCs), a crucial enzyme for fatty acid metabolism, has been shown to promote fatty acid oxidation and reduce body fat in animal models. Therefore, ACCs are attractive targets for structure-based inhibitor design, particularly the carboxyltransferase (CT) domain, which is the primary site for inhibitor interaction. We have cloned, expressed, and purified the CT domain of human ACC2 using baculovirus-mediated insect cell expression system. However, attempts to crystallize the human ACC2 CT domain have not been successful in our hands. Hence, we have been using the available crystal structure of yeast CT domain to design human ACC inhibitors. Unfortunately, as the selectivity of the lead series has increased against the full-length human enzyme, the potency against the yeast enzyme has decreased significantly. This loss of potency against the yeast enzyme correlated with a complete lack of binding of the human-specific compounds to crystals of the yeast CT domain. Here, we address this problem by converting nine key active site residues of the yeast CT domain to the corresponding human residues. The resulting humanized yeast ACC-CT (yCT-H9) protein exhibits biochemical and biophysical properties closer to the human CT domain and binding to human specific compounds. We report high resolution crystal structures of yCT-H9 complexed with inhibitors that show a preference for the human CT domain. These structures offer insights that explain the species selectivity of ACC inhibitors and may guide future drug design programs.     

Crystal structure of human insulin-like growth factor-1: detergent binding inhibits binding protein interactions

Despite efforts spanning considerably more than a decade, a high-resolution view of the family of proteins known as insulin-like growth factors (IGFs) has remained elusive. IGF-1 consists of three helical segments which are connected by a 12-residue linker known as the C-region. NMR studies of members of this family reveal a dynamic structure with a topology resembling insulin but little structural definition in the C-region. We have crystallized IGF-1 in the presence of the detergent deoxy big CHAPS, and determined its structure at 1.8 A resolution by multiwavelength anomalous diffraction, exploiting the anomalous scattering of a single bromide ion and six of the seven sulfur atoms of IGF-1. The structure reveals a well-defined conformation for much of the C-region, which extends away from the core of IGF-1 and has residues known to be involved in receptor binding prominently displayed in a type II beta-turn. In the crystal, these residues form a dimer interface, but analytical ultracentrifugation experiments demonstrate that at physiological concentrations IGF-1 is monomeric. A single detergent molecule contacts residues known to be important for IGF-1 binding protein (IGFBP) interactions. Biophysical and biochemical data show that the detergent binds to IGF-1 specifically and blocks binding of IGFBP-1 and IGFBP-3.     

Chronic exposure of diesel exhaust particles induces alveolar enlargement in mice

Background

Diesel exhaust particles (DEPs) are deposited into the respiratory tract and are thought to be a risk factor for the development of diseases of the respiratory system. In healthy individuals, the timing and mechanisms of respiratory tract injuries caused by chronic exposure to air pollution remain to be clarified.

Methods

We evaluated the effects of chronic exposure to DEP at doses below those found in a typical bus corridor in Sao Paulo (150 μg/m³). Male BALB/c mice were divided into mice receiving a nasal instillation: saline (saline; n = 30) and 30 μg/10 μL of DEP (DEP; n = 30). Nasal instillations were performed five days a week, over a period of 90 days. Bronchoalveolar lavage (BAL) was performed, and the concentrations of interleukin (IL)-4, IL-10, IL-13 and interferon-gamma (INF-γ) were determined by ELISA-immunoassay. Assessment of respiratory mechanics was performed. The gene expression of Muc5ac in lung was evaluated by RT-PCR. The presence of IL-13, MAC2+ macrophages, CD3+, CD4+, CD8+ T cells and CD20+ B cells in tissues was analysed by immunohistochemistry. Bronchial thickness and the collagen/elastic fibers density were evaluated by morphometry. We measured the mean linear intercept (Lm), a measure of alveolar distension, and the mean airspace diameter (D0) and statistical distribution (D2).

Results

DEP decreased IFN-γ levels in BAL (p = 0.03), but did not significantly alter IL-4, IL-10 and IL-13 levels. MAC2+ macrophage, CD4+ T cell and CD20+ B cell numbers were not altered; however, numbers of CD3+ T cells (p ≤ 0.001) and CD8+ T cells (p ≤ 0.001) increased in the parenchyma. Although IL-13 (p = 0.008) expression decreased in the bronchiolar epithelium, Muc5ac gene expression was not altered in the lung of DEP-exposed animals. Although respiratory mechanics, elastic and collagen density were not modified, the mean linear intercept (Lm) was increased in the DEP-exposed animals (p ≤ 0.001), and the index D2 was statistically different (p = 0.038) from the control animals.

Conclusion

Our data suggest that nasal instillation of low doses of DEP over a period of 90 days results in alveolar enlargement in the pulmonary parenchyma of healthy mice.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0172-z) contains supplementary material, which is available to authorized users.     

Biosynthesis of chlorophyll P-680 of photosystem II in greening leaves of plants adapted to heat shock

In etiolated pea and maize leaves illuminated after incubation at 38 degreesC, a new dark reaction was shown manifested in the bathochromic shift of spectral bands and accompanied by esterification of the product of protochlorophyllide photochemical reduction--Chld 684/676: Chld 684/676 --> Chl 688/680. After completion of the reaction a rapid (20-30 sec) quenching of the fluorescence of the reaction product (Chl 688/680) was observed. The reaction Chld 684/676 --> Chl 688/680 is inhibited under anaerobic conditions and in the presence of cyanide; the reaction accompanied by Chl 688/680 fluorescence quenching is not observed in pea mutants with impaired function of photosystem II reaction centers. The spectral properties of the formed Chl form with the absorption maximum at 680 nm, fluorescence quenching, and simultaneous synthesis of pheophytin suggest that the reaction is connected with the chlorophyll of photosystem II reaction center--P-680.     

Crystal structure of cyclophilin A complexed with a binding site peptide from the HIV-1 capsid protein.

The cellular protein, cyclophilin A (CypA), is incorporated into the virion of the type 1 human immunodeficiency virus (HIV-1) via a direct interaction with the capsid domain of the viral Gag polyprotein. We demonstrate that the capsid sequence 87His-Ala-Gly-Pro-Ile-Ala92 (87HAGPIA92) encompasses the primary cyclophilin A binding site and present an X-ray crystal structure of the CypA/HAGPIA complex. In contrast to the cis prolines observed in all previously reported structures of CypA complexed with model peptides, the proline in this peptide, Pro 90, binds the cyclophilin A active site in a trans conformation. We also report the crystal structure of a complex between CypA and the hexapeptide HVGPIA, which also maintains the trans conformation. Comparison with the recently determined structures of CypA in complexes with larger fragments of the HIV-1 capsid protein demonstrates that CypA recognition of these hexapeptides involves contacts with peptide residues Ala(Val) 88, Gly 89, and Pro 90, and is independent of the context of longer sequences.