The influence of Lys68 in decapeptide agonists of C5a on C5a receptor binding, activation and selectivity

The potent, conformationally biased C5a agonist peptide YSFKPMPLaR (C5a(65-74), Y65, F67, P69, P71, D-Ala73) was used as a template to gain insight into the nature and importance of lysine at position 68 in the peptide-receptor interaction. A panel of YSFKPMPLaR analogs with systematic substitutions for Lys68 was evaluated for C5a receptor (C5aR) binding affinity and activation in two well-characterized assay systems: human polymorphonuclear leukocytes (PMNs) and human fetal artery. In addition, we determined the activity of these new analogs in transfected rat basophilic leukemia (RBL) cells in which the Glu at position 199 of the C5aR (wtGlu199) was replaced by a Gin (C5aR-Gln199) or a Lys (C5aR-Lys199). Our results indicated that Lys68 in YSFKPMPLaR plays an important role in binding the C5aR expressed on PMNs and RBL cells. Furthermore, the data indicated that Lys68 interacted with Glu199 of the C5aR in PMNs and RBL cells. In human fetal artery, however, Lys68 substitutions had little or no effect on activity, which suggested that the receptor conformation may be different in this tissue. Thus, the interaction between Lys68 of the decapeptide agonist and Glu199 of the C5aR may be cell type-specific and may form the molecular basis for tissue-specific responses to C5a agonists.

Growth hormone binding protein correlates strongly with leptin and percentage body fat in GH-deficient adults, is increased by GH replacement but does not predict IGF-I response

GH-binding protein (GHBP) corresponds to the extracellular domain of the GH receptor (GHR) and has been shown to be closely related to body fat. This study aimed to examine the inter-relationship between GHBP, leptin and body fat, and to test the hypothesis that GHBP is modified by GH replacement in GH-deficient adults and predicts IGF-I response. Twenty adults, mean age 47 years (range 20-69) with proven GH deficiency were randomly allocated to either GH (up to 0.25 U/kg/week in daily doses) or placebo for 3 months before cross-over to the opposite treatment. Plasma GHBP and leptin were measured at baseline and 2, 4, 8 and 12 weeks after each treatment. Whole body composition was measured at baseline by dual-energy X-ray absorptiometry (DEXA). There was a strong correlation between baseline leptin and GHBP (r = 0.88, P < 0.0001) and between baseline GHBP and percentage body fat, (r = 0.83, P < 0.0001). Mean GHBP levels were higher on GH compared with placebo, 1.53 +/- 0.28 vs 1.41 +/- 0.25 nM, P = 0.049. There was no correlation between baseline IGF-I and GHBP (r = -0.049, P = 0.84), and GHBP did not predict IGF-I response to GH replacement. The close inter-relationship between GHBP, leptin and body fat suggests a possible role for GHBP in the regulation of body composition. GHBP is increased by GH replacement in GH-deficient adults, but does not predict biochemical response to GH replacement. (C) 1999 Churchill Livingstone.

The solution structure of the N-terminal zinc finger of GATA-1 reveals a specific binding face for the transcriptional co-factor FOG

Zinc fingers (ZnFs) are generally regarded as DNA-binding motifs. However, a number of recent reports have implicated particular ZnFs in the mediation of protein-protein interactions. The N-terminal ZnF of GATA-1 (NF) is one such finger, having been shown to interact with a number of other proteins, including the recently discovered transcriptional co-factor FOG. Here we solve the three-dimensional structure of the NF in solution using multidimensional H-1/N-15 NMR spectroscopy, and we use H-1/N-15 spin relation measurements to investigate its backbone dynamics. The structure consists of two distorted beta-hairpins and a single alpha-helix, and is similar to that of the C-terminal ZnF of chicken GATA-1. Comparisons of the NF structure with those of other C-4-type zinc binding motifs, including hormone receptor and LIM domains, also reveal substantial structural homology. Finally, we use the structure to map the spatial locations of NF residues shown by mutagenesis to be essential for FOG binding, and demonstrate that these residues all lie on a single face of the NE Notably, this face is well removed from the putative DNA-binding face of the NE an observation which is suggestive of simultaneous roles for the NF; that is, stabilisation of GATA-1 DNA complexes and recruitment of FOG to GATA-1-controlled promoter regions.

Crystal structure of human T cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins

Retroviral entry into cells depends on envelope glycoproteins, whereby receptor binding to the surface-exposed subunit triggers membrane fusion by the transmembrane protein (TM) subunit. We determined the crystal structure at 2.5-Angstrom resolution of the ectodomain of gp21, the TM from human T cell leukemia virus type 1. The gp21 fragment was crystallized as a maltose-binding protein chimera, and the maltose-binding protein domain was used to solve the initial phases by the method of molecular replacement. The structure of gp21 comprises an N-terminal trimeric coiled coil, an adjacent disulfide-bonded loop that stabilizes a chain reversal, and a C-terminal sequence structurally distinct from HIV type 1/simian immunodeficiency virus gp41 that packs against the coil in an extended antiparallel fashion. Comparison of the gp21 structure with the structures of other retroviral TMs contrasts the conserved nature of the coiled coil-forming region and adjacent disulfide-bonded loop with the variable nature of the C-terminal ectodomain segment. The structure points to these features having evolved to enable the dual roles of retroviral TMs: conserved fusion function and an ability to anchor diverse surface-exposed subunit structures to the virion envelope and infected cell surface. The structure of gp21 implies that the N-terminal fusion peptide is in close proximity to the C-terminal transmembrane domain and likely represents a postfusion conformation.

Targeting local tissues by transdermal application: Understanding drug physicochemical properties that best exploit protein binding and blood flow effects

The targeting of topically applied drug molecules into tissues below a site of application requires an understanding of the complex interrelationships between the drug, its formulation, the barrier properties of the skin, and the physiological processes occurring below the skin that are responsible for drug clearance from the site, tissue, and/or systemic distribution and eventual elimination. There is still a certain amount of controversy over the ability of topically applied drugs to penetrate into deeper tissues by diffusion or whether this occurs by redistribution in the systemic circulation. The major focus of our work in this area has been in determining how changes in drug structure and physicochemical properties, such as protein binding and lipophilicity, affect drug clearance into the local dermal microcirculation and lymphatics, as well as subsequent distribution into deeper tissues below an application site. The present study outlines our recent thinking on the drug molecule optimal physical attributes, in terms of plasma and tissue partitioning behaviour, that offer the greatest potential for deep tissue targeting. Drug Dev. Res. 46:309-315, 1999. (C) 1999 Wiley-Liss, Inc.

Binding of YY1 and Oct1 to a novel element that downregulates expression of IL-5 in human T cells

Background: IL-5 controls development of eosinophilia and has been shown to be involved in the pathogenesis of allergic diseases. In both atopic and nonatopic asthma, elevated IL-5 has been detected in peripheral blood and the airways. IL-5 is produced mainly by activated T cells, and its expression is regulated at the transcriptional level. Objective: This study focuses on the functional analysis of the human IL-5 (hIL-5) promoter and characterization of eis-regulatory elements and transcription factors involved in the suppression of IL-5 transcription in T cells. Methods: Methods used in this study include DNase I footprint assays, electrophoretic mobility shift assays, and functional analysis by mammalian cell transfection involving deletion analysis and site-directed mutagenesis. Results: We identified 5 protein binding regions (BRs) located within the proximal hIL-5 promoter. Functional analysis indicates that the BRs are involved in control of hIL-5 promoter activity. Two of these regions, BR3 and BR4 located at positions -102 to -73, have not previously been described as regulators of IL-5 expression in T cells. We show that the BR3 sequence contains a novel negative regulatory element located at positions -90 to -79 of the hIL-5 promoter, which binds Oct1, octamer-like, and YY1 nuclear factors. Substitution mutations, which abolished binding of these proteins to the BR3 sequence, significantly increased hIL-5 promoter activity in activated T cells. Conclusion: We suggest that Oct1, YY1, and octamer-like factors binding to the -90/-79 sequence within the proximal IL-5 promoter are involved in suppression of IL-5 transcription in T cells.

NMR studies of the low-density lipoprotein receptor-binding peptide of apolipoprotein E bound to dodecylphosphocholine micelles

Circular dichroism and NMR spectroscopy have been used to determine the structure of the low-density lipoprotein (LDL) receptor-binding peptide, comprising residues 130-152, of the human apolipoprotein E. This peptide has little persistent three-dimensional structure in solution, but when bound to micelles of dodecylphosphocholine (DPC) it adopts a predominantly alpha-helical structure. The three-dimensional structure of the DPC-bound peptide has been determined by using H-1-NMR spectroscopy: the structure derived from NOE-based distance constraints and restrained molecular dynamics is largely helical. The derived phi and psi angle order parameters show that the helical structure is well defined but with some flexibility that causes the structures not to be superimposable over the full peptide length. Deuterium exchange experiments suggest that many peptide amide groups are readily accessible to the solvent, but those associated with hydrophobic residues exchange more slowly, and this helix is thus likely to be positioned on the surface of the DPC micelles. In this conformation the peptide has one hydrophobic face and two that are rich in basic amino acid side chains. The solvent-exposed face of the peptide contains residues previously shown to be involved in binding to the LDL receptor.

Computational binding assays of antigenic peptides

Computer models can be combined with laboratory experiments for the efficient determination of (i) peptides that bind MHC molecules and (ii) T-cell epitopes. For maximum benefit, the use of computer models must be treated as experiments analogous to standard laboratory procedures. This requires the definition of standards and experimental protocols for model application. We describe the requirements for validation and assessment of computer models. The utility of combining accurate predictions with a limited number of laboratory experiments is illustrated by practical examples. These include the identification of T-cell epitopes from IDDM-, melanoma- and malaria-related antigens by combining computational and conventional laboratory assays. The success rate in determining antigenic peptides, each in the context of a specific HLA molecule, ranged from 27 to 71%, while the natural prevalence of MHC-binding peptides is 0.1-5%.

Identification of a new ligand binding domain in the al subunit of the inhibitory glycine receptor

Four discontinuous extracellular sequence domains have been proposed to form the ligand binding sites of the ligand-gated ion channel receptor superfamily. In this study, we investigated the role of 12 contiguous residues of the inhibitory glycine receptor that define the proposed loop A ligand binding domain; Using the techniques of site-directed mutagenesis and patch-clamp electrophysiology, four of the 12 residues were shown to have impaired ligand binding. Three mutants, I93A, A101H, and N102A, resulted in significant (17-44-fold) increases in the agonist EC50 values as compared with the wild-type glycine receptor, whereas Hill coefficients, I-max values, and antagonist affinity remained largely unaffected. Consideration of receptor efficacy values indicates that these residues are involved in ligand binding rather than channel activation. A fourth mutant, W94A, failed to give rise to any glycine-activated currents, although cell-surface expression was observed, suggesting that this residue may also be involved in agonist binding. These data provide the most extensive characterization of the loop A ligand binding domain available to date and define two new residue locations, Ile(93) and Asn(102), as contributing to the four-loop model of ligand binding.

Identification of residues involved in binding of IL5 to beta(com) using beta(IL3) and beta(com) chimeras

In mice there are two forms of the beta chain used in the IL3 receptor system, beta(com) and beta(IL3). beta(com) is used by the IL3, ILS and GM-CSF receptors whereas Pns is only used in the IL3 receptor. In this work an assay was developed to identify residues of beta(IL3) that restrict IL5 activity. It was found that such residues reside within the 2nd CRM of the molecule. Furthermore, when residues in the beta(IL3) B'-C' loop were replaced with beta(com) sequence a form of beta(IL3) was produced that was able to respond to IL5. This region is also responsible for IL3 binding to beta(IL3) in the absence of alpha chain. It is therefore an important structural motif of beta(com) and beta(IL3) responsible for both ligand interaction and specificity. (C) 1999 Federation of European Biochemical Societies.

Putative beta(4)-adrenoceptors in rat ventricle mediate increases in contractile force and cell Ca2+: comparison with atrial receptors and relationship to (-)-[H-3]-CGP 12177 binding

1 We identified putative beta(4)-adrenoceptors by radioligand binding, measured increases in ventricular contractile force by (-)-CGP 12177 and (+/-)-cyanopindolol and demonstrated increased Ca2+ transients by (-)-CGP 12177 in rat cardiomyocytes. 2 (-)-[H-3]-CGP 12177 labelled 13-22 fmol mg(-1) protein ventricular beta(1), beta(2)-adrenoceptors (pK(D) similar to 9.0) and 50-90 fmol mg(-1) protein putative beta(4)-adrenoceptors (pK(D) similar to 7.3). The affinity values (PKi) for (beta(1),beta(2)-) and putative beta(4)-adrenoceptors, estimated from binding inhibition, were (-)-propranolol 8.4, 5.7; (-)-bupranolol 9.7, 5.8; (+/-)-cyanopindolol 10.0,7.4. 3 In left ventricular papillary muscle, in the presence of 30 mu M 3-isobutyl-1-methylxanthine, (-)CGP 12177 and (+/-)-cyanopindolol caused positive inotropic effects, (pEC(50) (-)-CGP 12177, 7.6; (+/-)-cyanopindolol, 7.0) which were antagonized by (-)-bupranolol (pK(B) 6.7-7.0) and (-)-CGP 20712A (pK(B) 6.3-6.6). The cardiostimulant effects of(-)-CGP 12177 in papillary muscle, left and right atrium were antagonized by (+/-)-cyanopindolol (pK(i), 7.0-7.4). 4 (-)-CGP 12177 (1 mu M) in the presence of 200 nM (-)-propranolol increased Ca2+ transient amplitude by 56% in atrial myocytes, but only caused a marginal increase in ventricular myocytes. In the presence of 1 mu M 3-isobutyl-1-methylxanthine and 200 nM (-)-propranolol, 1 mu M (-)-CGP 12177 caused a 73% increase in Ca2+ transient amplitude in ventricular myocytes. (-)-CGP 12177 elicited arrhythmic transients in some atrial and ventricular myocytes. 5 Probably by preventing cyclic AMP hydrolysis, 3-isobutyl-1-methylxanthine facilitates the inotropic function of ventricular putative beta(4)-adrenoceptors. suggesting coupling to G(s) protein-adenylyl cyclase. The receptor-mediated increases in contractile force are related to increases of Ca2+ in atrial and ventricular myocytes. The agreement of binding affinities of agonists with cardiostimulant potencies is consistent with mediation through putative beta(4)-adrenoceptors labelled with (-)-[H-3]-CGP 12177.

Implication of the visual system in the regulation of activity cycles in the absence of solar light: 2-[I-125]iodomelatonin binding sites and melatonin receptor gene expression in the brains of demersal deep-sea gadiform fish

Relative eye size, gross brain morphology and central localization of 2-[I-125]iodomelatonin binding sites and melatonin receptor gene expression were compared in six gadiform fish living at different depths in the north-east Atlantic Ocean: Phycis blennoides (capture depth range 265-1260 m), Nezumia aequalis (445-1512 m), Coryphaenoides rupestris (706-1932 m), Trachyrincus murrayi (1010-1884 m), Coryphaenoides guentheri (1030 m) and Coryphaenoides (Nematonurus) armatus (2172-4787 m). Amongst these, the eye size range was 0.15-0.35 of head length with a value of 0.19 for C.(N.) armatus, the deepest species. Brain morphology reflected behavioural differences with well-developed olfactory regions in P.blennoides, T.murrayi and C. (N.) armatus and evidence of olfactory deficit in N. aequalis, C. rupestris and C. guentheri. All species had a clearly defined optic tectum with 2-[I-125] iodomelatonin binding and melatonin receptor gene expression localized to specific brain regions in a similar pattern to that found in shallow-water fish. Melatonin receptors were found throughout the visual structures of the brains of all species. Despite living beyond the depth of penetration of solar light these fish have retained central features associated with the coupling of cycles of growth, behaviour and reproduction to the diel light-dark cycle. How this functions in the deep sea remains enigmatic.