Low-Resolution Molecular Models Reveal the Oligomeric State of the PPAR and the Conformational Organization of Its Domains in Solution

The peroxisome proliferator-activated receptors (PPARs) regulate genes involved in lipid and carbohydrate metabolism, and are targets of drugs approved for human use. Whereas the crystallographic structure of the complex of full length PPARγ and RXRα is known, structural alterations induced by heterodimer formation and DNA contacts are not well understood. Herein, we report a small-angle X-ray scattering analysis of the oligomeric state of hPPARγ alone and in the presence of retinoid X receptor (RXR). The results reveal that, in contrast with other studied nuclear receptors, which predominantly form dimers in solution, hPPARγ remains in the monomeric form by itself but forms heterodimers with hRXRα. The low-resolution models of hPPARγ/RXRα complexes predict significant changes in opening angle between heterodimerization partners (LBD) and extended and asymmetric shape of the dimer (LBD-DBD) as compared with X-ray structure of the full-length receptor bound to DNA. These differences between our SAXS models and the high-resolution crystallographic structure might suggest that there are different conformations of functional heterodimer complex in solution. Accordingly, hydrogen/deuterium exchange experiments reveal that the heterodimer binding to DNA promotes more compact and less solvent-accessible conformation of the receptor complex

Medium Chain Fatty Acids Are Selective Peroxisome Proliferator Activated Receptor (PPAR) γ Activators and Pan-PPAR Partial Agonists

Thiazolidinediones (TZDs) act through peroxisome proliferator activated receptor (PPAR) γ to increase insulin sensitivity in type 2 diabetes (T2DM), but deleterious effects of these ligands mean that selective modulators with improved clinical profiles are needed. We obtained a crystal structure of PPARγ ligand binding domain (LBD) and found that the ligand binding pocket (LBP) is occupied by bacterial medium chain fatty acids (MCFAs). We verified that MCFAs (C8–C10) bind the PPARγ LBD in vitro and showed that they are low-potency partial agonists that display assay-specific actions relative to TZDs; they act as very weak partial agonists in transfections with PPARγ LBD, stronger partial agonists with full length PPARγ and exhibit full blockade of PPARγ phosphorylation by cyclin-dependent kinase 5 (cdk5), linked to reversal of adipose tissue insulin resistance. MCFAs that bind PPARγ also antagonize TZD-dependent adipogenesis in vitro. X-ray structure B-factor analysis and molecular dynamics (MD) simulations suggest that MCFAs weakly stabilize C-terminal activation helix (H) 12 relative to TZDs and this effect is highly dependent on chain length. By contrast, MCFAs preferentially stabilize the H2-H3/β-sheet region and the helix (H) 11-H12 loop relative to TZDs and we propose that MCFA assay-specific actions are linked to their unique binding mode and suggest that it may be possible to identify selective PPARγ modulators with useful clinical profiles among natural products

Hyperalgesic and edematogenic effects of peptides isolated from the venoms of honeybee (Apis mellifera) and neotropical social wasps (Polybia paulista and Protonectarina sylveirae)

Stings by bees and wasps, including Brazilian species, are a severe public health problem. The local reactions observed after the envenoming includes typical inflammatory response and pain. Several studies have been performed to identify the substances, including peptides that are responsible for such phenomena. The aim of the present study is to characterize the possible nociceptive (hyperalgesic) and edematogenic effects of some peptides isolated from the venoms of the honeybee (Apis mellifera) and the social wasps Polybia paulista and Protonectarina sylveirae, in addition to characterize some of the mechanisms involved in these phenomena. For this purpose, different doses of the peptides mellitin (Apis mellifera), Polybia-MP-I, N-2-Polybia-MP-I (Polybia paulista), Protonectarina-MP-NH2 and Protonectarina-MP-OH (Protonectarina sylveirae) were injected into the hind paw of mice. Hyperalgesia and edema were determined after peptide application, by using an electronic von Frey apparatus and a paquimeter. Carrageenin and saline were used as controls. Results showed that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH(2) and Protonectarina-MP-OH peptides produced a dose- and time-related hyperalgesic and edematogenic responses. Both phenomena are detected 2 h after melittin, Polybia-MP-I, N-2-Polybia-MP-I injection; their effects lasted until 8 h. In order to evaluate the role of prostanoids and the involvement of lipidic mediators in hyperalgesia induced by the peptides, indomethacin and zileuton were used. Results showed that zileuton blocked peptide-induced hyperalgesia and induced a decrease of the edematogenic response. on the other hand, indomethacin did not interfere with these phenomena. These results indicate that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH(2), and Protonectarina-MP-OH peptides could contribute to inflammation and pain induced by insect venoms.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Agelaia MP-I: A peptide isolated from the venom of the social wasp, Agelaia pallipes pallipes, enhances insulin secretion in mice pancreatic islets

Peptides isolated from animal venoms have shown the ability to regulate pancreatic beta cell function. Characterization of wasp venoms is important, since some components of these venoms present large molecular variability, and potential interactions with different signal transduction pathways. For example, the well studied mastoparan peptides interact with a diversity of cell types and cellular components and stimulate insulin secretion via the inhibition of ATP dependent K + (K ATP) channels, increasing intracellular Ca 2+ concentration. In this study, the insulin secretion of isolated pancreatic islets from adult Swiss mice was evaluated in the presence of synthetic Agelaia MP-I (AMP-I) peptide, and some mechanisms of action of this peptide on endocrine pancreatic function were characterized. AMP-I was manually synthesized using the Fmoc strategy, purified by RP-HPLC and analyzed using ESI-IT-TOF mass spectrometry. Isolated islets were incubated at increasing glucose concentrations (2.8, 11.1 and 22.2 mM) without (Control group: CTL) or with 10 μM AMP-I (AMP-I group). AMP-I increased insulin release at all tested glucose concentrations, when compared with CTL (P < 0.05). Since molecular analysis showed a potential role of the peptide interaction with ionic channels, insulin secretion was also analyzed in the presence of 250 μM diazoxide, a K ATP channel opener and 10 μM nifedipine, a Ca 2+ channel blocker. These drugs abolished insulin secretion in the CTL group in the presence of 2.8 and 11.1 mM glucose, whereas AMP-I also enhanced insulin secretory capacity, under these glucose conditions, when incubated with diazoxide and nifedipine. In conclusion, AMP-I increased beta cell secretion without interfering in K ATP and L-type Ca 2+ channel function, suggesting a different mechanism for this peptide, possibly by G protein interaction, due to the structural similarity of this peptide with Mastoparan-X, as obtained by modeling. © 2012 Elsevier Ltd

Shikimate Kinase (EC 2.7.1.71) from Mycobacterium tuberculosis: Kinetics and Structural Dynamics of a Potential Molecular Target for Drug Development

The enzymes of the shikimate pathway represent potential molecular targets for the development of non-toxic antimicrobial agents and anti-parasite drugs. One of the most promising of these enzymes is shikimate kinase (EC 2.7.1.71), which is responsible for the fifth step in the shikimate pathway. This enzyme phosphorylates shikimic acid to yield shikimate-3-phosphate, using ATP as a substrate. In this work, the conformational dynamics of the shikimate kinase from Mycobacterium tuberculosis was investigated in its apostate in solution. For this study, the enzyme was subjected to a gradient of temperatures from 15 degrees C to 45 degrees C in the presence or absence of deuterium oxide, and the amide H/D exchange was monitored using ESI-mass spectrometry. We observed: i) the phosphate binding domain in the apo-enzyme is fairly rigid and largely protected from solvent access, even at relatively high temperatures; ii) the shikimate binding domain is highly flexible, as indicated by the tendency of the apo-enzyme to exhibit large conformational changes to permit LID closure after the shikimate binding; iii) the nucleotide binding domain is initially conformationally rigid, which seems to favour the initial orientation of ADP/ATP, but becomes highly flexible at temperatures above 30 degrees C, which may permit domain rotation; iv) part of the LID domain, including the phosphate binding site, is partially rigid, while another part is highly flexible and accessible to the solvent.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Analysis of Agonist and Antagonist Effects on Thyroid Hormone Receptor Conformation by Hydrogen/Deuterium Exchange

Thyroid hormone receptors (TRs) are ligand-gated transcription factors with critical roles in development and metabolism. Although x-ray structures of TR ligand-binding domains (LBDs) with agonists are available, comparable structures without ligand (apo-TR) or with antagonists are not. It remains important to understand apo-LBD conformation and the way that it rearranges with ligands to develop better TR pharmaceuticals. In this study, we conducted hydrogen/deuterium exchange on TR LBDs with or without agonist (T(3)) or antagonist (NH(3)). Both ligands reduce deuterium incorporation into LBD amide hydrogens, implying tighter overall folding of the domain. As predicted, mass spectroscopic analysis of individual proteolytic peptides after hydrogen/deuterium exchange reveals that ligand increases the degree of solvent protection of regions close to the buried ligand-binding pocket. However, there is also extensive ligand protection of other regions, including the dimer surface at H10-H11, providing evidence for allosteric communication between the ligand-binding pocket and distant interaction surfaces. Surprisingly, C-terminal activation helix H12, which is known to alter position with ligand, remains relatively protected from solvent in all conditions suggesting that it is packed against the LBD irrespective of the presence or type of ligand. T(3), but not NH(3), increases accessibility of the upper part of H3-H5 to solvent, and we propose that TR H12 interacts with this region in apo-TR and that this interaction is blocked by T(3) but not NH(3.) We present data from site-directed mutagenesis experiments and molecular dynamics simulations that lend support to this structural model of apo-TR and its ligand-dependent conformational changes. (Molecular Endocrinology 25: 15-31, 2011)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)[300220/96-0]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[03/09462-5]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[06/00182-8]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[08/00078-1]National Institutes of Health (NIH)[DK41482]U.S. National Institutes of Health (NIH)National Institutes of Health (NIH)[51281]U.S. National Institutes of Health (NIH

Isolation and chemical characterization of PwTx-II: A novel alkaloid toxin from the venom of the spider Parawixia bistriata (Araneidae, Araneae)

Brazil has many species of spiders belonging to Araneidae family however, very little is known about the composition, chemical structure and mechanisms of action of the main venom components of these spiders. The main objective of this work was to isolate and to perform the chemical characterization of a novel beta-carboline toxin from the venom of the spider Parawixia bistriata, a typical species of the Brazilian 'cerrado'. The toxin was purified by RP-HPLC and structurally elucidated by using a combination of different spectroscopic techniques (UV, ESI-MS/MS and H-1 NMR), which permitted the assignment of the molecular structure of a novel spider venom toxin, identified as 1-4-guanidinobutoxy-6-hydroxy-1,2,3,4-tetrahydro-beta-carboline, and referred to here as PwTx-II. This compound is toxic to insects (LD50 = 12 +/- 3 eta g/mg honeybee), neurotoxic, convulsive and lethal to rats (LD50 = 9.75 mg/kg of male Wistar rat). (c) 2005 Elsevier Ltd. All rights reserved

Mode of peroxisome proliferator-activated receptor gamma activation by luteolin

The peroxisome proliferator-activated receptor γ (PPARγ) is a target for treatment of type II diabetes and other conditions. PPARγ full agonists, such as thiazolidinediones (TZDs), are effective insulin sensitizers and anti-inflammatory agents, but their use is limited by adverse side effects. Luteolin is a flavonoid with anti-inflammatory actions that binds PPARγ but, unlike TZDs, does not promote adipocyte differentiation. However, previous reports suggested variously that luteolin is a PPARγ agonist or an antagonist. We show that luteolin exhibits weak partial agonist/antagonist activity in transfections, inhibits several PPARγ target genes in 3T3-L1 cells (LPL, ORL1, and CEBPα) and PPARγ-dependent adipogenesis, but activates GLUT4 to a similar degree as rosiglitazone, implying gene-specific partial agonism. The crystal structure of the PPARγ ligand-binding domain (LBD) reveals that luteolin occupies a buried ligand-binding pocket (LBP) but binds an inactive PPARγ LBD conformer and occupies a space near the β-sheet region far from the activation helix (H12), consistent with partial agonist/antagonist actions. A single myristic acid molecule simultaneously binds the LBP, suggesting that luteolin may cooperate with other ligands to bind PPARγ, and molecular dynamics simulations show that luteolin and myristic acid cooperate to stabilize the Ω-loop among H2′, H3, and the β-sheet region. It is noteworthy that luteolin strongly suppresses hypertonicity-induced release of the pro-inflammatory interleukin-8 from human corneal epithelial cells and reverses reductions in transepithelial electrical resistance. This effect is PPARγ-dependent. We propose that activities of luteolin are related to its singular binding mode, that anti-inflammatory activity does not require H12 stabilization, and that our structure can be useful in developing safe selective PPARγ modulators816788799COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPSem informação2007/58443-4; 2010/08680-