The ups and downs of growth hormone secretagogue receptor signaling

The growth hormone secretagogue receptor (GHSR) has emerged as one of the most fascinating molecules from the perspective of neuroendocrine control. GHSR is mainly expressed in the pituitary and the brain, and plays key roles regulating not only growth hormone secretion but also food intake, adiposity, body weight, glucose homeostasis and other complex functions. Quite atypically, GHSR signaling displays a basal constitutive activity that can be up- or downregulated by two digestive system-derived hormones: the octanoylated-peptide ghrelin and the liver-expressed antimicrobial peptide 2 (LEAP2), which was recently recognized as an endogenous GHSR ligand. The existence of two ligands with contrary actions indicates that GHSR activity can be tightly regulated and that the receptor displays the capability to integrate such opposing inputs in order to provide a balanced intracellular signal. This article provides a summary of the current understanding of the biology of ghrelin, LEAP2 and GHSR and discusses the reconceptualization of the cellular and physiological implications of the ligand-regulated GHSR signaling, based on the latest findings.Fil: Cornejo, María Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Mustafá, Emilio Román. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Cassano, Daniela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Banères, Jean Louis. Université Montpellier II; FranciaFil: Raingo, Jesica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Perello, Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentin

Mycobacteriophage-antibiotic therapy promotes enhanced clearance of drug-resistant Mycobacterium abscessus.

Infection by multidrug-resistant Mycobacterium abscessus is increasingly prevalent in cystic fibrosis (CF) patients, leaving clinicians with few therapeutic options. A compassionate study showed the clinical improvement of a CF patient with a disseminated M. abscessus (GD01) infection, following injection of a phage cocktail, including phage Muddy. Broadening the use of phage therapy in patients as a potential antibacterial alternative necessitates the development of biological models to improve the reliability and successful prediction of phage therapy in the clinic. Herein, we demonstrate that Muddy very efficiently lyses GD01 in vitro, an effect substantially increased with standard drugs. Remarkably, this cooperative activity was retained in an M. abscessus model of infection in CFTR-depleted zebrafish, associated with a striking increase in larval survival and reduction in pathological signs. The activity of Muddy was lost in macrophage-ablated larvae, suggesting that successful phage therapy relies on functional innate immunity. CFTR-depleted zebrafish represent a practical model to rapidly assess phage treatment efficacy against M. abscessus isolates, allowing the identification of drug combinations accompanying phage therapy and treatment prediction in patients. This article has an associated First Person interview with the first author of the paper

Systematic analysis of the entire second extracellular loop of the V 1a vasopressin receptor :Key residues, conserved throughout a G-protein-coupled receptor family, identified

The roles of extracellular residues of G-protein-coupled receptors (GPCRs) are not well defined compared with residues in transmembrane helices. Nevertheless, it has been established that extracellular domains of both peptide-GPCRs and amine-GPCRs incorporate functionally important residues. Extracellular loop 2 (ECL2) has attracted particular interest, because the x-ray structure of bovine rhodopsin revealed that ECL2 projects into the binding crevice within the transmembrane bundle. Our study provides the first comprehensive investigation into the role of the individual residues comprising the entire ECL2 domain of a small peptide-GPCR. Using the V1a vasopressin receptor, systematic substitution of all of the ECL2 residues by Ala generated 30 mutant receptors that were characterized pharmacologically. The majority of these mutant receptor constructs (24 in total) had essentially wild-type ligand binding and intracellular signaling characteristics, indicating that these residues are not critical for normal receptor function. However, four aromatic residues Phe189, Trp206, Phe209, and Tyr218 are important for agonist binding and receptor activation and are highly conserved throughout the neurohypophysial hormone subfamily of peptide-GPCRs. Located in the middle of ECL2, juxtaposed to the highly conserved disulfide bond, Trp206 and Phe209 project into the binding crevice. Indeed, Phe209 is part of the Cys-X-X-X-Ar (where Ar is an aromatic residue) motif, which is well conserved in both peptide-GPCRs and amine-GPCRs. In contrast, Phe189 and Tyr218, located at the extreme ends of ECL2, may be important for determining the position of the ECL2 cap over the binding crevice. This study provides mechanistic insight into the roles of highly conserved ECL2 residues

LEAP2 Impairs the Capability of the Growth Hormone Secretagogue Receptor to Regulate the Dopamine 2 Receptor Signaling

The growth hormone secretagogue receptor (GHSR) signals in response to ghrelin, but also acts via ligand-independent mechanisms that include either constitutive activation or interaction with other G protein-coupled receptors, such as the dopamine 2 receptor (D2R). A key target of GHSR in neurons is voltage-gated calcium channels type 2.2 (CaV2.2). Recently, the liver-expressed antimicrobial peptide 2 (LEAP2) was recognized as a novel GHSR ligand, but the mechanism of action of LEAP2 on GHSR is not well understood. Here, we investigated the role of LEAP2 on the canonical and non-canonical modes of action of GHSR on CaV2.2 function. Using a heterologous expression system and patch-clamp recordings, we found that LEAP2 impairs the reduction of CaV2.2 currents induced by ghrelin-evoked and constitutive GHSR activities, acting as a GHSR antagonist and inverse agonist, respectively. We also found that LEAP2 prevents GHSR from modulating the effects of D2R signaling on CaV2.2 currents, and that the GHSRbinding N-terminal region LEAP2 underlies these effects. Using purified labeled receptors assembled into lipid nanodiscs and Forster Resonance Energy Transfer (FRET) assessments, we found that the N-terminal region of LEAP2 stabilizes an inactive conformation of GHSR that is dissociated from Gq protein and, consequently, reverses the effect of GHSR on D2R-dependent Gi activation. Thus, our results provide critical molecular insights into the mechanism mediating LEAP2 modulation of GHSR.Instituto Multidisciplinario de Biología Celula

G-protein-coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent

G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A2A receptor (A2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR-SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (∼5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A2AR controls. The A2AR-SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR-SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([3H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms

Salomó Saporta: un mercader judío ante la Inquisición valenciana

Inquisitorial persecution against Jews has not been systematically approached by historiography, because investigations into the repression of inquisitorial courts in transit between the fifteenth and sixteenth centuries have focused on the Convert group. And while it is true that the modern Inquisition used some prosecutions of the Hebrews in an exemplary way to highlight the danger they posed to the Christian community, and that they were later collected by the investigators, the case of Salomó Saporta has been studied and commented as a isolated case. For this reason, perhaps it is time to raise the need to study the coercive mechanisms against the peninsular Jewish community in a more general way. A modest first step may be the analysis of the persecution suffered by Salomó Saporta during the second half of the 1480s.La persecución inquisitorial contra los judíos no ha sido un tema abordado sistemáticamente por la historiografía, porque las investigaciones sobre la represión de los tribunales inquisitoriales en el tránsito entre los siglos XV y XVI se han centrado en el grupo judeoconverso. Y si bien es cierto que la Inquisición moderna utilizó algunos procesamientos a hebreos de manera ejemplarizante para resaltar el peligro que representaban para la comunidad cristiana, y que han sido recogidos posteriormente por los investigadores, el caso de Salomó Saporta ha sido estudiado y comentado como un caso aislado. Por esa razón quizás sea el momento de plantear la necesidad de estudiar los mecanismos coercitivos contra la comunidad judía peninsular de manera más general. Un primer paso, modesto, puede ser el análisis de la persecución que sufrió Salomó Saporta durante la segunda mitad de la década de 1480

Artificial membranes for membrane protein purification, functionality and structure studies.

Membrane proteins represent one of the most important targets for pharmaceutical companies. Unfortunately, technical limitations have long been a major hindrance in our understanding of the function and structure of such proteins. Recent years have seen the refinement of classical approaches and the emergence of new technologies that have resulted in a significant step forward in the field of membrane protein research. This review summarizes some of the current techniques used for studying membrane proteins, with overall advantages and drawbacks for each method

The history of a repression : the Judeo-Christian converts in the Kingdom of Valencia in the early days of the Spanish Inquisition (1461-1530)

L’étude de l’Inquisition dans le district de Valence reposait essentiellement sur l’analyse menée par l’historien Ricardo García Cárcel en 1976. Nous avons voulu par l’étude exhaustive de différents documents : abécédaires inquisitoriaux, procès, mais aussi documents comptables, documents notariés, établir une nouvelle liste des condamnés entre 1478, date de l’implantation du Saint-Office à Valence et 1530, période où le filon judéo-convers se tarit, laissant place à des nouvelles stratégies et à la persécution de nouveaux groupes dissidents. À Valence, les années 1520-1530 marquent le déclin de la région au profit d’une Castille conquérante, dominatrice en Espagne et dans le monde. Le nouveau registre que nous avons établi, riche de 3 094 condamnés en grande majorité judéo-convers (93,39 %), nous a servi de fondement pour dresser les contours de ce que fut la répression dans cette région et comprendre le rôle d’une Inquisition qui, entre urgences financières du monarque et uniformisation religieuse et culturelle, bouleversa l’équilibre d’une communauté judéo-converse, de plus en plus intégrée à la société vieille-chrétienne à laquelle elle appartenait depuis sa conversion. À travers l’analyse des comptes du receveur des biens confisqués, limitée aux familles judéo-converses de trois des villes principales du royaume, Gandía, Xàtiva et Segorbe, nous avons voulu déterminer le niveau social de cette communauté et savoir quel fut l’impact de cette répression dans une région qui perdait ses prérogatives et son pouvoir au profit de la nouvelle monarchie des Habsbourg.The study of the Spanish Inquisition in Valencia has up until now depended primarily on the 1976 analysis by historian Ricardo García Cárcel. Through exhaustive investigation of different documents in archives in Madrid and Valencia – lists of convicted persons, trials, but also accounting and notarial records –, our aim was to establish a new list of Inquisition victims between 1478, when the Holy Office was established in Valencia, and 1530, when the massive persecution of converts from Judaism began to be replaced by other strategies and repression of other dissident groups. The period of the 1520s and 1530s saw the decline of the Kingdom of Valencia in favour of the dominant Castille, then in the process of becoming a world power. The new list that we have drawn up includes the names of 3,094 victims, most of whom (93.39%) were conversos. It forms the basis for describing the characteristics of this repression and understanding the part played by the Inquisition which, between the growing financial needs of the king and the drive to standardise religion and culture, disrupted a community which, since its conversion, had become increasingly integrated into the ancient Christian society. Analysis of receivers’ ledgers of confiscated goods from converso families from three of the major cities of the kingdom— Gandia, Xativa and Segorbe—, offers insight into the socio-economic level of this community, as well as the impact of this repression in a region which was losing its prerogatives and its power to the new Habsburg monarchy