Exploring Ligand Binding to Calcitonin Gene-Related Peptide Receptors

Class B1 G protein-coupled receptors (GPCRs) are important targets for many diseases, including cancer, diabetes, and heart disease. All the approved drugs for this receptor family are peptides that mimic the endogenous activating hormones. An understanding of how agonists bind and activate class B1 GPCRs is fundamental for the development of therapeutic small molecules. We combined supervised molecular dynamics (SuMD) and classic molecular dynamics (cMD) simulations to study the binding of the calcitonin gene-related peptide (CGRP) to the CGRP receptor (CGRPR). We also evaluated the association and dissociation of the antagonist telcagepant from the extracellular domain (ECD) of CGRPR and the water network perturbation upon binding. This study, which represents the first example of dynamic docking of a class B1 GPCR peptide, delivers insights on several aspects of ligand binding to CGRPR, expanding understanding of the role of the ECD and the receptor-activity modifying protein 1 (RAMP1) on agonist selectivity

The circularly permuted globin domain of androglobin exhibits atypical heme stabilization and nitric oxide interaction

In the decade since the discovery of androglobin, a multi-domain hemoglobin of metazoans associated with ciliogenesis and spermatogenesis, there has been little advance in the knowledge of the biochemical and structural properties of this unusual member of the hemoglobin superfamily. Using a method for aligning remote homologues, coupled with molecular modelling and molecular dynamics, we have identified a novel structural alignment to other hemoglobins. This has led to the first stable recombinant expression and characterization of the circularly permuted globin domain. Exceptional for eukaryotic globins is that a tyrosine takes the place of the highly conserved phenylalanine in the CD1 position, a critical point in stabilizing the heme. A disulfide bond, similar to that found in neuroglobin, forms a closed loop around the heme pocket, taking the place of androglobin's missing CD loop and further supporting the heme pocket structure. Highly unusual in the globin superfamily is that the heme iron binds nitric oxide as a five-coordinate complex similar to other heme proteins that have nitric oxide storage functions. With rapid autoxidation and high nitrite reductase activity, the globin appears to be more tailored toward nitric oxide homeostasis or buffering. The use of our multi-template profile alignment method to yield the first biochemical characterisation of the circularly permuted globin domain of androglobin expands our knowledge of the fundamental functioning of this elusive protein and provides a pathway to better define the link between the biochemical traits of androglobin with proposed physiological functions

Deconvoluting the Molecular Control of Binding and Signaling at the Amylin 3 Receptor: RAMP3 Alters Signal Propagation through Extracellular Loops of the Calcitonin Receptor

Amylin is coexpressed with insulin in pancreatic islet β-cells and has potent effects on gastric emptying and food intake. The effect of amylin on satiation has been postulated to involve AMY3 receptors (AMY3R) that are heteromers of the calcitonin receptor (CTR) and receptor activity-modifying protein 3 (RAMP3). Understanding the molecular control of signaling through the AMY3R is thus important for peptide drug targeting of this receptor. We have previously used alanine scanning mutagenesis to study the contribution of the extracellular surface of the CTR to binding and signaling initiated by calcitonin (CT) and related peptides (Dal Maso, E., et al. (2019) The molecular control of calcitonin receptor signaling. ACS Pharmacol. Transl. Sci.2, 31–51). That work revealed ligand- and pathway-specific effects of mutation, with extracellular loops (ECLs) 2 and 3 particularly important in the distinct propagation of signaling mediated by individual peptides. In the current study, we have used equivalent alanine scanning of ECL2 and ECL3 of the CTR in the context of coexpression with RAMP3 to form AMY3Rs, to examine functional affinity and efficacy of peptides in cAMP accumulation and extracellular signal-regulated kinase (ERK) phosphorylation (pERK). The effect of mutation was determined on representatives of the three major distinct classes of CT peptide, salmon CT (sCT), human CT (hCT), and porcine CT (pCT), as well as rat amylin (rAmy) or human α-CGRP (calcitonin gene-related peptide, hCGRP) whose potency is enhanced by RAMP interaction. We demonstrate that the dynamic nature of CTR ECL2 and ECL3 in propagation of signaling is fundamentally altered when complexed with RAMP3 to form the AMY3R, despite only having predicted direct interactions with ECL2. Moreover, the work shows that the role of these loops in receptor signaling is highly peptide dependent, illustrating that even subtle changes to peptide sequence may change signaling output downstream of the receptor

Preclinical and clinical characterization of fibroblast-derived neuregulin-1 on trastuzumab and pertuzumab activity in HER2-positive breast cancer

[Purpose]: To characterize expression of neuregulin-1 (NRG1), an HER3 ligand, in HER2-positive breast cancer and its relation with the efficacy of trastuzumab with or without pertuzumab.[Experimental Design]: Characterization of NRG1 expression in tumor cell lines, in tumor specimens, and in cancer-associated fibroblasts (CAFs). Patient-derived CAFs were used to investigate NRG1 impact on the activity of trastuzumab with or without pertuzumab in HER2-positive breast cancer cells. The relationship between NRG1 expression and pathologic response to anti-HER2–based neoadjuvant therapy was assessed in a retrospective patient cohort and in the NeoSphere trial.[Results]: NRG1 was expressed in HER2-positive breast cancer–derived fibroblasts at significantly higher levels than in cancer cells. NRG1 and the conditioned media (CM) from CAFs phosphorylated HER3 and AKT in cancer cells and mediated trastuzumab resistance. Stable genetic depletion of NRG1 from CAFs overcame trastuzumab resistance. Pertuzumab effectively suppressed trastuzumab resistance mediated by either NRG1 or CAF's CM. NRG1 engaged an epithelial-to-mesenchymal transition that was prevented by trastuzumab and pertuzumab. In clinical samples, stromal and/or tumor cell expression of NRG1 determined by immunohistochemistry was uncommon (13.2%) yet significantly linked with residual disease following trastuzumab-based neoadjuvant therapy. In the NeoSphere trial, the magnitude of the difference of pathologic complete response rates favoring the pertuzumab arm was higher in the NRG1-high group.[Conclusions]: CAF-derived NRG1 mediates trastuzumab resistance through HER3/AKT, which might be reverted by pertuzumab. In patients with HER2-positive breast cancer, high expression of NRG1 was associated to poor response to trastuzumab, but not in combination with pertuzumab.This work is supported by ISCIII (CIBERONC CB16/12/00481, CB16/12/00241, PI18/00382, PI18/00006, PI18/01219 and by Generalitat de Catalunya (2017 SGR 507). S. Menendez is supported by Department de Salut, Generalitat de Catalunya (PERIS SLT006/17/00040). MARBiobanc is supported by ISCiii/FEDER (PT17/0015/0011) and by “Xarxa de Bancs de tumors” sponsored by Pla Director d’ Oncologia de Catalunya (XBTC) and Fundacion Jimenez Díaz Biobanks Platform by PT13/0010/0012 grant. Ministry of Economy and Competitiveness of Spain (BFU2015-71371-R) and the CRIS Cancer Foundation provides support to A. Pandiella. Work carried out in our laboratories receive support from the European Community through the Regional Development Funding Program (FEDER). J.C. Montero is funded by the ISCIII through a Miguel Servet program (CPII17/00015) and receives research support from the same institution (PI18/00796). J. Albanell is supported by Breast Cancer Research Foundation (BCRF20-08), Instituto de Salud Carlos III Project Reference number AC15/00062 and the EC under the framework of the ERA-NET TRANSCAN-2 initiative co-financed by FEDER, Instituto de Salud Carlos III (CB16/12/00449 and PI19/01181), and Asociacion Espanola Contra el Cáncer (AECC)

Development of the transgenic sugarcane event TUC 87-3RG resistant to glyphosate

La variedad comercial de caña de azúcar RA 87-3 se transformó mediante biobalística con una construcción genética que porta el gen epsps de la cepa CP4 de Agrobacterium tumefaciens, y el gen nptII que confieren resistencia a glifosato y resistencia a kanamicina/geneticina, respectivamente. Las líneas transformadas fueron multiplicadas en invernadero y se evaluó la resistencia al herbicida utilizando diferentes concentraciones de glifosato (3, 4 y 8 l/ha). Las líneas resistentes al herbicida (RH) se evaluaron en campo para confirmar la resistencia a glifosato (3 l/ha) y realizar un análisis preliminar del comportamiento de estas con respecto al cultivar parental. Todas las líneas transformadas mantuvieron la resistencia al herbicida pero muchas mostraron cambios fenotípicos. Las diez líneas RH que resultaron muy parecidas a la variedad RA 87-3 se analizaron fenotípica y genéticamente utilizando los nueve descriptores morfológicos obligatorios propuestos por la “International Union for the Protection of New Varieties of Plants” (UPOV) y con 339 marcadores moleculares, respectivamente. Seis líneas de las anteriores presentaron cambios morfológicos y genéticos menores y fueron seleccionadas para ensayos en campo durante dos ciclos vegetativos (caña planta y soca 1), en dos áreas de producción de Argentina. Las seis líneas RH presentaron características agronómicas, industriales y de composición química indistinguibles respecto del cultivar parental. La herencia estable del gen CP4 epsps fue confirmada mediante ensayos de RT-qPCR y Southern blot en diferentes generaciones clonales (caña planta y soca 1). Los estudios confirmaron la utilidad de la transformación genética como una herramienta complementaria a la mejora clásica, y destaca la ventaja del uso de los descriptores de UPOV junto con los marcadores moleculares para una selección temprana de eventos transgénicos que tengan un alto parecido con el genotipo parental. Tomando en cuenta los resultados, una de las seis líneas estudiadas se seleccionó para una posible liberación comercial, la cual debe ser sometida a evaluación por parte de los entes regulatorios (CONABIA, SENASA y DNMA).The commercial variety of sugarcane RA 87-3 was transformed by biobalistic with a genetic construct that carries the epsps gene of the CP4 strain of Agrobacterium tumefaciens, and with the nptII gene that confer resistance to glyphosate and resistance to kanamycin / geneticin, respectively. Transformed lines were multiplied in greenhouse and resistance to the herbicide was evaluated using different concentrations of glyphosate (3, 4 and 8 l / ha). Resistant lines to the herbicide (RH) were evaluated in the field to confirm resistance to glyphosate and to carry out a preliminary analysis of their behavior with respect to the parental cultivar. All transformed lines maintained resistance to the herbicide but many showed phenotypic changes. Ten RH lines that were very similar to the RA 87-3 variety were analyzed phenotypically and genetically using the 9 compulsory morphological markers proposed by The International Union for the Protection of New Varieties of Plants (UPOV) and with 339 molecular markers, respectively. Six lines of the previous ones presented minor morphological and genetic changes and were selected for field trials during two vegetative cycles (cane plant and ratoon 1), in two production areas of Argentina. The six RH lines presented agronomic, industrial and chemical composition characteristics indistinguishable from the parental cultivar. The stable inheritance of the CP4 epsps gene was confirmed by RT-qPCR and Southern blot assays in different clonal generations (growth cycles). Our studies confirmed the usefulness of genetic transformation as a complementary tool to classical improvement and highlight the advantage of the use of UPOV descriptors together with molecular markers for an early selection of transgenic events that have a high similarity with the parental genotype. Taking into account the results, one of the six lines evaluated was proposed for a possible commercial release, under the requirements of regulatory affairs offices (CONABIA, SENASA and DNMA).Fil: Noguera, Aldo Sergio. Gobierno de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial Obispo Colombres; ArgentinaFil: Enrique, Ramón Atanasio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Provincia de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial "Obispo Colombres" (p). Instituto de Tecnología Agroindustrial del Noroeste Argentino; ArgentinaFil: Ostengo, Santiago. Gobierno de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial Obispo Colombres; ArgentinaFil: Perera, María Francisca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Provincia de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial "Obispo Colombres" (p). Instituto de Tecnología Agroindustrial del Noroeste Argentino; ArgentinaFil: Racedo, Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Provincia de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial "Obispo Colombres" (p). Instituto de Tecnología Agroindustrial del Noroeste Argentino; ArgentinaFil: Chayle Costilla, Diego Roberto. Gobierno de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial Obispo Colombres; ArgentinaFil: Zozzi. Silvia. Gobierno de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial Obispo Colombres; ArgentinaFil: Cuenya, María Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Provincia de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial "Obispo Colombres" (p). Instituto de Tecnología Agroindustrial del Noroeste Argentino; ArgentinaFil: Filippone, María Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Provincia de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial "Obispo Colombres" (p). Instituto de Tecnología Agroindustrial del Noroeste Argentino; ArgentinaFil: Welin, Bjorn. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Provincia de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial "Obispo Colombres" (p). Instituto de Tecnología Agroindustrial del Noroeste Argentino; ArgentinaFil: Castagnaro, Atilio Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Provincia de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial "Obispo Colombres" (p). Instituto de Tecnología Agroindustrial del Noroeste Argentino; Argentin

The Molecular Control of Calcitonin Receptor Signaling

The calcitonin receptor (CTR) is a class B G protein-coupled receptor (GPCR) that responds to the peptide hormone calcitonin (CT). CTs are clinically approved for the treatment of bone diseases. We previously reported a 4.1 Å structure of the activated CTR bound to salmon CT (sCT) and heterotrimeric Gs protein by cryo-electron microscopy (Liang, Y.-L., et al. Phase-plate cryo- EM structure of a class B GPCR-G protein complex. Nature 2017, 546, 118–123). In the current study, we have reprocessed the electron micrographs to yield a 3.3 Å map of the complex. This has allowed us to model extracellular loops (ECLs) 2 and 3, and the peptide N-terminus that previously could not be resolved. We have also performed alanine scanning mutagenesis of ECL1 and the upper segment of transmembrane helix 1 (TM1) and its extension into the receptor extracellular domain (TM1 stalk), with effects on peptide binding and function assessed by cAMP accumulation and ERK1/2 phosphorylation. These data were combined with previously published alanine scanning mutagenesis of ECL2 and ECL3 and the new structural information to provide a comprehensive 3D map of the molecular surface of the CTR that controls binding and signaling of distinct CT and related peptides. The work highlights distinctions in how different, related, class B receptors may be activated. The new mutational data on the TM1 stalk and ECL1 have also provided critical insights into the divergent control of cAMP versus pERK signaling and, collectively with previous mutagenesis data, offer evidence that the conformations linked to these different signaling pathways are, in many ways, mutually exclusive. This study furthers our understanding of the complex nature of signaling elicited by GPCRs and, in particular, that of the therapeutically important class B subfamily

An extensive review on phenolic compounds and their potential estrogenic properties on skin physiology

Polyphenolic compounds constitute a diverse group of natural components commonly occurring in various plant species, known for their potential to exert both beneficial and detrimental effects. Additionally, these polyphenols have also been implicated as endocrine-disrupting (ED) chemicals, raising concerns about their widespread use in the cosmetics industry. In this comprehensive review, we focus on the body of literature pertaining to the estrogenic properties of ED chemicals, with a particular emphasis on the interaction of isoflavones with estrogen receptors. Within this review, we aim to elucidate the multifaceted roles and effects of polyphenols on the skin, exploring their potential benefits as well as their capacity to act as ED agents. By delving into this intricate subject matter, we intend to provoke thoughtful consideration, effectively opening a Pandora’s box of questions for the reader to ponder. Ultimately, we invite the reader to contemplate whether polyphenols should be regarded as friends or foes in the realm of skincare and endocrine disruption