Presence Of Exogenous Ligands Research Articles

The Role of Heterogeneous Nucleation in the Activation of Blood Cells

The processes of receptors clusterization on the surface of blood cells are considered in the framework of nucleation theory. In the absence of ligands, the characteristic time of homogeneous nucleation during receptors clusterization was assumed to be much longer than the cell life. Varying the constants of the kinetics of bond formation and breaking during receptor clustering, it was possible to describe the activation conditions experimentally observed for several types of blood cells. It was shown that the characteristic time of heterogeneous nucleation leading to the receptors clusterization in the presence of exogenous ligands can be less than one minute. Parametric diagrams showing the effect of several ligands examined on the receptor system are constructed. Possible clinical implications of the results are discussed.

Роль гетерогенной нуклеации в активации клеток крови

The processes of receptors clustering on the surface of blood cells are considered in the nucleation theory framework. In the absence of ligands, the characteristic time of homogeneous nucleation of receptors clustering was assumed to be much longer than a cell's life. By varying the kinetics of the bonds' formation and breaking during receptor clustering, it was possible to describe the activation conditions experimentally observed for several types of blood cells. It was shown that the heterogeneous nucleation characteristic time leading to receptors clustering in the presence of exogenous ligands can be less than one minute. Parametric diagrams are constructed that display the effect of a number of examined ligands on the receptor system. The possible clinical significance of the results is discussed.

Early Post-stroke Activation of Vascular Endothelial Growth Factor Receptor 2 Hinders the Receptor 1-Dependent Neuroprotection Afforded by the Endogenous Ligand.

Vascular endothelial growth factor (VEGF) has long been connected to the development of tissue lesion following ischemic stroke. Contradictory findings either situate VEGF as a promoter of large infarct volumes or as a potential attenuator of damage due to its well documented neuroprotective capability. The core of this discrepancy mostly lies on the substantial number of pleiotropic functions driven by VEGF. Mechanistically, these effects are activated through several VEGF receptors for which various closely related ligands exist. Here, we tested in an experimental model of stroke how the differential activation of VEGF receptors 1 and 2 would modify functional and histological outcomes in the acute phase post-ischemia. We also assessed whether VEGF-mediated responses would involve the modulation of inflammatory mechanisms and how this trophic factor acted specifically on neuronal receptors. We produced ischemic infarcts in adult rats by transiently occluding the middle cerebral artery and induced the pharmacological inhibition of VEGF receptors by i.c.v. administration of the specific VEGFR2 inhibitor SU1498 and the pan-VEGFR blocker Axitinib. We evaluated the neurological performance of animals at 24 h following stroke and the occurrence of brain infarctions analyzed at the gross metabolic and neuronal viability levels. We also assessed the induction of peripheral pro- and anti-inflammatory cytokines in the cerebrospinal fluid and blood and assessed the polarization of activated microglia. Finally, we studied the direct involvement of cortical neuronal receptors for VEGF with in vitro assays of excitotoxic damage. Preferential VEGFR1 activation by the endogenous ligand promotes neuronal protection and prevents the presentation of large volume infarcts that highly correlate with neurological performance, while the concomitant activation of VEGFR2 reduces this effect, even in the presence of exogenous ligand. This process partially involves the polarization of microglia to the state M2. At the cellular level, neurons also responded better to the preferential activation of VEGFR1 when challenged to N-methyl-D-aspartate-induced excitotoxicity. Endogenous activation of VEGFR2 hinders the neuroprotective mechanisms mediated by the activation of VEGFR1. The selective modulation of these concurrent processes might enable the development of therapeutic approaches that target specific VEGFR1-mediated signaling during the acute phase post-stroke.

Abstract 5031: R-spondin potentiates Wnt/β-catenin signaling in Ewing sarcoma cells.

Abstract Proper regulation of Wnt signaling is highly context-dependent and critical for growth and cellular maintenance. It is well-established that dysregulation of the canonical Wnt/β-catenin pathway contributes to the development and progression of numerous malignancies. Despite this, there has been little evidence to support a role for canonical Wnt signaling in Ewing sarcoma (ES) pathogenesis. Recent studies have shown that when members of the leucine-rich G-protein coupled receptor (LGR) family, namely LGR4 and LGR5, are bound by their ligands, R-spondins, they potentiate Wnt/β-catenin activity. Importantly, recent studies from our lab have shown that some ES tumors and cell lines express high levels of LGR5. In addition, R-spondin is highly expressed in the developing bone, which is the primary site of ES in children. Since ES cells do not exhibit canonical Wnt/β-catenin activity in standard culture conditions, we hypothesized that cues from the tumor microenvironment may be required for activation of this pathway. In particular, we hypothesized that R-spondins would potentiate Wnt activity in LGR-expressing ES cells. To test this hypothesis, ES cells were cultured in the presence of WNT3A and/or R-spondin 2 (Rspo2). Immunocytochemistry was used to determine β-catenin nuclear localization, and Wnt transcriptional activity was determined by flow cytometry and luciferase activity using cell lines transduced with lentiviral 7xTCF-GFP and 7xTCF-luciferase reporters, respectively. qRT-PCR was used to confirm transcription of the Wnt target gene AXIN2. Our results show that exogenous WNT3A induces β-catenin nuclear localization in ES cells and is associated with induction of TCF-mediated transcriptional activity and AXIN2 expression. Furthermore, Wnt/β-catenin activation is potentiated in the presence of Rspo2, and ES cells lines with the highest expression of LGRs show the strongest potentiation. Interestingly, however, exogenous WNT3A and Rspo2 did not induce Wnt activity in all cells. Together these data confirm that in the presence of exogenous ligand, Wnt/β-catenin signaling can be robustly activated in a subpopulation of ES cells and that signaling is potently enhanced in the presence of Rspo2 and LGRs. To our knowledge, this is the first evidence of R-spondins influencing Wnt activity in a non-epithelial cancer. These studies support the concept that R-spondin expression in the tumor microenvironment may contribute to ES pathogenesis by modulating Wnt/β-catenin signaling. Citation Format: Elisabeth A. Pedersen, Christopher A. Scannell, Elizabeth R. Lawlor. R-spondin potentiates Wnt/β-catenin signaling in Ewing sarcoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5031. doi:10.1158/1538-7445.AM2013-5031

Bacteriogenic Manganese Oxides

Microorganisms control the redox cycling of manganese in the natural environment. Although the homogeneous oxidation of Mn(II) to form manganese oxide minerals is slow, solid MnO(2) is the stable form of manganese in the oxygenated portion of the biosphere. Diverse bacteria and fungi have evolved the ability to catalyze this process, producing the manganese oxides found in soils and sediments. Other bacteria have evolved to utilize MnO(2) as a terminal electron acceptor in respiration. This Account summarizes the properties of Mn oxides produced by bacteria (bacteriogenic MnO(2)) and our current thinking about the biochemical mechanisms of bacterial Mn(II) oxidation. According to X-ray absorption spectroscopy and X-ray scattering studies, the MnO(2) produced by bacteria consists of stacked hexagonal sheets of MnO(6) octahedra, but these particles are extremely small and have numerous structural defects, particularly cation vacancies. The defects provide coordination sites for binding exogenous metal ions, which can be adsorbed to a high loading. As a result, bacterial production of MnO(2) influences the bioavailability of these metals in the natural environment. Because of its high surface area and oxidizing power, bacteriogenic MnO(2) efficiently degrades biologically recalcitrant organic molecules to lower-molecular-mass compounds, spurring interest in using these properties in the bioremediation of xenobiotic organic compounds. Finally, bacteriogenic MnO(2) is reduced to soluble Mn(II) rapidly in the presence of exogenous ligands or sunlight. It can therefore help to regulate the bioavailability of Mn(II), which is known to protect organisms from superoxide radicals and is required to assemble the water-splitting complex in photosynthetic organisms. Bioinorganic chemists and microbiologists have long been interested in the biochemical mechanism of Mn(IV) oxide production. The reaction requires a two-electron oxidation of Mn(II), but genetic and biochemical evidence for several bacteria implicate multicopper oxidases (MCOs), which are only known to engage one-electron transfers from substrate to O(2). In experiments with the exosporium of a Mn(II)-oxidizing Bacillus species, we could trap the one-electron oxidation product, Mn(III), as a pyrophosphate complex in an oxygen-dependent reaction inhibited by azide, consistent with MCO catalysis. The Mn(III) pyrophosphate complex can further act as a substrate, reacting in the presence of the exosporium to produce Mn(IV) oxide. Although this process appears to be unprecedented in biology, it is reminiscent of the oxidation of Fe(II) to form Fe(2)O(3) in the ferritin iron storage protein. However, it includes a critical additional step of Mn(III) oxidation or disproportionation. We shall continue to investigate this biochemically unique process with purified enzymes.

Changing the Ligation of the Distal [4Fe4S] Cluster in NiFe Hydrogenase Impairs Inter- and Intramolecular Electron Transfers

In NiFe hydrogenases, electrons are transferred from the active site to the redox partner via a chain of three Iron-Sulfur clusters, and the surface-exposed [4Fe4S] cluster has an unusual His(Cys)3 ligation. When this Histidine (H184 in Desulfovibrio fructosovorans) is changed into a cysteine or a glycine, a distal cubane is still assembled but the oxidative activity of the mutants is only 1.5 and 3% of that of the WT, respectively. We compared the activities of the WT and engineered enzymes for H2 oxidation, H+ reduction and H/D exchange, under various conditions: (i) either with the enzyme directly adsorbed onto an electrode or using soluble redox partners, and (ii) in the presence of exogenous ligands whose binding to the exposed Fe of H184G was expected to modulate the properties of the distal cluster. Protein film voltammetry proved particularly useful to unravel the effects of the mutations on inter and intramolecular electron transfer (ET). We demonstrate that changing the coordination of the distal cluster has no effect on cluster assembly, protein stability, active-site chemistry and proton transfer; however, it slows down the first-order rates of ET to and from the cluster. All-sulfur coordination is actually detrimental to ET, and intramolecular (uphill) ET is rate determining in the glycine variant. This demonstrates that although [4Fe4S] clusters are robust chemical constructs, the direct protein ligands play an essential role in imparting their ability to transfer electrons.

Cleavage of the iron‐methionine bond in c‐type cytochromes: Crystal structure of oxidized and reduced cytochrome c2 from Rhodopseudomonas palustris and its ammonia complex

AbstractThe three‐dimensional structures of the native cytochrome c2 from Rhodopseudomonas palustris and of its ammonia complex have been obtained at pH 4.4 and pH 8.5, respectively. The structure of the native form has been refined in the oxidized state at 1.70 Å and in the reduced state at 1.95 Å resolution. These are the first high‐resolution crystal structures in both oxidation states of a cytochrome c2 with relatively high redox potential (+350 mV). The differences between the two oxidation states of the native form, including the position of internal water molecules, are small. The unusual six‐residue insertion Gly82‐Ala87, which precedes the heme binding Met93, forms an isolated 310‐helix secondary structural element not previously observed in other c‐type cytochromes. Furthermore, this cytochrome shows an external methionine residue involved in a strained folding near the exposed edge of the heme. The structural comparison of the present cytochrome c2 with other c‐type cytochromes has revealed that the presence of such a residue, with torsion angles ϕ and ξ of approximately −140 and −130°, respectively, is a typical feature of this family of proteins. The refined crystal structure of the ammonia complex, obtained at 1.15 Å resolution, shows that the sulphur atom of the Met93 axial ligand does not coordinate the heme iron atom, but is replaced by an exogenous ammonia molecule. This is the only example so far reported of an X‐ray structure with the heme iron coordinated by an ammonia molecule. The detachment of Met93 is accompanied by a very localized change in backbone conformation, involving mainly the residues Lys92, Met93, and Thr94. Previous studies under typical denaturing conditions, including high‐pH values and the presence of exogenous ligands, have shown that the detachment of the Met axial ligand is a basic step in the folding/unfolding process of c‐type cytochromes. The ammonia adduct represents a structural model for this important step of the unfolding pathway. Factors proposed to be important for the methionine dissociation are the strength of the H‐bond between the Met93 and Tyr66 residues that stabilizes the native form, and the presence in this bacterial cytochrome c2 of the rare six‐residue insertion in the helix 310 conformation that increases Met loop flexibility.

Cleavage of the iron-methionine bond in c-type cytochromes: crystal structure of oxidized and reduced cytochrome c(2) from Rhodopseudomonas palustris and its ammonia complex.

The three-dimensional structures of the native cytochrome c(2) from Rhodopseudomonas palustris and of its ammonia complex have been obtained at pH 4.4 and pH 8.5, respectively. The structure of the native form has been refined in the oxidized state at 1.70 A and in the reduced state at 1.95 A resolution. These are the first high-resolution crystal structures in both oxidation states of a cytochrome c(2) with relatively high redox potential (+350 mV). The differences between the two oxidation states of the native form, including the position of internal water molecules, are small. The unusual six-residue insertion Gly82-Ala87, which precedes the heme binding Met93, forms an isolated 3(10)-helix secondary structural element not previously observed in other c-type cytochromes. Furthermore, this cytochrome shows an external methionine residue involved in a strained folding near the exposed edge of the heme. The structural comparison of the present cytochrome c(2) with other c-type cytochromes has revealed that the presence of such a residue, with torsion angles phi and psi of approximately -140 and -130 degrees, respectively, is a typical feature of this family of proteins. The refined crystal structure of the ammonia complex, obtained at 1.15 A resolution, shows that the sulphur atom of the Met93 axial ligand does not coordinate the heme iron atom, but is replaced by an exogenous ammonia molecule. This is the only example so far reported of an X-ray structure with the heme iron coordinated by an ammonia molecule. The detachment of Met93 is accompanied by a very localized change in backbone conformation, involving mainly the residues Lys92, Met93, and Thr94. Previous studies under typical denaturing conditions, including high-pH values and the presence of exogenous ligands, have shown that the detachment of the Met axial ligand is a basic step in the folding/unfolding process of c-type cytochromes. The ammonia adduct represents a structural model for this important step of the unfolding pathway. Factors proposed to be important for the methionine dissociation are the strength of the H-bond between the Met93 and Tyr66 residues that stabilizes the native form, and the presence in this bacterial cytochrome c(2) of the rare six-residue insertion in the helix 3(10) conformation that increases Met loop flexibility.

Activation of peroxisome proliferator-activated receptors (PPARs) by their ligands and protein kinase A activators.

The nuclear peroxisome proliferator-activated receptors (PPARs) alpha, beta, and gamma activate the transcription of multiple genes involved in lipid metabolism. Several natural and synthetic ligands have been identified for each PPAR isotype but little is known about the phosphorylation state of these receptors. We show here that activators of protein kinase A (PKA) can enhance mouse PPAR activity in the absence and the presence of exogenous ligands in transient transfection experiments. Activation function 1 (AF-1) of PPARs was dispensable for transcriptional enhancement, whereas activation function 2 (AF-2) was required for this effect. We also show that several domains of PPAR can be phosphorylated by PKA in vitro. Moreover, gel retardation experiments suggest that PKA stabilizes binding of the liganded PPAR to DNA. PKA inhibitors decreased not only the kinase-dependent induction of PPARs but also their ligand-dependent induction, suggesting an interaction between both pathways that leads to maximal transcriptional induction by PPARs. Moreover, comparing PPAR alpha knockout (KO) with PPAR alpha WT mice, we show that the expression of the acyl CoA oxidase (ACO) gene can be regulated by PKA-activated PPAR alpha in liver. These data demonstrate that the PKA pathway is an important modulator of PPAR activity, and we propose a model associating this pathway in the control of fatty acid beta-oxidation under conditions of fasting, stress, and exercise.

Activation of Peroxisome Proliferator-Activated Receptors (PPARs) by Their Ligands and Protein Kinase A Activators

The nuclear peroxisome proliferator-activated receptors (PPARs) α, β, and γ activate the transcription of multiple genes involved in lipid metabolism. Several natural and synthetic ligands have been identified for each PPAR isotype but little is known about the phosphorylation state of these receptors. We show here that activators of protein kinase A (PKA) can enhance mouse PPAR activity in the absence and the presence of exogenous ligands in transient transfection experiments. Activation function 1 (AF-1) of PPARs was dispensable for transcriptional enhancement, whereas activation function 2 (AF-2) was required for this effect. We also show that several domains of PPAR can be phosphorylated by PKA in vitro. Moreover, gel retardation experiments suggest that PKA stabilizes binding of the liganded PPAR to DNA. PKA inhibitors decreased not only the kinase-dependent induction of PPARs but also their ligand-dependent induction, suggesting an interaction between both pathways that leads to maximal transcriptional induction by PPARs. Moreover, comparing PPARα knockout (KO) with PPARα WT mice, we show that the expression of the acyl CoA oxidase (ACO) gene can be regulated by PKA-activated PPARα in liver. These data demonstrate that the PKA pathway is an important modulator of PPAR activity, and we propose a model associating this pathway in the control of fatty acidβ -oxidation under conditions of fasting, stress, and exercise.

Processing of the transforming growth factor β type I and II receptors. Biosynthesis and ligand-induced regulation.

In Fig. 4 of the above mentioned manuscript we identified an ∼55-kDa 35S-labeled band as the mature type I TGF-β receptor which was not altered in its turnover by exogenous (10 ng/ml) recombinant TGFβ1. For this experiment, receptor turnover was assessed in the absence of glycosidases. In a recent paper, Dr. Wellset al. (1Wells R.G. Yankelev H. Lin H.Y. Lodish H.F. J. Biol. Chem. 1997; 272: 11444-11451Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) identify two species of type I receptors: an Endo H-sensitive population with a half-life of about 3 h and an Endo H-resistant population. By subtracting the amount of Endo H-sensitive receptor from the total amount (N-glycosidase F-treated) of TGF-βRI, the authors estimate that no more than 50% of the pulse-labeled TGF-βRI is converted to an Endo H-resistant form. The half-life of the latter could be estimated in excess of 2.5 h. The metabolic stability of the Endo H-sensitive receptor was not altered by exogenous TGFβ1 (Fig. 4B). However, since the total receptor population was less stable in the presence of exogenous ligand, the authors correctly infer that the Endo H-resistant, mature TGF-βRI is less stable in the presence of TGFβ1 (Fig. 4C). Therefore, it is likely that the type I receptor band in our paper represents the Endo H-sensitive population and not the total TGF-βRI as pointed out by Wells et al. (1Wells R.G. Yankelev H. Lin H.Y. Lodish H.F. J. Biol. Chem. 1997; 272: 11444-11451Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Hence, our conclusion that the half-life of TGF-βRI is not appreciably affected by exogenous TGF-β1 should apply only to the Endo H-sensitive TGF-βRI as correctly assessed by Wells et al.(1Wells R.G. Yankelev H. Lin H.Y. Lodish H.F. J. Biol. Chem. 1997; 272: 11444-11451Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). C. L. A. was aware of the paper by Wells et al. at the time of submission ((1997)J. Biol. Chem. 272, 6423–6427). Regretfully this was not acknowledged as it should have been in the latter manuscript. My formal apologies to Dr. Wells et al.

ChemInform Abstract: Electron Paramagnetic Resonance Studies of the Intramolecular Coordination in Superstructured Iron(III) Porphyrins.

AbstractEPR spectra of Fe(III) complexes of both face hindered porphyrins (basket‐handle porphyrins) (I), in which O or N ligand donors are incorporated into one of the two handles, are reported in the absence or presence of exogenous ligands.

Iron-molybdenum cofactor from nitrogenase. Modified extraction methods as probes for composition.

Five modifications of the preparative procedure for isolating iron-molybdenum cofactor (FeMoco) from the molybdenum-iron (MoFe) protein of Azotobacter vinelandii nitrogenase have been developed. This variety of isolation methods has established that no single component of the original isolation protocol, i.e. Tris, Cl-, citrate, HPO4(2-), N,N-dimethylformamide, and N-methylformamide, is essential for the effective isolation and/or structural stability of FeMoco, although any of them may act as ligands to FeMoco when present. The acid-bse status (effective pH) of the extracting solvent is a key adjustable parameter in the isolation procedure. The new procedures produced FeMoco with yields, metal analysis, charge, EPR spectrum, and specific activity (after reconstituting crude extracts from A. vinelandii UW45 mutant cells) essentially identical with FeMoco isolated by the original procedure. After purification, FeMoco apparently contains molybdenum, iron, and sulfide in a 1:7:4 ratio with N-methylformamide as a ligand but no amino acid residues, common sugars, coenzyme A, or lipoic acid. Reaction with o-phenanthroline allows quantitation of both adventitious and FeMoco-associated iron. Correlations of total activity after UW45 reconstitution with molybdenum, total iron, and o-phenanthroline-resistant iron contents show that only the last gives a consistent relationship of 35 +/- 5 nmol of C2H4/min/ng atom of Fe. Both o-phenanthroline and EDTA interact with FeMoco to abolish its EPR signal in reactions reversible by additions of Fe2+ or Zn2+, respectively. These and related reactions point against the presence of an endogenous organic component in FeMoco and toward the presence of exogenous ligands and imply a relatively labile coordination sphere whose nature may be determinable by a systematic investigation.