117 research outputs found
The Structural Determinants of Insulin-Like Peptide 3 Activity
Insulin-like peptide 3 (INSL3) is a hormone and/or paracrine factor which is a member of the relaxin peptide family. It has key roles as a fertility regulator in both males and females. The receptor for INSL3 is the leucine rich repeat (LRR) containing G-protein coupled receptor 8 (LGR8) which is now known as relaxin family peptide receptor 2 (RXFP2). Receptor activation by INSL3 involves binding to the LRRs in the large ectodomain of RXFP2 by residues within the B-chain of INSL3 as well as an interaction with the transmembrane exoloops of the receptor. Although the binding to the LRRs is well characterized the features of the peptide and receptor involved in the exoloop interaction are currently unknown. This study was designed to determine the key INSL3 determinants for RXFP2 activation. A chimeric peptide approach was first utilized to demonstrate that the A-chain is critical for receptor activation. Replacement of the INSL3 A-chain with that from the related peptides INSL5 and INSL6 resulted in complete loss of activity despite only minor changes in binding affinity. Subsequent replacement of specific A-chain residues with those from the INSL5 peptide highlighted that the N-terminus of the A-chain of INSL3 is critical for its activity. Remarkably, replacement of the entire N-terminus with four or five alanine residues resulted in peptides with near native activity suggesting that specific residues are not necessary for activity. Additionally removal of two amino acids at the C-terminus of the A-chain and mutation of Lys-8 in the B-chain also resulted in minor decreases in peptide activity. Therefore we have demonstrated that the activity of the INSL3 peptide is driven predominantly by residues 5–9 in the A-chain, with minor additional contributions from the two C-terminal A-chain residues and Lys-8 in the B-chain. Using this new knowledge, we were able to produce a truncated INSL3 peptide structure which retained native activity, despite having 14 fewer residues than the parent peptide
Moyamoya Disease in Pregnancy: Management after Intracranial Bypass Grafting
Moyamoya disease (MD) is a chronic, progressive cerebrovascular disease distinguished by bilateral stenosis or occlusion of the arteries around the circle of Willis with resulting prominent arterial collateral circulation. We describe a pregnant woman in whom this diagnosis was confirmed by cerebral angiogram and treated with bilateral superficial temporal artery-middle cerebral artery (STA-MCA) bypass grafting prior to conception. The patient was managed with strict blood pressure monitoring and low-dose aspirin antepartum, intrapartum, and postpartum. The patient presented in spontaneous labor at term and underwent a spontaneous vaginal delivery without complications
Examination of relaxin and its receptors expression in pig gametes and embryos
<p>Abstract</p> <p>Background</p> <p>Relaxin is a small peptide also known as pregnancy hormone in many mammals. It is synthesized by both male and female tissues, and its secretions are found in various body fluids such as plasma serum, ovarian follicular fluid, utero-oviduct secretions, and seminal plasma of many mammals, including pigs. However, the presence and effects of relaxin in porcine gametes and embryos are still not well-known. The purpose of this study was to assess the presence of relaxin and its receptors RXFP1 and RXFP2 in pig gametes and embryos.</p> <p>Methods</p> <p>Immature cumulus-oocyte complexes (COCs) were aspirated from sows' ovaries collected at the abattoir. After <it>in vitro</it>-maturation, COCs were <it>in vitro</it>-fertilized and cultured. For studies, immature and mature COCs were separately collected, and oocytes were freed from their surrounding cumulus cells. Denuded oocytes, cumulus cells, mature boar spermatozoa, zygotes, and embryos (cleaved and blastocysts) were harvested for temporal and spatial gene expression studies. Sections of ovary, granulosa and neonatal porcine uterine cells were also collected to use as controls.</p> <p>Results</p> <p>Using both semi-quantitative and quantitative PCRs, relaxin transcripts were not detected in all tested samples, while RXFP1 and RXFP2 mRNA were present. Both receptor gene products were found at higher levels in oocytes compared to cumulus cells, irrespective of the maturation time. Cleaved-embryos contained higher levels of RXFP2 mRNA, whereas, blastocysts were characterized by a higher RXFP1 mRNA content. Using western-immunoblotting or <it>in situ </it>immunofluorescence, relaxin and its receptor proteins were detected in all samples. Their fluorescence intensities were consistently more important in mature oocytes than immature ones. The RXFP1 and RXFP2 signal intensities were mostly located in the plasma membrane region, while the relaxin ones appeared homogeneously distributed within the oocytes and embryonic cells. Furthermore, spermatozoa displayed stronger RXFP2 signal than RXFP1 after western-immunoblotting.</p> <p>Conclusion</p> <p>All together, our findings suggest potential roles of relaxin and its receptors during oocyte maturation, early embryo development, and beyond.</p
Development of a scaffold displaying exoloops of RXFP1
Relaxin family peptide receptor 1 (RXFP1), the cognate receptor for relaxin, is a G-protein coupled receptor (GPCR) possessing a unique extracellular region consisting of a domain of 10 leucine rich repeats (LRRs) linked to an N-terminal low density lipoprotein Class A module. Relaxin binds to its receptor primarily by a high affinity interaction with the LRRs. An additional low-affinity interaction has been proposed to occur between relaxin and the the exoloops (ELs) of the transmembrane domain, however the molecular detail of this interaction remains undefined. While site directed mutagenesis and subsequent functional characterisation of these mutants traditionally allows identification of residues contributing to receptor function, in this case results are complicated by the presence of the high affinity binding site in the LRRs. To create a tool to investigate the low-affinity interaction, a protein scaffold system displaying exoloops 1 and 2 from RXFP1 was designed. This was achieved by inserting RXFP1 exoloops 1 and 2 into the native loops of a thermostabilised 6 kDa GB1 protein creating EL1/EL2-GB1. This protein has been expressed and purified in milligram quantities and used in conjunction with biophysical techniques such as NMR to explore relaxin binding to the exoloops of RXFP1
Real‐time examination of cAMP activity at relaxin family peptide receptors using a BRET ‐based biosensor
Distinct but overlapping binding sites of agonist and antagonist at the relaxin family peptide 3 (RXFP3) receptor
The relaxin-3 neuropeptide activates the relaxin family peptide 3 (RXFP3) receptor to modulate stress, appetite and cognition. RXFP3 shows promise as a target for treating neurological disorders, but realization of its clinical potential requires development of smaller RXFP3-specific drugs that can penetrate the blood brain barrier. Designing such drugs is challenging and requires structural knowledge of agonist- and antagonist-binding modes. Here, we used structure-activity data for relaxin-3 and a peptide RXFP3 antagonist termed R3 B1-22R, to guide receptor mutagenesis and develop models of their binding modes. RXFP3 residues were alanine substituted individually and in combination and tested in cell-based binding and functional assays to refine models of agonist and antagonist binding to active- and inactive-state homology models of RXFP3, respectively. These models suggested that both agonists and antagonists interact with RXFP3 via similar residues in their B-chain central helix. The models further suggested that the B-chain Trp-27 inserts into the binding pocket of RXFP3 and interacts with Trp-138 and Lys-271, the latter through a salt bridge with the C-terminal carboxyl group of Trp-27 in relaxin-3. R3 B1-22R, which does not contain Trp-27, used a non-native Arg-23 residue to form cation-pi and salt bridge interactions with Trp-138 and Glu-141 in RXFP3, explaining a key contribution of Arg-23 to affinity. Overall, relaxin-3 and R3 B1-22R appear to share similar binding residues but may differ in binding modes, leading to active and inactive RXFP3 conformational states, respectively. These mechanistic insights may assist structure-based drug design of smaller relaxin-3 mimetics to manage neurological disorders
Earthquake forecasting in Italy, before and after Umbria-Marche seismic sequence 1997. A review of the earthquake occurrence modeling at different spatio-temporal-magnitude scales.
The main goal of this work is to review the scientific researches carried out before and after the Umbria-Marche sequence related to the earthquake forecasting/prediction in Italy. In particular, I focus the attention on models that aim addressing three main practical questions: was (is) Umbria-Marche a region with high probability of occurrence of a destructive earthquake? Was a precursory activity recorded before the mainshock(s)? What was our capability to model the spatio-temporal-magnitude evolution of that seismic sequence? The models are reviewed pointing out what we have learned after the Umbria-Marche earthquakes, in terms of physical understanding of earthquake occurrence process, and of improving our capability to forecast earthquakes and to track in real-time seismic sequences
The Concise Guide to PHARMACOLOGY 2015/16:Ligand-gated ion channels
The Concise Guide to PHARMACOLOGY 2015/16 provides concise overviews of the key properties of over 1750 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.13349/full. Ligand-gated ion channels are one of the eight major pharmacological targets into which the Guide is divided, with the others being: ligand-gated ion channels, voltage-gated ion channels, other ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The Concise Guide is published in landscape format in order to facilitate comparison of related targets. It is a condensed version of material contemporary to late 2015, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in the previous Guides to Receptors & Channels and the Concise Guide to PHARMACOLOGY 2013/14. It is produced in conjunction with NC-IUPHAR and provides the official IUPHAR classification and nomenclature for human drug targets, where appropriate. It consolidates information previously curated and displayed separately in IUPHAR-DB and GRAC and provides a permanent, citable, point-in-time record that will survive database updates
THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: G protein-coupled receptors
The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15538. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate
The Concise Guide to PHARMACOLOGY 2015/16:Transporters
The Concise Guide to PHARMACOLOGY 2015/16 provides concise overviews of the key properties of over 1750 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.13355/full. G protein-coupled receptors are one of the eight major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ligand-gated ion channels, voltage-gated ion channels, other ion channels, nuclear hormone receptors, catalytic receptors and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The Concise Guide is published in landscape format in order to facilitate comparison of related targets. It is a condensed version of material contemporary to late 2015, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in the previous Guides to Receptors & Channels and the Concise Guide to PHARMACOLOGY 2013/14. It is produced in conjunction with NC-IUPHAR and provides the official IUPHAR classification and nomenclature for human drug targets, where appropriate. It consolidates information previously curated and displayed separately in IUPHAR-DB and GRAC and provides a permanent, citable, point-in-time record that will survive database updates
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