Peroxisome proliferator-activated receptor structures: ligand specificity, molecular switch and interactions with regulators.

Peroxisome proliferator-activated receptors (PPARs) compose a family of nuclear receptors that mediate the effects of lipidic ligands at the transcriptional level. In this review, we highlight advances in the understanding of the PPAR ligand binding domain (LBD) structure at the atomic level. The overall structure of PPARs LBD is described, and important protein ligand interactions are presented. Structure-activity relationships between isotypes structures and ligand specificity are addressed. It is shown that the numerous experimental three-dimensional structures available, together with in silico simulations, help understanding the role played by the activating function-2 (AF-2) in PPARs activation and its underlying molecular mechanism. The relation between the PPARs constitutive activity and the intrinsic stability of the active conformation is discussed. Finally, the interactions of PPARs LBD with co-activators or co-repressors, as well as with the retinoid X receptor (RXR) are described and considered in relation to PPARs activation.

Force-induced globule-coil transition in laminin binding protein and its role for viral-cell membrane fusion.

The specific interactions of the pairs laminin binding protein (LBP)-purified tick-borne encephalitis viral surface protein E and certain recombinant fragments of this protein, as well as West Nile viral surface protein E and certain recombinant fragments of that protein, are studied by combined methods of single-molecule dynamic force spectroscopy (SMDFS), enzyme immunoassay and optical surface waves-based biosensor measurements. The experiments were performed at neutral pH (7.4) and acid pH (5.3) conditions. The data obtained confirm the role of LBP as a cell receptor for two typical viral species of the Flavivirus genus. A comparison of these data with similar data obtained for another cell receptor of this family, namely human αVβ3 integrin, reveals that both these receptors are very important. Studying the specific interaction between the cell receptors in question and specially prepared monoclonal antibodies against them, we could show that both interaction sites involved in the process of virus-cell interaction remain intact at pH 5.3. At the same time, for these acid conditions characteristic for an endosome during flavivirus-cell membrane fusion, SMDFS data reveal the existence of a force-induced (effective already for forces as small as 30-70 pN) sharp globule-coil transition for LBP and LBP-fragments of protein E complexes. We argue that this conformational transformation, being an analog of abrupt first-order phase transition and having similarity with the famous Rayleigh hydrodynamic instability, might be indispensable for the flavivirus-cell membrane fusion process. Copyright © 2014 John Wiley & Sons, Ltd.

Protein-binding site at the immunoglobulin mu membrane polyadenylylation signal: possible role in transcription termination.

mRNAs specifying immunoglobulin mu and delta heavy chains are encoded by a single large, complex transcription unit (mu + delta gene). The transcriptional activity of delta gene segments in terminally differentiated, IgM-secreting B lymphocytes is 10-20 times lower than in earlier B-lineage cells expressing delta mRNA. We find that transcription of the mu + delta gene in IgM-secreting murine myeloma cells terminates within a region of 500-1000 nucleotides immediately following the mu membrane (mu m) polyadenylylation site. Transcription decreases only minimally through this region in murine cell lines representative of earlier stages in B-cell development. A DNA fragment containing the mu m polyadenylylation signal gives protein-DNA complexes with different mobilities in gel retardation assays with nuclear extracts from myeloma cells than with nuclear extracts from earlier B-lineage cells. However, using a recently developed "footprinting" procedure in which protein-DNA complexes resolved in gel retardation assays are subjected to nucleolytic cleavage while still in the polyacrylamide gel, we find that the DNA sequences protected by factors from the two cell types are indistinguishable. The factor-binding site on the DNA is located 5' of the mu m polyadenylylation signal AATAAA and includes the 15-nucleotide-long A + T-rich palindrome CTGTAAACAAATGTC. This type of palindromic binding site exhibits orientation-dependent activity consistent with the reported properties of polymerase II termination signals. This binding site is followed by two sets of directly repeated DNA sequences with different helical conformation as revealed by their reactivity with the chemical nuclease 1,10-phenanthroline-copper. The close proximity of these features to the signals for mu m mRNA processing may reflect a linkage of the processes of developmentally regulated mu m polyadenylylation and transcription termination.

Role of CD8 in aberrant function of cytotoxic T lymphocytes.

Using H-2Kd-restricted photoprobe-specific cytotoxic T lymphocyte (CTL) clones, which permit assessment of T cell receptor (TCR)-ligand interactions by TCR photoaffinity labeling, we observed that the efficiency of antigen recognition by CTL was critically dependent on the half-life of TCR-ligand complexes. We show here that antigen recognition by CTL is essentially determined by the frequency of serial TCR engagement, except for very rapid dissociations, which resulted in aberrant TCR signaling and antagonism. Thus agonists that were efficiently recognized exhibited rapid TCR-ligand complex dissociation, and hence a high frequency of serial TCR engagement, whereas the opposite was true for weak agonists. Surprisingly, these differences were largely accounted for by the coreceptor CD8. While it was known that CD8 substantially decreases TCR-ligand complex dissociation, we observed in this study that this effect varied considerably among ligand variants, indicating that epitope modifications can alter the CD8 contribution to TCR-ligand binding, and hence the efficiency of antigen recognition by CTL.

Immuno-electron microscopic identification of human estrogen receptor-DNA complexes at the estrogen-responsive element and in the first intron of a Xenopus vitellogenin gene.

Using an extract of nuclei from the estrogen-responsive human breast cancer cell line MCF-7, protein-DNA complexes were assembled in vitro at the 5' end of the Xenopus laevis vitellogenin gene B2 that is normally expressed in liver after estrogen induction. The complexes formed were analyzed by electron microscopy after labeling by the indirect colloidal gold immunological method using a monoclonal antibody specific for the human estrogen receptor. As identified by its interaction with protein A-gold, the antibody was found linked to two protein-DNA complexes, the first localized at the estrogen responsive element of the gene and the second in intron I, thus proving a direct participation of the receptor in these two complexes. The procedure used allows the visualization and rapid localization of specific transcription factors bound in vitro to a promoter or any other gene region.

Electron microscopic visualization of protein-DNA interactions at the estrogen responsive element and in the first intron of the Xenopus laevis vitellogenin gene.

Stable protein-DNA complexes can be assembled in vitro at the 5' end of Xenopus laevis vitellogenin genes using extracts of nuclei from estrogen-induced frog liver and visualized by electron microscopy. Complexes at the three following sites can be identified on the gene B2: the transcription initiation site, the estrogen responsive element (ERE) and in the first intron. The complex at the transcription initiation site is stabilized by dinucleotides and thus represents a ternary transcription complex. The formation of the complexes at the two other sites is enhanced by estrogen and is reduced by tamoxifen, an antagonist of estrogen, while this latter effect is reversed by adding an excess of hormone. No sequence homology is apparent between the site containing the ERE and the binding site in intron I and functional tests in MCF-7 cells suggest that these two sites are not equivalent. Finally, we made use of previously characterized deletion mutants of the 5' flanking region of the gene B1, a close relative of the gene B2, to demonstrate that the 13-bp palindromic core element of the ERE is involved in the formation of the complexes observed upstream of the transcription initiation site.

Studies on some Metal Complexes of Dithio Ligands

The thesis deals with our studies on the synthesis and elucidation of structure of some metal complexes of dithio ligands, such as the dithiocarbamates, xanthates and 2-aminocyclopent-1-ene-1-dithiocarboxylate and its N-alkyl derivatives.2-Aminocyclopent-1-ene-1-dithiocarboxylate (ACDA) is an interesting ligand, because of its potential dual capability of bonding between the metal and the ligand. Since the earlier reports on the complexes of ACDA contain contradictory ideas on the nature of its bonding, it was thought worthwhile to undertake a detailed and systematic study of these type of complexes. As the ACDA complexes have very low solubilities in solvents like chloroform, we have used the isopropyl derivative of ACDA as the ligand. The increased solubility of these complexes have made it possible to investigate their NMR and solution electronic spectra.The complexes of this ligand have not yet been reported in the literature.We have synthesised some new mixed ligand complexes of dithiocarbamates by reacting bis(dithiocarbamato)-µ-dichloro dicopper complexes (obtained by the reaction of mixed benzoic dithiocarbamic anhydride and copper(II) chloride) with ACDA or its N-alkyl derivatives.Interactions of metal halides with the mixed anhydrides formed from benzoylchloride and xanthates have also been investigated. Novel complexes of the type, [Cu2(RXant)CI] (R=i-Bu, i-Pr, n-Bu or n-Pr)) have been isolated from the reaction of copper(II) chloride and the mixed anhydride, and these reactions appear to be like the clock reactions reported in the literature.It also deal with the characterisation of the complexes of the type [Hg(R2d t c )X] (X=Cl, Br or 1),[Cu3(R2dtc)6][Cu2Br6] and [Cu(R2dtc)Cl2](R2=Me2,Et2,Pip,Morph or Pyrr)respectively, synthesised by simple and novel routes, different from those reported earlier.

Immersive Projection for biochemical pattern matching

In all biological processes, protein molecules and other small molecules interact to function and form transient macromolecular complexes. This interaction of two or more molecules can be described by a docking event. Docking is an important phase for structure-based drug design strategies, as it can be used as a method to simulate protein-ligand interactions. Various docking programs exist that allow automated docking, but most of them have limited visualization and user interaction. It would be advantageous if scientists could visualize the molecules participating in the docking process, manipulate their structures and manually dock them before submitting the new conformations to an automated docking process in an immersive environment, which can help stimulate the design/docking process. This also could greatly reduce docking time and resources. To achieve this, we propose a new virtual modelling/docking program, whereby the advantages of virtual modelling programs and the efficiency of the algorithms in existing docking programs will be merged.

The binding site distance test score: a robust method for the assessment of predicted protein binding sites

We propose a novel method for scoring the accuracy of protein binding site predictions – the Binding-site Distance Test (BDT) score. Recently, the Matthews Correlation Coefficient (MCC) has been used to evaluate binding site predictions, both by developers of new methods and by the assessors for the community wide prediction experiment – CASP8. Whilst being a rigorous scoring method, the MCC does not take into account the actual 3D location of the predicted residues from the observed binding site. Thus, an incorrectly predicted site that is nevertheless close to the observed binding site will obtain an identical score to the same number of nonbinding residues predicted at random. The MCC is somewhat affected by the subjectivity of determining observed binding residues and the ambiguity of choosing distance cutoffs. By contrast the BDT method produces continuous scores ranging between 0 and 1, relating to the distance between the predicted and observed residues. Residues predicted close to the binding site will score higher than those more distant, providing a better reflection of the true accuracy of predictions. The CASP8 function predictions were evaluated using both the MCC and BDT methods and the scores were compared. The BDT was found to strongly correlate with the MCC scores whilst also being less susceptible to the subjectivity of defining binding residues. We therefore suggest that this new simple score is a potentially more robust method for future evaluations of protein-ligand binding site predictions.

Equilibrium studies on mixed ligand complex formation of Co(II), Ni(II), Cu(II) and Zn(II) with N-(2-hydroxybenzyl)-L-histidine (H(2)hb-L-his) and typical N,N donor ligands: Crystal structure of [Ni(hb-L-his)(bipyridine)]center dot H2O complex

Equilibrium study on complex formation of Co(II), Ni(II), Cu(II) and Zn(II), hereafter M(II), with the quadridentate (O-, N, O-, N) donor ligand, N-(2-hydroxybenzyl)-L-histidine (H(2)hb-L-his, hereafter H2L), in the absence and in the presence of typical (N, N) donor bidentate ligands, 1,10 phenanthroline(phen), 2, 2'-bipyridine(bipy), ethylenediamine(en), hereafter B, in aqueous solution at 25 +/- 1 degrees C was done at a fixed ionic strength, I = 0.1 mol dm(-3) (NaNO3) by combined pH-metric, UV-Vis and EPR measurements provide evidence for the formation of mononuclear and dinuclear binary and mixed ligand complexes of the types: M(L), M(L)(2)(2-), M-2(L)(2+), M-2(H-1L)(+), M(L)(B), (B)M(H-1L)M(B)(+). The imidazole moiety of the ligand is found to act as a bridging bidentate ligand in the dinuclear M-2(L)(2+), M-2(H-1L)(+) and (B)M(H-1L)M(B)(+) complexes, using its N-3 atom and N1-H deprotonated moiety. Stability constants of the complexes provide evidence of discrimination of Cu(II) from the other M(II) ions by this ligand. Solid complexes: [Ni(L)(H2O)(2)] (1), [Cu(L)(H2O)] (2), and [Ni(L)(bipy)] (.) H2O (3) have been isolated and characterized by various physicochemical studies. Single crystal X-ray diffraction of the ternary complex, 3, shows an octahedral [(O-,N,N,O-)(N,N)] geometry with extensive pi-pi stacking of the aromatic rings and H-bonding with imidazole (N1-H), secondary amino N-atom, the lattice H2O molecule, and the carboxylate and phenolate O-atoms. (c) 2006 Elsevier B.V. All rights reserved.

Palladium and platinum complexes of 2-(2 '-carboxyphenylazo)-4-methylphenol: synthesis, structure and spectral properties

Reaction of 2-(2'-carboxyphenylazo)-4-methylphenol (H2L) with [M(PPh3)(2)Cl-2] (M = Pd, Pt) affords mixed-ligand complexes of type [M(PPh3)(L)]. Structures of both the complexes have been determined by X-ray crystallography. Both the complexes are square planar, where the 2-(2'-carboxyphenylazo)-4-methylphenol is coordinated to the metal center, via dissociation of the two acidic protons, as a dianionic tridentate O,N,O-donor, and the fourth position is occupied by the triphenylphosphine. These complexes show intense MLCT transitions in the visible region.

Characterization of protein-polyphenol interactions

Dietary polyphenols have received attention for their biologically significant functions as antioxidants, anticarcinogens or antimutagens, which have led to their recognition as potential nutraceuticals. Polyphenols also characteristically possess a significant binding affinity for proteins, which can lead to the formation of soluble and insoluble protein-polyphenol complexes. Questions remain concerning whether and to what extent the protein-polyphenol interaction influences functionality. For example, is the formation of protein-polyphenol complexes an obstacle to the nutritional bioavailability of either species? This article discusses the development of suitable methodologies to investigate the physicochemical basis of protein-polyphenol interactions and the influence of structure-activity relationships on binding affinities. (C) 2003 Elsevier Ltd. All rights reserved.

Metal complexes with atropisomeric sulfur ligands in asymmetric hydroformylation X-ray structure of [Rh2(μ-biphes)(cod)2] (H2biphes = 4,4′-biphenanthrene-3,3′-dithiol)

The addition of the atropisomeric racemic sulfur compound 4,4′-biphenanthrene-3,3′-dithiol (H2 biphes) to a dichloromethane solution of [{M(μ-OMe)(cod)}2] (M = Rh, Ir, cod = cycloocta-1,5-diene) afforded the dithiolate-bridged complexes [{Rh2(μ-biphes)(cod)2}n] (n = 2 5 or n = 1 6) and [{Ir2(μ-biphes)(cod)2}n]·nCH2Cl27. When 1,1′-binaphthalene-2,2′-dithiol (H2 binas) reacted with [{Ir(μ-OMe)(cod)}2], complex [Ir2(μ-binas)(cod)2] 8 was obtained. Complexes 5 and 6 reacted with carbon monoxide to give the dinuclear tetracarbonyl complex [Rh2(μ-biphes)(CO)4] 9. The reaction of 9 with PR3 provided the mixed-ligand complexes [{Rh2(μ-biphes)(CO)2(PR3)2}2] · xCH2Cl2 (R = Ph, x = 2 10, C6H11, x = 1 11) and [{Rh2(μ-biphes)(CO)3(PR3)}2] · CH2Cl212 (R = OC6H4But-o). The crystal structure of 6 was determined by X-ray diffraction. Reaction of the dithioether ligand Me2biphes with [Rh(cod)2]ClO4 in CH2Cl2 solution afforded the cationic complex [Rh(cod)(Me2biphes)]ClO4 · CH2Cl213. Asymmetric hydroformylation of styrene was performed using the complexes described. The extent of aldehyde conversion ranges from 53 to 100%, with selectivities towards branched aldehydes in the range 51 to 96%. The enantioselectivities were quite low and did not exceed 20%.

Protease-activated receptor 2: activation, signalling and function

PARs (protease-activated receptors) are a family of four G-protein-coupled receptors for proteases from the circulation, inflammatory cells and epithelial tissues. This report focuses on PAR(2), which plays an important role in inflammation and pain. Pancreatic (trypsin I and II) and extrapancreatic (trypsin IV) trypsins, mast cell tryptase and coagulation factors VIIa and Xa cleave and activate PAR(2). Proteases cleave PAR(2) to expose a tethered ligand that binds to the cleaved receptor. Despite this irreversible activation, PAR(2) signalling is attenuated by beta-arrestin-mediated desensitization and endocytosis, and by lysosomal targeting and degradation, which requires ubiquitination of PAR(2). beta-Arrestins also act as scaffolds for the assembly of multi-protein signalling complexes that determine the location and function of activated mitogen-activated protein kinases. Observations of PAR(2)-deficient mice support a role for PAR(2) in inflammation, and many of the effects of PAR(2) activators promote inflammation. Inflammation is mediated in part by activation of PAR(2) in the peripheral nervous system, which results in neurogenic inflammation and hyperalgesia.

Syntheses, structures and efficient catalysis for C–C coupling of some benzaldehyde thiosemicarbazone complexes of palladium

Reaction of the 4-R-benzaldehyde thiosemicarbazones (denoted in general as L-R; R = OCH(3), CH(3), H, Cl and NO(2)) with trans-[Pd(PPh(3))(2)Cl(2)] afforded a group of mixed-ligand complexes (denoted in general as 1-R) incorporating a N,S-coordinated thiosemicarbazone. a triphenylphosphine and a chloride. Similar reaction with Na(2)[PdCl(4)] afforded a family of bis-thiosemicarbazone complexes (denoted in general as 2-R), where each ligand is N,S-coordinated. Crystal structures of 1-CH(3), 1-NO(2), 2-OCH(3), 2-NO(2) and L-NO(2) have been determined. In all the complexes the thiosemicarbazones are coordinated to the metal center, via dissociation of the acidic proton, as bidentate N,S-donors forming five-membered chelate rings. With reference to the structure of the uncoordinated thiosemicarbazone, this coordination mode is associated with a conformational change around the C=N bond. All the 1-R and 2-R complexes display intense absorptions in the visible region. Catalytic activity of the 1-R and 2-R complexes towards some C-C coupling reactions (e.g. Suzuki, Heck and Sonogashira) has been examined and while both are found to be efficient catalysts, 1-R is much better catalyst than 2-R.