scholarly journals Structure-Based Functional Analysis of a Hormone Belonging to an Ecdysozoan Peptide Superfamily: Revelation of a Common Molecular Architecture and Residues Possibly for Receptor Interaction

2021 ◽  
Vol 22 (20) ◽  
pp. 11142
Author(s):  
Yun-Ru Chen ◽  
Nai-Wan Hsiao ◽  
Yi-Zong Lee ◽  
Shiau-Shan Huang ◽  
Chih-Chun Chang ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Receptor Binding ◽  
Binding Sites ◽  
Disulfide Bonds ◽  
Solution Structure ◽  
Receptor Interaction ◽  
Resonance Spectroscopy ◽  
Molecular Architecture ◽  
Major Factors

A neuropeptide (Sco-CHH-L), belonging to the crustacean hyperglycemic hormone (CHH) superfamily and preferentially expressed in the pericardial organs (POs) of the mud crab Scylla olivacea, was functionally and structurally studied. Its expression levels were significantly higher than the alternative splice form (Sco-CHH) in the POs, and increased significantly after the animals were subjected to a hypo-osmotic stress. Sco-CHH-L, but not Sco-CHH, significantly stimulated in vitro the Na+, K+-ATPase activity in the posterior (6th) gills. Furthermore, the solution structure of Sco-CHH-L was resolved using nuclear magnetic resonance spectroscopy, revealing that it has an N-terminal tail, three α-helices (α2, Gly9−Asn28; α3, His34−Gly38; and α5, Glu62−Arg72), and a π-helix (π4, Cys43−Tyr54), and is structurally constrained by a pattern of disulfide bonds (Cys7–Cys43, Cys23–Cys39, and Cys26–Cys52), which is characteristic of the CHH superfamily-peptides. Sco-CHH-L is topologically most similar to the molt-inhibiting hormone from the Kuruma prawn Marsupenaeus japonicus with a backbone root-mean-square-deviation of 3.12 Å. Ten residues of Sco-CHH-L were chosen for alanine-substitution, and the resulting mutants were functionally tested using the gill Na+, K+-ATPase activity assay, showing that the functionally important residues (I2, F3, E45, D69, I71, and G73) are located at either end of the sequence, which are sterically close to each other and presumably constitute the receptor binding sites. Sco-CHH-L was compared with other members of the superfamily, revealing a folding pattern, which is suggested to be common for the crustacean members of the superfamily, with the properties of the residues constituting the presumed receptor binding sites being the major factors dictating the ligand–receptor binding specificity.

scholarly journals Structure-based functional study of a peptide of an ecdysozoan superfamily: reveling a common molecular architecture and receptor-interacting residues

2020 ◽  
Author(s):  
Yun-Ru Chen ◽  
Nai-Wan Hsiao ◽  
Shiau-Shan Huang ◽  
Chih-Chun Chang ◽  
Yi-Zong Lee ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Receptor Binding ◽  
Binding Sites ◽  
Disulfide Bonds ◽  
Solution Structure ◽  
Functional Study ◽  
Resonance Spectroscopy ◽  
Molecular Architecture ◽  
Major Factors

ABSTRACTA neuropeptide (Sco-CHH-L), belonging to the crustacean hyperglycemic hormone (CHH) superfamily and preferentially expressed in the pericardial organs (POs) of the mud crab Scylla olivacea, was functionally and structurally studied. Its expression levels were significantly higher than the alternative splice form (Sco-CHH) in the POs and increased significantly after animals were subjected to a hypo-osmotic stress. Sco-CHH-L, but not Sco-CHH, significantly stimulated in vitro the Na+, K+-ATPase activity in the posterior (6th) gills. Furthermore, solution structure of Sco-CHH-L was resolved using nuclear magnetic resonance spectroscopy revealing that it has an N-terminal tail, three α-helices (α2, Gly9−Asn28; α3, His34−Gly38; α5, Glu62−Arg72), and a π-helix (π4, Cys43−Tyr53) and is structurally constrained by a pattern of disulfide bonds (Cys7-Cys43, Cys23-Cys39, Cys26-Cys52), which is characteristic of the CHH superfamily-peptides. Sco-CHH-L is topologically most similar to the molt-inhibiting hormone from the Kuruma prawn Marsupenaeus japonicus with a backbone root-mean-square-deviation of 3.12 Å. Ten residues of Sco-CHH-L were chosen for alanine-substituted and the resulting mutants were functionally tested using the gill Na+, K+-ATPase activity assay, showing that the functionally important residues (I2, F3, E45, D69, I71, G73) are located at either end of the sequence, which are sterically close to each other and presumably constitutes the receptor binding sites. Sco-CHH-L was compared with other members of the superfamily revealing a molecular architecture, which is suggested to be common for the crustacean members of the superfamily, with the properties of the residues constituting the presumed receptor binding sites being the major factors dictating the ligand-receptor binding specificity.

scholarly journals Substitution of lysine for arginine at position 42 of human transforming growth factor-alpha eliminates biological activity without changing internal disulfide bonds.

1989 ◽  
Vol 9 (9) ◽  
pp. 4083-4086 ◽  
Author(s):  
D Defeo-Jones ◽  
J Y Tai ◽  
G A Vuocolo ◽  
R J Wegrzyn ◽  
T L Schofield ◽  
...  
Keyword(s):  
Biological Activity ◽  
Growth Factor ◽  
Receptor Binding ◽  
Disulfide Bonds ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Receptor Interaction ◽  
Composition Analysis ◽  
Transforming Growth Factor Alpha ◽  
Factor Alpha

Transforming growth factor-alpha (TGF-alpha) is a growth-promoting protein that binds to the epidermal growth factor (EGF) receptor. To identify critical residues that govern TGF-alpha-EGF receptor binding, we prepared site-specific substitution mutants of TGF-alpha. Mutant proteins were tested in receptor-binding and mitogenesis assays. Semiconservative substitutions at positions 4, 12, 18, and 45 decreased biological activity 2.1- to 14-fold. The conservative substitution of lysine for arginine at position 42 completely eliminated biological activity. Amino acid composition analysis of proteolytic fragments from TGF-alpha and the Lys-42 mutant indicated that these proteins contained the same disulfide bonds. These studies suggest that arginine 42 may be a contact point for TGF-alpha-EGF receptor interaction.

A comparison of in vivo and in vitro neuroimaging of 5-HT1A receptor binding sites in the cat brain

2006 ◽  
Vol 31 (3) ◽  
pp. 226-232 ◽  
Author(s):  
Nicolas Aznavour ◽  
Latifa Rbah ◽  
Lucienne Léger ◽  
Colette Buda ◽  
Jean-Pierre Sastre ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Binding Sites ◽  
Cat Brain

Localization and characterization of endothelin receptor binding sites in the rat brain visualized by in vitro autoradiography4

Neuroscience ◽  
1991 ◽  
Vol 42 (1) ◽  
pp. 245-260 ◽  
Author(s):  
M. Kohzuki ◽  
S.Y. Chai ◽  
G. Paxinos ◽  
A. Karavas ◽  
D.J. Casley ◽  
...  
Keyword(s):  
Rat Brain ◽  
Receptor Binding ◽  
Binding Sites ◽  
Endothelin Receptor

scholarly journals A domain of the even-skipped protein represses transcription by preventing TFIID binding to a promoter: repression by cooperative blocking.

1995 ◽  
Vol 15 (9) ◽  
pp. 4683-4693 ◽  
Author(s):  
R J Austin ◽  
M D Biggin
Keyword(s):  
Glucocorticoid Receptor ◽  
Rna Polymerase Ii ◽  
Receptor Binding ◽  
Binding Sites ◽  
In Vitro System ◽  
Cooperative Interactions ◽  
General Transcription Factors ◽  
Promoter Dna ◽  
Template Dna

We examined the mechanism by which the C-terminal 236 amino acids of the even-skipped protein (region CD) repress transcription. A fusion protein, CDGB, was created that contains region CD fused to the glucocorticoid receptor DNA binding domain. This protein repressed transcription in an in vitro system containing purified fractions of the RNA polymerase II general transcription factors, and repression was dependent upon the presence of high-affinity glucocorticoid receptor binding sites in the promoter. Repression by CDGB was prevented when the promoter DNA was preincubated with TFIID or TBP, whereas preincubation of the template DNA with CDGB prevented TFIID binding. Together, these results strongly imply that CDGB represses transcription by inhibiting TFIID binding, and further experiments suggested a mechanism by which this may occur. Region CD can mediate cooperative interactions between repressor molecules such that molecules bound at the glucocorticoid receptor binding sites stabilize binding of additional CDGB molecules to low-affinity binding sites throughout the basal promoter. Binding to some of these low-affinity sites was shown to contribute to repression. Further experiments suggested that the full-length eve protein also represses transcription by the same mechanism. We speculate that occupancy of secondary sites within the basal promoter by CDGB or the eve protein inhibits subsequent TFIID binding to repress transcription, a mechanism we term cooperative blocking.

Autoradiographic detection of kinin receptors in the human medulla of control, hypertensive, and diabetic donors

2002 ◽  
Vol 80 (4) ◽  
pp. 249-257 ◽  
Author(s):  
Hudson de Sousa Buck ◽  
Brice Ongali ◽  
Gaétan Thibault ◽  
Charles J Lindsey ◽  
Réjean Couture
Keyword(s):  
Receptor Binding ◽  
Binding Sites ◽  
Specific Binding ◽  
Inferior Olivary Nucleus ◽  
Binding Studies ◽  
Specific Binding Sites ◽  
Kinin Receptors ◽  
Medullary Nuclei ◽  
Inferior Olivary

Kinins have been elected to the status of central neuromediators. Their effects are mediated through the activation of two G-protein-coupled receptors, denoted B1 and B2. Functional and binding studies suggested that B1 and B2 receptors are upregulated in the medulla and spinal cord of hypertensive and diabetic rats. The aim of this study was to localize and quantify kinin receptors in post-mortem human medulla obtained from normotensive, hypertensive, and diabetic subjects, using in vitro receptor autoradiography with the radioligands [125I]HPP-HOE140 (B2 receptor) and [125I]HPP[des-Arg10]-HOE140 (B1 receptor). Data showed specific binding sites for B2 receptor (0.4–1.5 fmol/mg tissue) in 11 medullary nuclei from 4 control specimens (paratrigeminal > ambiguus > cuneate, gelatinous layer of the caudal spinal trigeminal nucleus > caudal and interpolar spinal trigeminal, external cuneate, solitary tract > hypoglossal > gracile > inferior olivary nuclei). Increased density of B2 receptor binding sites was observed in seven medullary nuclei of four hypertensive specimens (paratrigeminal > external cuneate > interpolar and caudal spinal trigeminal, gracile, inferior olivary > hypoglossal nuclei). B2 receptor binding sites were seemingly increased in the same medullary nuclei of two diabetic specimens. Specific binding sites for B1 receptor (1.05 and 1.36 fmol/mg tissue) were seen only in the inferior olivary nucleus in two out of the ten studied specimens. The present results support a putative role for kinins in the regulation of autonomic, nociceptive, and motor functions at the level of the human medulla. Evidence is also provided that B2 receptors are upregulated in medullary cardiovascular centers of subjects afflicted of cardiovascular diseases.Key words: bradykinin, hypertension, diabetes, human brain.

scholarly journals Characterization and localization of Ac-SDKP receptor binding sites using125I-labeled Hpp-Aca-SDKP in rat cardiac fibroblasts

2007 ◽  
Vol 292 (2) ◽  
pp. H984-H993 ◽  
Author(s):  
Jia L. Zhuo ◽  
Oscar A. Carretero ◽  
Hongmei Peng ◽  
Xiao C. Li ◽  
Domenico Regoli ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Binding Sites ◽  
Collagen Synthesis ◽  
Cardiac Fibroblasts ◽  
Biologically Active ◽  
Inhibition Constant ◽  
Maximal Effect ◽  
Ang Ii ◽  
Protein Dissociation

We have shown that the tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) inhibited endothelin-1 (ET-1)-induced cell proliferation and collagen synthesis in cultured rat cardiac fibroblasts (CFs) and reduced left ventricle collagen deposition in rats with aldosterone (salt)- and ANG II-induced hypertension. However, it is not known whether these effects are mediated by receptor binding sites specific for Ac-SDKP. We hypothesized that Ac-SDKP exerts antifibrotic effects by binding to specific receptor sites in cultured rat CFs, which mediate the inhibitory effects of Ac-SDKP on ET-1-stimulated collagen synthesis. Ac-SDKP binding sites in rat CFs and hearts were characterized by a specific radioligand,125I-labeled 3-( p-hydroxyphenyl)-propionic acid (or desaminotyrosine) (Hpp)-Aca-SDKP, a biologically active analog of Ac-SDKP.125I-labeled Hpp-Aca-SDKP bound to rat CFs and fractionated membranes with similar affinities and specificity in a concentration- and time-dependent fashion. Scatchard plot analyses revealed a single class of high-affinity Hpp-Aca-SDKP binding sites (maximal binding: 1,704 ± 198 fmol/mg protein; dissociation constant: 3.3 ± 0.6 nM).125I-labeled Hpp-Aca-SDKP binding in CFs was displaced by unlabeled native peptide Ac-SDKP (inhibition constant: 0.69 ± 0.15 nM) and the analog Hpp-Aca-SDKP (inhibition constant: 10.4 ± 0.2 nM) but not the unrelated peptide ANG II or ET-1 (10 μM). In vitro, both Ac-SDKP and Hpp-Aca-SDKP inhibited ET-1-stimulated collagen synthesis in CFs in a dose-dependent fashion, reaching a maximal effect at 1 nM (control: 7.5 ± 0.4, ET-1: 19.9 ± 1.2, ET-1+SDKP: 7.7 ± 0.4, ET-1+Hpp-Aca-SDKP: 9.7 ± 0.1 μg/mg protein; P < 0.001). Ac-SDKP also significantly attenuated ET-1-induced increases in intracellular calcium and MAPK ERK1/2 phosphorylation in CFs. In the rat heart, in vitro autoradiography revealed specific125I-labeled Hpp-Aca-SDKP binding throughout the myocardium, primarily interstitially. We believe that these results demonstrate for the first time that Hpp-Aca-SDKP is a functional ligand specific for Ac-SDKP receptor binding sites and that both Ac-SDKP and Hpp-Aca-SDKP exert antifibrotic effects by binding to Ac-SDKP receptors in rat CFs.

Sign in / Sign up

Export Citation Format

Share Document