PTCHD1 Binds Cholesterol but Not Sonic Hedgehog, Suggesting a Distinct Cellular Function

Deleterious mutations in the X-linked Patched domain-containing 1 (PTCHD1) gene may account for up to 1% of autism cases. Despite this, the PTCHD1 protein remains poorly understood. Structural similarities to Patched family proteins point to a role in sterol transport, but this hypothesis has not been verified experimentally. Additionally, PTCHD1 has been suggested to be involved in Hedgehog signalling, but thus far, the experimental results have been conflicting. To enable a variety of biochemical and structural experiments, we developed a method for expressing PTCHD1 in Spodoptera frugiperda cells, solubilising it in glycol-diosgenin, and purifying it to homogeneity. In vitro and in silico experiments show that PTCHD1 function is not interchangeable with Patched 1 (PTCH1) in canonical Hedgehog signalling, since it does not repress Smoothened in Ptch1−/− mouse embryonic fibroblasts and does not bind Sonic Hedgehog. However, we found that PTCHD1 binds cholesterol similarly to PTCH1. Furthermore, we identified 13 PTCHD1-specific protein interactors through co-immunoprecipitation and demonstrated a link to cell stress responses and RNA stress granule formation. Thus, our results support the notion that despite structural similarities to other Patched family proteins, PTCHD1 may have a distinct cellular function

Activation of the Gi protein-RHOA axis by non-canonical Hedgehog signaling is independent of primary cilia.

Primary cilia are solitary organelles that emanate from the plasma membrane during growth arrest in almost all mammalian cells. The canonical Hedgehog (HH) pathway requires trafficking of the G protein-coupled receptor SMOOTHENED (SMO) and the GLI transcription factors to the primary cilium upon binding of a HH ligand to PATCHED1. However, it is unknown if activation of the small GTPase RHOA by SMO coupling to heterotrimeric Gi proteins, a form of non-canonical HH signaling, requires localization of SMO in the primary cilium. In this study, we compared RHOA and Gi protein stimulation by activation of SMO or sphingosine 1-phosphate receptor (S1P) receptors in WT and KIF3A-deficient mouse embryonic fibroblasts that lack primary cilia. We found that activation of SMO in response to Sonic HH (SHH) or purmorphamine (PUR), a small molecule agonist of SMO, stimulates Gi proteins and RHOA independently of the presence of primary cilia, similar to the effects of S1P. However, while S1P induced a fast activation of AKT that is sensitive to the Gi inhibitor pertussis toxin, HH pathway activators did not significantly activate AKT, suggesting that RHOA activation is not downstream of AKT. Our findings demonstrate that early events in some forms of non-canonical HH signaling occur in extraciliary membranes, which might be particularly relevant for actively-cycling cells, for some cancers characterized by loss of primary cilia, and in ciliopathies

Coupling of Smoothened to inhibitory G proteins reduces voltage-gated K+ currents in cardiomyocytes and prolongs the cardiac action potential duration

Smoothened (SMO), the central transducer of Hedgehog signaling, is coupled to heterotrimeric Gi proteins in many cell types, including cardiomyocytes. In this study, we report that activation of SMO with Sonic Hedgehog (SHH) or a small agonist, purmorphamine, rapidly causes a prolongation of the action potential duration that is sensitive to a SMO inhibitor. In contrast, neither of the SMO agonists prolonged the action potential in cardiomyocytes from transgenic GiCT/TTA mice, in which Gi signaling is impaired, suggesting that the effect of SMO is mediated by Gi proteins. Investigation of the mechanism underlying the change in action potential kinetics revealed that activation of SMO selectively reduces outward voltage-gated K⁺ repolarizing (Kv) currents in isolated cardiomyocytes and that it induces a downregulation of membrane levels of Kv4.3 in cardiomyocytes and intact hearts from wild type but not from GiCT/TTA mice. Moreover, perfusion of intact hearts with Shh or purmorphamine increased the ventricular repolarization time (QT interval) and induced ventricular arrhythmias. Our data constitute the first report that acute, non-canonical Hh signaling mediated by Gi proteins regulates K⁺ currents density in cardiomyocytes and sensitizes the heart to the development of ventricular arrhythmias

Ptch2/Gas1 and Ptch1/Boc differentially regulate Hedgehog signalling in murine primordial germ cell migration.

Gas1 and Boc/Cdon act as co-receptors in the vertebrate Hedgehog signalling pathway, but the nature of their interaction with the primary Ptch1/2 receptors remains unclear. Here we demonstrate, using primordial germ cell migration in mouse as a developmental model, that specific hetero-complexes of Ptch2/Gas1 and Ptch1/Boc mediate the process of Smo de-repression with different kinetics, through distinct modes of Hedgehog ligand reception. Moreover, Ptch2-mediated Hedgehog signalling induces the phosphorylation of Creb and Src proteins in parallel to Gli induction, identifying a previously unknown Ptch2-specific signal pathway. We propose that although Ptch1 and Ptch2 functionally overlap in the sequestration of Smo, the spatiotemporal expression of Boc and Gas1 may determine the outcome of Hedgehog signalling through compartmentalisation and modulation of Smo-downstream signalling. Our study identifies the existence of a divergent Hedgehog signal pathway mediated by Ptch2 and provides a mechanism for differential interpretation of Hedgehog signalling in the germ cell niche

The systematic annotation of the three main GPCR families in Reactome

Reactome is an open-source, freely available database of human biological pathways and processes. A major goal of our work is to provide an integrated view of cellular signalling processes that spans from ligand–receptor interactions to molecular readouts at the level of metabolic and transcriptional events. To this end, we have built the first catalogue of all human G protein-coupled receptors (GPCRs) known to bind endogenous or natural ligands. The UniProt database has records for 797 proteins classified as GPCRs and sorted into families A/1, B/2 and C/3 on the basis of amino accid sequence. To these records we have added details from the IUPHAR database and our own manual curation of relevant literature to create reactions in which 563 GPCRs bind ligands and also interact with specific G-proteins to initiate signalling cascades. We believe the remaining 234 GPCRs are true orphans. The Reactome GPCR pathway can be viewed as a detailed interactive diagram and can be exported in many forms. It provides a template for the orthology-based inference of GPCR reactions for diverse model organism species, and can be overlaid with protein–protein interaction and gene expression datasets to facilitate overrepresentation studies and other forms of pathway analysis

Importancia del agua en la hidratación de la población española: documento FESNAD 2010

For any healthy individual, thirst is an appropriate sign to drink water, except for babies, sportsmen, and most of ill and elderly people. In these instances, it is convenient to schedule appropriate times to drink water since great demands and the physiological mechanisms that determine thirst in these situations may condition water unbalances with important consequences for health and the physical and intellectual performance. The human body has a number of mechanisms that allow keeping constant the water content by adjusting intakes and wastes. Water balance is determined by intake (consumed water, beverages, and water contained in foods) and wastes (urine, stools, the skin, and expired air from the lungs). Failure of these mechanisms and subsequent impairments in water balance may produce severe disarrangements that may threaten somebody's life. In the present document, we analyze the evidences regarding the factors conditioning water needs in the different life stages and physiological situations, as well as the consequences of water unbalance under different situations. A proper hydration may be achieved by feeding and the use of water and other liquids. Although water is the beverage by excellence and represents the ideal way of restoring the losses and get hydrated, we should be aware that, from the very beginning, we have sought other liquid sources with hydration properties. In the last decades we have increased the consumption of different beverages, with a proliferation of sugar-containing beverages. Since excessive sugar consumption has been related to obesity and other chronic conditions, it is evident that the use of these caloric beverages should be rationalized, especially in children. In this document all the considerations regarding hydration are presented and different recommendations are expose

Functional divergence in the role of N-linked glycosylation in smoothened signaling

The G protein-coupled receptor (GPCR) Smoothened (Smo) is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh) pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Gαi. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. Herein, we present a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, we found that all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Gαi, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, we postulate a profound shift in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice