Addition Of Exogenous Ligands Research Articles

MR1 expression and surface translocation in airway epithelial cells from COPD or smoker lungs is altered following exposure to IFNγ or MAIT cells.

Abstract MAIT cells, a highly prevalent subset of T cells restricted by the MHC Class I-like molecule MR1, play a key role in early response to bacterial infection. Chronic obstructive pulmonary disease (COPD) pathogenesis is associated with airway inflammation, increased infiltration by CD8+ T lymphocytes, and infection-driven exacerbations. Cigarette smoke (CS), a leading cause of COPD, leads to impaired immune function of airway epithelial cells (AECs) and decreases surface expression of classical MHC Class I molecules. However, the impact of CS and COPD on MR1 expression and airway MAIT cell function is unknown. We have demonstrated that exposure to CS alters MAIT cell responses to primary AECs from healthy, COPD, or smoker lungs. Here, we examined whether CS impacts expression of MR1 mRNA or surface translocation of MR1 in these primary AEC populations as a possible mechanism for this dysregulation. Short-term treatment of AECs with CS or infection with S. pneumoniae (SP) did not alter MR1 expression in any of the donor AEC groups. However, incubating CS-treated or SP-infected AECs with MAIT cells increased MR1 expression. This upregulation may be mediated through IFNγ signaling pathways, as treatment with IFNγ alone resulted in MR1 upregulation. Our data further suggest MAIT cell- and IFNγ-mediated MR1 upregulation is altered in COPD cells. Additionally, we observed increased baseline surface expression of MR1 protein in smoker AECs and reduced surface translocation upon addition of exogenous ligand. COPD- and CS-mediated dysregulation of MR1 expression could impact MAIT cell function in airways, resulting in inflammation or reduced early response to bacteria, contributing to lower airway colonization and COPD exacerbations.

Effect of Membrane Environment on the Ligand-Binding Properties of the Terminal Oxidase Cytochrome bd-I from Escherichia coli.

Cytochrome bd-I is a terminal oxidase of the Escherichia coli respiratory chain. This integral membrane protein contains three redox-active prosthetic groups (hemes b558, b595, and d) and couples the electron transfer from quinol to molecular oxygen to the generation of proton motive force, as one of its important physiological functions. The study was aimed at examining the effect of the membrane environment on the ligand-binding properties of cytochrome bd-I by absorption spectroscopy. The membrane environment was found to modulate the ligand-binding characteristics of the hemoprotein in both oxidized and reduced states. Absorption changes upon the addition of exogenous ligands, such as cyanide or carbon monoxide (CO), to the detergent-solubilized enzyme were much more significant and heterogeneous than those observed with the membrane-bound enzyme. In the native membranes, both cyanide and CO interacted mainly with heme d. An additional ligand-binding site (heme b558) appeared in the isolated enzyme, as was evidenced by more pronounced changes in the absorption in the Soret band. This additional reactivity could also be detected after treatment of E. coli membranes with a detergent. The observed effect did not result from the enzyme denaturation, since reconstitution of the isolated enzyme into azolectin liposomes restored the ligand-binding pattern close to that observed for the intact membranes.

Impact of copper ligand mutations on a cupredoxin with a green copper center

Mononuclear cupredoxins contain a type 1 copper center with a trigonal or tetragonal geometry usually maintained by four ligands, a cystein, two histidines and a methionine. The recent discovery of new members of this family with unusual properties demonstrates, however, the versatility of this class of proteins. Changes in their ligand set lead to drastic variation in their metal site geometry and in the resulting spectroscopic and redox features. In our work, we report the identification of the copper ligands in the recently discovered cupredoxin AcoP. We show that even though AcoP possesses a classical copper ligand set, it has a highly perturbed copper center. In depth studies of mutant's properties suggest a high degree of constraint existing in the copper center of the wild type protein and even the addition of exogenous ligands does not lead to the reconstitution of the initial copper center. Not only the chemical nature of the axial ligand but also constraints brought by its covalent binding to the protein backbone might be critical to maintain a green copper site with high redox potential. This work illustrates the importance of experimentally dissecting the molecular diversity of cupredoxins to determine the molecular determinants responsible for their copper center geometry and redox potential.

Constitutive activation of LXR in macrophages regulates metabolic and inflammatory gene expression: identification of ARL7 as a direct target

Ligand activation of liver X receptors (LXRs) has been shown to impact both lipid metabolism and inflammation. One complicating factor in studies utilizing synthetic LXR agonists is the potential for pharmacologic and receptor-independent effects. Here, we describe an LXR gain-of-function system that does not depend on the addition of exogenous ligand. We generated transgenic mice expressing a constitutively active VP16-LXRα protein from the aP2 promoter. These mice exhibit increased LXR signaling selectively in adipose and macrophages. Analysis of gene expression in primary macrophages derived from two independent VP16-LXRα transgenic lines confirmed the ability of LXR to drive expression of genes involved in cholesterol efflux and fatty acid synthesis. Moreover, VP16-LXRα expression also suppressed the induction of inflammatory genes by lipopolysaccharide to a comparable degree as synthetic agonist. We further utilized VP16-LXRα-expressing macrophages to identify and validate new targets for LXRs, including the gene encoding ADP-ribosylation factor-like 7 (ARL7). ARL7 has previously been shown to transport cholesterol to the membrane for ABCA1-associated removal and thus may be integral to the LXR-dependent efflux pathway. We show that the ARL7 promoter contains a functional LXRE and can be transactivated by LXRs in a sequence-specific manner, indicating that ARL7 is a direct target of LXR. These findings provide further support for an important role of LXRs in the coordinated regulation of lipid metabolic and inflammatory gene programs in macrophages.

Site-Isolation and Recycling of PdCl2using PDMS Thimbles

Macroscopic thimbles composed of polydimethylsiloxane (PDMS) were used to site-isolate PdCl(2) from the products of Wacker-Tsuji oxidations and Pd-mediated homocouplings. The reactions were completed on the interior of hollow thimbles, and the organic products were isolated by their selective flux to the exterior of the thimbles. Although organic molecules had high flux through the walls of the thimbles, PdCl(2) remained encapsulated as a result of its polar structure that rendered it insoluble in PDMS. In two examples, less than 0.002% of the PdCl(2) added to the interior of the thimble partitioned to the exterior; thus, over 99.998% remained encapsulated on the interior of the thimble. Because it was encapsulated, this catalyst was readily recycled five times for the Wacker-Tsuji oxidation of styrene. A sequential reaction was also completed where p-methylstyrene was oxidized to 4'-methyl-acetophenone by PdCl(2) on the interior of a thimble and then fluxed to the exterior to react with phenylmagnesium bromide to yield 1-phenyl-1-p-tolyl-ethanol. This method site-isolated PdCl(2) catalysts without requiring them to be rendered heterogeneous, the addition of exogenous ligands, or any modifications to the catalyst. The catalyst was site-isolated by affecting its environment rather than by altering its ligand structure.

Conformation-Sensitive Steroid and Fatty Acid Sites in the Transmembrane Domain of the Nicotinic Acetylcholine Receptor

The mechanism by which some hydrophobic molecules such as steroids and free fatty acids (FFA) act as noncompetitive inhibitors of the nicotinic acetylcholine receptor (AChR) is still not known. In the present work, we employ Förster resonance energy transfer (FRET) between the intrinsic fluorescence of membrane-bound Torpedo californica AChR and the fluorescent probe Laurdan using the decrease in FRET efficiency (E) caused by steroids and FFA to identify potential sites of these hydrophobic molecules. Structurally different steroids produced similar changes (DeltaE) in FRET, and competition studies between them demonstrate that they occupy the same site(s). They also share their binding site(s) with FFA. Furthermore, the FRET conditions define the location of the sites at the lipid-protein interface. Endogenous production of FFA by controlled phospholipase A2 enzymatic digestion of membrane phospholipids yielded DeltaE values similar to those obtained by addition of exogenous ligand. This finding, together with the preservation of the sites in membranes subjected to controlled proteolysis of the extracellular AChR moiety with membrane-impermeable proteinase K, further refines the topology of the sites at the AChR transmembrane domain. Agonist-induced desensitization resulted in the masking of the sites observed in the absence of agonist, thus demonstrating the conformational sensitivity of FFA and steroid sites in the AChR.

Distinct Amino Acid Residues May Be Involved in Coactivator and Ligand Interactions in Hepatocyte Nuclear Factor-4α

Hepatocyte nuclear factor-4 (HNF-4) is a transcription factor of the nuclear hormone receptor superfamily that is constitutively active without the addition of exogenous ligand. Crystallographic analysis of the HNF-4alpha and HNF-4gamma ligand binding domains (LBDs) demonstrated the presence of endogenous ligands that may act as structural cofactors for HNF-4. It was also proposed by crystallographic studies that a combination of ligand and coactivator might be required to lock the receptor in its active state. We previously showed that mutations in amino acid residues Ser-181 and Met-182 in H3, Leu-219 and Leu-220 and Arg-226 in H5, Ileu-338 in H10, and Ileu-346 in H11, which line the LBD pocket in HNF-4alpha and come in contact with the ligand, impair its transactivation potential. In the present study, physical and functional interaction assays were utilized with two different coactivators, PGC-1 and SRC-3, to address the role of coactivators in HNF-4 function. We show that the integrity of the hinge (D) domain of HNF-4alpha and the activation function (AF)-2 activation domain region are critical for coactivation. Surprisingly, a different mode of coactivation is observed among the LBD point mutants that lack transcriptional activity. In particular, coactivation is maintained in mutants Ser-181, Arg-226, and Ile-346 but is abolished in mutants Met-182, Leu-219, and Ile-338. Physical interactions confirm this pattern of activation, implying that distinct amino acid residues may be involved in coactivator and ligand interactions, although some residues may be critical for both functions. Our results provide evidence and expand predictions based on the crystallographic data as to the role of coactivators in HNF-4alpha constitutive transcriptional activity.

Spatiotemporal retinoid-X receptor activation detected in live vertebrate embryos.

Most studies on the nuclear retinoid-X receptor (RXR) have focused on its role as a heterodimeric partner but less about its own activation pattern during development and the distribution of potential endogenous ligands. The aim of this study is to visualize the distribution of activated RXRalpha in live transgenic Xenopus laevis embryos across a wide range of developmental stages. We adopted a nuclear receptor-Gal4 fusion/upstream activation sequence-based reporter system for our assay. Strong activation of the RXRalpha ligand-binding domain was observed in a segment of the spinal cord just posterior to the hindbrain. This activation is first detected in neurula stage embryos and persists up to swimming tadpole stages, after which activation strongly declines. Addition of exogenous ligands, such as 9-cis retinoic acid or all-trans retinoic acid, expands the activation of RXR throughout the spinal cord but not in the brain, whereas the RXR-specific ligand LG268 expanded the Gal4-RXR activation into the brain and olfactory epithelia. Treatment with the RAR-specific ligand 4-(E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid or thyroid hormone had no effect on Gal4-RXR activation, whereas these compounds significantly increased their corresponding Gal4/receptor fusion proteins under similar conditions. Embryos expressing a Gal4-RXR fusion protein with a deletion in the ligand-dependent activation domain (AF2) show no reporter gene activation. The results shown in this paper reveal a specific activation pattern for Gal4-RXRalpha specifically in the developing spinal cord and suggest the existence of RXR ligand "hot-spots" in this region.

Magnetic and optical properties of copper-substituted alcohol dehydrogenase: a bisthiolate copper (II) complex.

Replacement of the catalytic Zn(II) in horse liver alcohol dehydrogenase (HLADH) with copper produces a mononuclear Cu(II) chromophore with a ligand set consisting of two cysteine sulphurs, one histidine nitrogen plus one further atom. The fourth ligand to the metal ion and the conformation of the protein may be altered by addition of exogenous ligands and/or the cofactor NADH. Absorbance, CD, low-temperature magnetic CD (MCD) and EPR spectra are presented of copper-substituted HLADH samples in both 'open' and 'closed' conformations and in the presence and absence of the exogenous ligands pyrazole and DMSO. The EPR spectra indicate a strong, predominantly axial field about the copper(II) ion with high copper-thiol (cysteine) covalence. The optical and MCD spectra are interpreted in terms of four d-d transitions to low energy, also reflecting the axial ligand field, and four charge-transfer transitions to copper(II) between 30000 and 16000 cm-1 arising from the two cysteine sulphur atoms which give two pairs of oppositely signed MCD C-terms. These transitions are polarized mainly in the axial plane defined by Cys-46, Cys-174 and His-67. The binary complex formed with pyrazole displays quite different EPR and optical spectra which can be understood in terms of a rotation of the copper hole-orbital away from the axial plane thus decreasing sharply the copper-thiol covalence. The magneto-optical spectra in the presence and absence of DMSO are indistinguishable.

Construction of a bisaquo heme enzyme and binding by exogenous ligands.

The crystal structure of the His-175-->Gly (H175G) mutant of cytochrome-c peroxidase (EC 1.11.1.5), missing its only heme ligand, reveals that the histidine is replaced by solvent to give a bisaquo heme protein. This protein retains some residual activity, which can be stimulated or inhibited by addition of exogenous ligands. Structural analysis confirms the binding of imidazole to the heme at the position of the wild-type histidine ligand. This imidazole complex reacts readily with hydrogen peroxide to produce a radical species with novel properties. However, reactivation in this complex is incomplete (approximately 5%), which, in view of the very similar structures of the wild-type and the H175G/imidazole forms, implies a critical role for tethering of the axial ligand in catalysis. This study demonstrates the feasibility of constructing heme enzymes with no covalent link to the protein and with unnatural ligand replacements. Such enzymes may prove useful in studies of electron transfer mechanisms and in the engineering of novel heme-based catalysts.

Electronic properties of the dissimilatory sulphite reductase from Desulfovibrio vulgaris (Hildenborough): comparative studies of optical spectra and relative reduction potentials for the [Fe4S4]-sirohaem prosthetic centres

The dissimilatory sulphite reductase (desulfoviridin) from the sulphate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) displays distinct optical and redox characteristics relative to the haem subunit of Escherichia coli assimilatory sulphite reductase. For high-spin pentaco-ordinate desulfoviridin there is minimal change in the absorbance of the oxidized chromophores both after reduction or after addition of exogenous ligands. A ligand-metal charge-transfer band approximately 702 nm is observed in both the oxidized and one-electron-reduced enzyme. E.p.r. spectroscopy has been used to define the relative reduction potentials for sirohaem and [Fe4S4] centres (delta E0 = Es0-Ec0) as a function of sirohaem axial co-ordination. Typically delta E0 lies in a range from -10 to -50 mV. These results show a correlation with the sigma-donor or pi-acceptor properties of the ligand and stand in sharp contrast with estimates for the E. coli enzyme. The electronic properties of the coupled [Fe4S4]-sirohaem redox centre common to both nitrite- and sulphite-reducing enzymes are apparently strongly dependent on the environment generated by protein side chains.

Vanadium chemistry of ascidians

1. 1. Addition of exogenous ligands, 2,2′-bipyridine and 1,10-phenanthroline, to a variety of aplousobranch and phlebobranch ascidians, oproduced deep purple oxo-bridged binuclear complexes of bis(chelate)vanadium(III) within compartment (including signet ring) and some unpigmented morula cells confirming the presence of vanadium(III) in these cells. 2. 2. Treatment with acetylacetone formed [VO(acac) 2] and [V(acac) 3]. 3. 3. Electron paramagnetic resonance studies confirmed the presence of vanadium(IV) in all vanadium-containing Aplousobranchs, and little or no vanadium(IV) in the Phlebobranchs, and identified two types of vanadium(IV) binding to organic ligands with the ‘oxidized’ Phlebobranchs generally of Type I and the Aplousobranchs generally of Type II with e.p.r. parameters: Type I— g x 1.992, g y 1.987, g z 1.967, A x 53.01, A y 51.95, A z 155.47 × 10 4 cm −1; Type II— g y 1.980, g y 1.985, g z 1.951, A x 59.08, A y 55.72, A x 166.68 × 10 4 cm −1. 4. 4. Electron microscopic investigation of the morula cells of the phlebobranch, Ecteinascidia nexa, showed that the intracellular globules were rich in vanadium.

Comparison of peripheral blood lymphocyte C3 receptor capping properties in patients with chronic lymphocytic leukemia and lymphoma

Bacteria-antibody complexes were used to study capping of receptors for the activated third component of complement on peripheral blood lymphocytes. The impairment of C3d receptor capping improves in patients with chronic lymphocytic leukemia responding to therapy, but this does not necessarily correspond to the fall in peripheral lymphocytosis. Following lymphapheresis, increased capping appears to be due to selective removal of noncapping cells, but the mechanisms of improvement remain unexplained following other therapeutic measures. Lymphoma capping at 37 degrees C was normal in untreated patients but impaired after treatment, suggesting that this is related to the therapy itself. A high proportion of lymphocytes had receptors in the capped configuration in the basal state before incubation. In non-Hodgkin's lymphoma this was unrelated to treatment status, but it occurred only in untreated patients with Hodgkin's lymphoma with no further increment in capping after incubation. These receptors may exist in the aggregated state without the addition of exogenous ligand.