Literatura académica sobre el tema "G-Hedgehog"

Enteric protozoan parasites, such as Blastocystis sp., Balantioides coli, Cryptosporidium spp., and Giardia duodenalis, may have implications for both animal and human health.Transmitted through the fecal–oral route, these parasites cause symptoms such as diarrhea, abdominal pain, and weight loss. This study investigated the presence of these enteric protozoan parasites and genetically characterized them in hedgehogs from Portugal. A total of 110 hedgehog stool samples were collected. Molecular detection methods showed an overall occurrence of protozoa in 1.82% (2/110 95% CI: 0.22–6.41) of hedgehogs, with Blastocystis being found in one hedgehog and Cryptosporidium being found in another. No evidence for the presence of B. coli or G. duodenalis was found. This study suggests that there is a need to stay aware of hedgehogs as potential hosts of enteric protozoa. Ongoing research and surveillance efforts are recommended to explore practical prevention and control strategies. The results contribute to the limited knowledge of these parasites in Portuguese hedgehog populations and underscore their potential relevance to both veterinary and public health.

Background: Hedgehog pathway plays a crucial role in the neovascularisation and angiogenesis during the embryonic stage in humans. Three genes of hedgehog protein isolated from humans are Sonic hedgehog, Desert hedgehog and Indian hedgehog gene. Two G-protein coupled receptors identified in the sonic hedgehog pathway served as patched receptor and smoothened receptor. Materials and Methods: Particularly, sonic hedgehog gene plays a versatile role in cellular homeostasis and can be a novel therapeutic target in the prevention of cardiovascular disorders. Further various sonic hedgehog modulators have been reported working as futuristic drug molecules in the modulation of cardiovascular dysfunctions. Results: However, there was limited literature availability that has summarized the possible mechanism of targeting Sonic hedgehog signaling pathway. Conclusion: Thus, the present review is aimed at exploring the role of targeting sonic hedgehog protein signaling and modulators as well as to enlighten that how targeting sonic hedgehog protein involves in the amelioration of atherosclerosis, ischemic heart diseases, vascular endothelial dysfunction, heart failure and congenital heart diseases.

We tested the effects of 18 models of robotic lawn mowers in collision with dead European hedgehogs and quantified the results into six damage categories. All models were tested on four weight classes of hedgehogs, each placed in three different positions. None of the robotic lawn mowers tested was able to detect the presence of dependent juvenile hedgehogs (<200 g) and all models had to touch the hedgehogs to detect them. Some models caused extensive damage to the hedgehog cadavers, but there were noteworthy differences in the degree of harm inflicted, with some consistently causing no damage. Our results showed that the following technical features significantly increased the safety index of the robotic lawn mowers: pivoting blades, skid plates, and front wheel drive. Based on these findings, we encourage future collaboration with the manufacturers of robotic lawn mowers to improve the safety for hedgehogs and other garden wildlife species.

This study reported that a 15-month-old intact female African pygmy hedgehog (Atelerix albiventris) was presented to IPB University Veterinary Teaching Hospital to evaluate a 1.5 cm × 1.5 cm × 0.5 cm mass on the left ventral thorax, three days after the mass was first discovered by the owner. Overgrown nails were observed on all four toes of each of the four limbs of the hedgehog. The body weight was 150 g, and the axial temperature was 36.5°C. The heart and respiratory rates were 180 per minute and 40 per minute, respectively. After a thorough physical examination, the patient was diagnosed with subcutaneous abscesses and overgrown nails. The abscess was surgically resected using a local anesthetic. The overgrown nails were trimmed to prevent further injury and the recurrence of abscesses. Following surgery, the hedgehog was discharged directly from the hospital and treated using Amoxicillin 15 mg/kg per oral every 12 hours for seven days, daily routine wound cleaning with sodium chloride, and topical powder of neomycin sulfate 5 mg/g and bacitracin 250 IU/g. The patient responded effectively to the systemic treatment, and medical signs and symptoms resolved. The hedgehog fully recovered from the subcutaneous abscess 14 days after the first hospital visit. No recurrence was reported in the subsequent month after resolution.

Smoothened, a heptahelical membrane protein, functions as the transducer of Hedgehog signaling. The kinases that modulate Smoothened have been thoroughly analyzed in flies. However, little is known about how phosphorylation affects Smoothened in vertebrates, mainly, because the residues, where Smoothened is phosphorylated are not conserved from Drosophila to vertebrates. Given its molecular architecture, Smoothened signaling is likely to be regulated in a manner analogous to G protein–coupled receptors (GPCRs). Previously, it has been shown, that arrestins and GPCR kinases, (GRKs) not only desensitize G protein–dependent receptor signaling but also function as triggers for GPCR trafficking and formation of signaling complexes. Here we describe that a GRK contributes to Smoothened-mediated signaling in vertebrates. Knockdown of the zebrafish homolog of mammalian GRK2/3 results in lowered Hedgehog transcriptional responses, impaired muscle development, and neural patterning. Results obtained in zebrafish are corroborated both in cell culture, where zGRK2/3 phosphorylates Smoothened and promotes Smoothened signal transduction and in mice where deletion of GRK2 interferes with neural tube patterning. Together, these data suggest that a GRK functions as a vertebrate kinase for Smoothened, promoting Hedgehog signal transduction during early development.

Muscles are composed of several fibre types, the precise combination of which determines muscle function. Whereas neonatal and adult fibre type is influenced by a number of extrinsic factors, such as neural input and muscle load, there is little knowledge of how muscle cells are initially determined in the early embryo. In the zebrafish, fibres of the slow twitch class arise from precociously specified myoblasts that lie close to the midline whereas the remainder of the myotome differentiates as fast myosin expressing muscle. In vivo evidence has suggested the Sonic Hedgehog glycoprotein, secreted from the notochord, controls the formation of slow twitch and fast twitch muscle fates. Here we describe an in vitro culture system that we have developed to test directly the ability of zebrafish myoblasts to respond to exogenous Sonic Hedgehog peptide. We find that Sonic Hedgehog peptide can control the binary cell fate choice of embryonic zebrafish myoblasts in vitro. We have also used this culture system to assay the relative activities of different Hedgehog-family proteins and to investigate the possible involvement of heterotrimeric G-proteins in Hedgehog signal transduction.

ABSTRACT Deregulation of the Sonic hedgehog pathway has been implicated in an increasing number of human cancers. In this pathway, the seven-transmembrane (7TM) signaling protein Smoothened regulates cellular proliferation and differentiation through activation of the transcription factor Gli. The activity of mammalian Smoothened is controlled by three different hedgehog proteins, Indian, Desert, and Sonic hedgehog, through their interaction with the Smoothened inhibitor Patched. However, the mechanisms of signal transduction from Smoothened are poorly understood. We show that a kinase which regulates signaling by many “conventional” 7TM G-protein-coupled receptors, G protein-coupled receptor kinase 2 (GRK2), participates in Smoothened signaling. Expression of GRK2, but not catalytically inactive GRK2, synergizes with active Smoothened to mediate Gli-dependent transcription. Moreover, knockdown of endogenous GRK2 by short hairpin RNA (shRNA) significantly reduces signaling in response to the Smoothened agonist SAG and also inhibits signaling induced by an oncogenic Smoothened mutant, Smo M2. We find that GRK2 promotes the association between active Smoothened and β-arrestin 2. Indeed, Gli-dependent signaling, mediated by coexpression of Smoothened and GRK2, is diminished by β-arrestin 2 knockdown with shRNA. Together, these data suggest that GRK2 plays a positive role in Smoothened signaling, at least in part, through the promotion of an association between β-arrestin 2 and Smoothened.

Progressive osseous heteroplasia (POH) is an ultrarare genetic disease of progressive ectopic ossification caused by heterozygous inactivating mutations of GNAS, the gene encoding the alpha subunit of the G-stimulatory protein of adenylyl cyclase (Gs?). Extensive ossification of the deep connective tissues can result in ankylosis of affected joints and growth retardation of involved limbs. Inhibition of main molecular signaling, Hedgehog (Hh) pathway, by pharmacological methods may reduce the severity of ectopic bone formation in POH patients. Hh inhibitors currently used or known for other conditions may be potential candidate drugs for treating this debilitating disease. In this study, three potential Hedgehog pathway inhibitors such as arsenic trioxide, statin, and vitamin D and their combinations were tested on subcutaneous mesenchymal progenitor (SMP) cells of G?s f/f mice model for possible therapeutic application for POH. The combination of these three drugs at their significantly reduced concentrations retained anti-osteogenic activity in SMP cells with aberrant Hedgehog activity. In that light, we propose here a potential new approach of the drug combination in order to reduce potential toxicity, the side effect and increase success rate for Hh inhibitors drug repositioning.

The Hedgehog signalling pathway is an essential developmental pathway present in all bilaterians that is involved in embryogenesis, body patterning and stem cell homeostasis. Dysregulation of the Hh pathway leads to various kinds of cancer, such as basal cell carcinoma and medulloblastoma. Smoothened (SMO), a Frizzled-type G-protein coupled receptor (GPCR), is the essential transmembrane signal transducer within the Hh pathway, conveying the signal from the upstream transmembrane protein, Patched1 (Ptc1), to the downstream intracellular proteins. The mechanisms by which SMO transmits the Hh signal from the extracellular environment, through the plasma membrane and to the intracellular proteins are not known. In this thesis, I present my work into the structural and functional characterisation of the extracellular and transmembrane domains (TMD) of human SMO in order to better understand the molecular mechanisms of its signal transduction. The extracellular region of SMO contains a highly conserved cysteine-rich domain (CRD) and a linker domain (LD). I present the first crystal structure of the CRD, LD and TMD of SMO, which is also the first crystal structure of a GPCR with a large functional extracellular domain. This structure revealed a domain architecture for SMO that enables regulation of its transmembrane domain by its extracellular domains. It also revealed a cholesterol molecule bound to the CRD, which we subsequently determined to be a new endogenous small-molecule agonist for SMO. I present five further structures of SMO bound to different small molecule agonists and antagonists. Together, these structures demonstrate that the position of the CRD relative to the TMD reflects the activation state of SMO. We also generated nanobodies against the extracellular region of SMO in order to stabilise its conformation. These studies not only improve our understanding of the workings of a key transmembrane protein within a fundamental signalling pathway but will also aid efforts to develop better therapeutics for an important cancer target.

The Hedgehog (Hh) signaling pathway is an essential and highly conserved pathway required for embryonic development and adult tissues homeostasis in both invertebrates and vertebrates. In humans, abnormal Hh signaling leads to numerous congenital diseases and deregulation of Hh signaling is associated with certain aspects of tumorigenesis. Therefore, a complete characterization of this pathway may help us to better understand the role of Hh signaling under physiological and pathological conditions. A key transducer of Hh signaling is Smoothened (Smo), a seven-pass transmembrane protein and distant member of the G-protein-coupled receptor (GPCR) family. In the absence of the secreted Hh protein, Smo is inhibited by another membrane protein, the Hh-receptor Patched (Ptc). Hh binding to Ptc releases this inhibition and triggers accumulation of Smo at the cell surface. Phosphorylation of Smo by Protein kinase A (PKA) and additional kinases allows Smo to adopt an active conformation, which turns the Hh signaling pathway on and ultimately allows the expression of Hh target genes. G-protein-coupled receptor kinases (GRKs) are known to phosphorylate active GPCRs and promote homologous desensitization of GPCR signaling. Smo is a functional GPCR in Drosophila and several recent studies suggest that GRKs participate in Hh signaling in both invertebrates and vertebrates. To address the role of GRKs in Drosophila Hh signaling, we analyzed flies mutant for gprk2, one of two GRKs in flies. Interestingly, gprk2 mutant flies display defects typical of impaired Hh signaling, suggesting that Gprk2 is required for transducing the Hh signal. Further studies, however, showed that Gprk2 also plays a negative role in Hh signaling by promoting Smo desensitization, which is consistent with typical GRK biology. To explain the dual and contradictory role of Gprk2 in Hh signaling, we hypothesized that heterotrimeric G-protein-dependent signaling may be affected in gprk2 mutants through misregulation of Smo and other GPCRs. Consistent with this idea we find that basal cAMP levels are abnormally low in gprk2 mutants. Cellular cAMP concentrations control the activity of PKA, the key activator of Smo. With genetic experiments, we confirmed that cAMP levels and PKA activity are limiting for Hh target gene expression in gprk2 mutants. In conclusion, our results suggest that Gprk2 regulates Hh signaling in two ways. On one hand, it limits Hh signaling by promoting homologous desensitization of Smo. On the other hand, it is also required to keep cellular cAMP concentrations in a physiological range that is required for normal Hh signaling. We speculate that the abnormally low cAMP levels in gprk2 mutants are not only caused by misregulation of Smo, but also by a more global misregulation of GPCR signaling, suggesting that Hh signalling is affected by signalling cross-talk.
La voie de signalisation Hedgehog (Hh) est une cascade de signalisation essentielle et hautement conservée, requise lors du développement embryonnaire et le maintien de l'homéostasie dans les tissus adultes, tant chez les invertébrés que chez les vertébrés. Chez l'homme, une signalisation anormale de cette voie de signalisation entraîne de nombreux défauts congénitaux, et est associée à certains aspects de la tumorigénèse. Ainsi, une caractérisation complète de cette cascade signalitique nous aiderait à mieux comprendre le rôle de Hh dans des conditions physiologiques et pathologiques. La protéine Smoothened (Smo) est une composante importante de la signalisation par Hh. Smo est composée de sept domaines transmembranaires et fait partie de la famille de "G-protein-coupled receptor" (GPCR). En absence de Hh, Smo est inhibée par la protéine membranaire "Hh-receptor Patched" (Ptc). La liaison de Hh à Ptc relâche l'inhibition qu'exerce cette dernière sur Smo, qui s'accumule alors à la surface de la cellule. La phosphorylation de Smo par la Protéine kinase A (PKA) ou d'autres kinases permet à Smo d'adopter sa conformation dite active, qui enclenche alors la cascade signalitique qui induit l'expression des gènes cibles de Hh.Il a été démontré que les protéines "G-protein-coupled receptor kinases" (GRKs) phosphorylent les GPCRs actives et promouvoient ainsi la désensibilisation à leur signalisation. De récentes études suggèrent que les GRKs participent à la signalisation par Hh chez les invertébrés et chez les vertébrés. Afin de mieux comprendre le role des GRKs dans la signalisation de Hh chez la drosophile, nous avons analysé des mouches mutantes pour gprk2, l'une des deux GRKs présentes dans ce modèle. Surprenament, les mouches mutantes pour gprk2 présentent des défauts qui sont typiques à la voie de signalisation Hh. Ceci suggère que Gprk2 est requise pour transmettre les signaux de signalisation en aval de Hh. Toutefois, de plus amples études ont révélées que Gprk2 régule aussi de façon negative cette voie de signalisation en induisant la désensibilisation à Smo. Afin d'expliquer les rôles contradictoires de Gprk2 dans la voie de signalisation Hh, nous avons émis l'hypothèse que les défauts de signalisation par les GPCRs observés chez les mouches mutantes pour gprk2 sont dûs à une mauvaise regulation de Smo et d'autres GPCRs. Ainsi, nous avons découvert que les niveaux basals d'AMPc sont anormalement bas chez les mutants de gprk2. Les concentrations intracellulaires d'AMPc régulent l'activité de la PKA, qui elle-même régule Smo. À l'aide d'expériences génétiques, nous avons confirmé que les bas niveaux d'AMPc et de l'activité de la PKA limitent l'expresssion des gènes cibles de Hh chez les mutants de gprk2. En conclusion, nous résultats suggèrent que Gprk2 régule la signalisaiton de Hh de deux façons distinctes. D'une part, elle régule la signalisation en amont de Hh en induisant la désensibilisation à Smo. D'autre part, Gprk2 est requise afin de maintenir les niveaux d'AMPc à des niveaux physiologiques, ce qui est nécessaire pour une signalisation normale de Hh. Nos résultats suggèrent que les niveaux anormalement faible d'AMPc chez les mutants de gprk2 ne sont pas dûs uniquement à une mauvaise regulation de Smo, mais à une mauvaise regulation plus globale de GPCRs en general. Ainsi, la signalisation par Hh semble être régulée par différentes boucles de rétroactivation.

El cerebelo es el órgano encargado de integrar las vías sensitivas y las motoras del sistema nervioso controlando, por ejemplo, los movimientos involuntarios del cuerpo. Su desarrollo comienza en la etapa embrionaria, en la que tiene lugar la formación del órgano y de sus principales capas. Conforme avanza el desarrollo, tiene lugar un crecimiento del órgano debido a una elevada proliferación de los precursores de las células granulares (PNGCs), localizados en su capa más externa. Su desarrollo finaliza en la etapa postnatal, en la que el cerebelo aumenta aun más su tamaño debido al mantenimiento de esta elevada actividad mitogénica. Alcanza su madurez cuando los PNGCs se diferencian y migran hacia la capa más interna. El morfógeno Sonic Hedgehog (Shh) es el responsable de la proliferación de los PNGCs. Esta proteína activa al complejo de receptores constituído por Patched (Ptch) y Smoothened (Smo), que desencadenaran una cascada de señalización dando lugar a la división celular. La actividad de Shh puede ser regulada negativamente por la proteína quinasa A dependiente de AMP cíclico (PKA). En su estado basal, la subunidad catalítica de PKA (PKA-C) se encuentra inhibida por su asociación con la subunidad reguladora (R-PKA). En la presente tesis se demuestra que en presencia de Shh la subunidad PKA-C inactiva se localiza en la base del cilio de PNGCs, estructura celular necesaria para que la señalización de Shh tenga lugar y donde se acumulan varias moléculas de la vía como Smo y Ptch. Cuando los PNGCs son deprivados de Shh tiene lugar una activación y una parcial dispersión de PKA-C de la base del cilio. Mostramos que la subunidad reguladora RII (PKA-RII) de PKA es la responsable del anclaje de la holoenzima a través de su unión a las proteínas de anclaje de PKA (AKAPs). La disrupción de la interacción entre PKA y AKAPs inhibe la actividad de Shh bloqueando la proliferación de los PNGCs. Estos resultados, por lo tanto, demuestran que el pool de PKA localizado en la base del cilio juega un papel esencial en la integración de la transducción de la señal de Shh. En la segunda parte de la presente tesis estudiamos la activación de proteínas G heterotriméricas mediante Smo. Aunque en su secuencia Smo sugiere que pertenece a la familia de los receptores acoplados a proteína G (GPCR), únicamente en Drosophila melanogaster se demostró que la proteína Gαi es necesaria para la actividad de Hedgehog (Hh), mientras que las evidencias de la implicación de las proteínas G como efectoras de la vía de Shh de los vertebrados no son muy claras. En este trabajo demostramos que la inducción de la proliferación de PNGCs se potencia por la expresión de formas activas de proteínas de la clase Gαi/o (Gαi1, Gαi2, Gαi3 y Gαo) pero no por las de la clase Gα12 de proteínas G. Además, observamos que los miembros de la familia de Gαi poseen un patrón de expresión específico en el cerebelo en desarrollo: únicamente Gαi2 y Gαi3 se encuentran altamente expresados en la parte más externa de la capa granular externa, donde tiene lugar la proliferación de los PNGCs. En concordancia con este resultado observamos que la proliferación de los PNGCs se reduce de manera significativa mediante la eliminación de Gαi2 y Gαi3 pero no cuando silenciamos a otros miembros de la familia de Gαi/o. Finalmente, los resultados de la presente tesis demuestran que las subunidades Gαi2 y Gαi3 se localizan en el cilio primario cuando son sobre-expresados en cultivos de PCGCs. Por lo tanto, los efectos proliferativos de Shh en estas células están mediados por la activación combinada de las proteínas Gαi2 y Gαi3 de proteínas G.
During cerebellum development, clonal expansion of cerebellar granular neuronal precursors (CGNPs) takes place in response to Sonic Hedgehog (Shh) signalling pathway. Shh transduces its signals through the Patched (Ptc) and Smoothened (Smo) receptor complex, delivering activated forms of Gli (Ci) transcription factors to the nucleus. PKA is a known negative regulator of the CGNPs mitogenic activity by inducing Gli2/3 phosphorylation and their subsequent degradation by the proteasome generating potent transcriptional repressors of the pathway. In mammals, Shh pathway requires and takes place at a specific cellular structure: the primary cilum, where many of the proteins of the pathway localize. In this thesis we demonstrated that PKA-C subunit accumulates at the base of the primary cilium through PKA-RII subunit that joins to an unknown AKAP. In the absence of Shh, PKA activates and spreads from the primary cilium base and phosphorylates its target proteins. PKA needs to be accumulated at this specific location, since the inhibition of PKA localization to the cilium base reduces CGNPs mitosis. In the second part of the present thesis we demonstrate that any active member of the Gai/o but not of the Ga12/13 family of heterotrimeric G proteins can induce CGNPs proliferation at low but non active doses of Shh. We also show that Gai2 and Gai3 are the only expressed proteins of this family in the external granular layer of the developing cerebellum (EGL), where proliferation takes place. These proteins are the specific members responsible for Shh activity as their ablation by RNAi has a synergic effect in reducing CGNPs mitogenic activity. Moreover, these subunits can exclusively accumulate at the cilium shaft. All together these results indicate that Gai2 and Gai3 are the two specific members that mediate Shh pathway in this cells.

La voie de signalisation Hedgehog constitue l’une des plus importantes voies dans le développement embryonnaire et la prolifération des cellules souches chez l’adulte. Cette voie implique deux protéines membranaires, Patched et Smoothened, dont les dysfonctionnements sont associés à de très nombreux cancers. Durant ma thèse, j’ai mis au point l’expression hétérologue du récepteur humain Smoothened (hSmo) et sa purification pour une caractérisation structurale et fonctionnelle. L’expression de hSmo a été réalisée dans la levure Saccharomyces cerevisiae. Utilisant la technique SPR, j’ai démontré que hSmo exprimé à la membrane plasmique de la levure est dans sa conformation native capable de fixer son antagoniste. J’ai ensuite mis au point la purification de hSmo et testé de nouvelles classes de surfactants. J’ai ainsi trouvé les meilleures conditions qui stabilisent le récepteur hSmo en solution après purification. La caractérisation d’une mutation ponctuelle au niveau de la 3ème boucle intracellulaire combinée à l’utilisation des nouveaux surfactants ont permis d’améliorer la stabilité de hSmo en solution. Les conditions que j’ai mises au point permettront l’étude structurale de hSmo et les essais de cristallisation. D’autre part, les stratégies développées en SPR permettront la recherche des partenaires protéiques cytoplasmiques de ce récepteur, encore inconnus à ce jour, afin de mieux comprendre la signalisation en aval de Smoothened. Les données structurales ainsi que la découverte des partenaires protéiques cytoplasmiques de hSmo permettront l’élaboration de nouvelles thérapies anticancéreuses
The Hedgehog pathway is one of the most important pathways in embryogenesis and in proliferation of adult stem cells. This pathway involves two transmembrane receptors, Patched and Smoothened whose dysfunctions have been linked to many human diseases including cancers. This study reports expression and purification of the human GPCR Smoothened, for structure-function relationship characterization. Therefore I developed the heterologous expression of Human Smoothened (hSmo) in the yeast S. Cerevisiae. Using SPR technology, I showed that hSmo, expressed at the plasma membrane of yeast, is in its native conformation able to bind its antagonist, cyclopamine (CPN). Then, I developed the purification of hSmo by affinity chromatography and tested new surfactants. Results show that the new surfactants stabilize hSmo in solution after purification and are preserve antagonist-binding ability of Smo suggesting that purified hSmo maintains its native conformation in solution. In addition, characterization of a single mutation of Smoothened (hSmoG435R) combined to one of the surfactants, revealed an enhanced stability of the receptor. These established conditions will be useful for crystallization assays. SPR strategies developed in this study will also be used for the research of hSmo’s cytoplasmic partners. Together, structural and functional data will contribute to the better understanding of Smo signaling and to the development of new cancer therapies

Abstract There is an entry in Time Flies (1885), ‘A Reading Diary’, by Christina G. Rossetti, where time flies back and returns her from her mid-fifties to her childhood. The mouse and the insect revive, as mortally stark as the groundhog of Richard Eberhart’s poem or the hedgehog of Beckett’s Company. But one would never divine from the scholar’s paraphrase in 1987 that such an incident could put the fear of God into anyone: Rossetti recalls her first experience of death when, in early childhood, she found a dead mouse in the grounds of a cottage.