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Milano, Italy

Kuhn C.,Axxam SpA | Meyerhof W.,German Institute of Human Nutrition
Methods in Cell Biology | Year: 2013

The superfamily of G protein-coupled receptors (GPCRs) mediates numerous physiological processes, including neurotransmission, cell differentiation and metabolism, and sensory perception. In recent years, it became evident that these receptors might function not only as monomeric receptors but also as homo- or heteromeric receptor complexes. The family of TAS1R taste receptors are prominent examples of GPCR dimerization as they act as obligate functional heteromers: TAS1R1 and TAS1R3 combine to form an umami taste receptor, while the combination of TAS1R2 and TAS1R3 is a sweet taste receptor. So far, TAS2Rs, a second family of ~. 25 taste receptors in humans that mediates responses to bitter compounds, have been shown to function on their own, but if they do so as receptor monomers or as homomeric receptors still remains unknown. Using two different experimental approaches, we have recently shown that TAS2Rs can indeed form both homomeric and heteromeric receptor complexes. The employed techniques, coimmunoprecipitations and bioluminescence resonance energy transfer (BRET), are based on different principles and complement each other well and therefore provided compelling evidences for TAS2R oligomerization. Furthermore, we have adapted the protocols to include a number of controls and for higher throughput to accommodate the investigation of a large number of receptors and receptor combinations. Here, we present the protocols in detail. © 2013 Elsevier Inc.

Haddock S.H.D.,Monterey Bay Aquarium Research Institute | Mastroianni N.,Axxam SpA | Christianson L.M.,Monterey Bay Aquarium Research Institute
Proceedings of the Royal Society B: Biological Sciences | Year: 2010

Genes for the family of green-fluorescent proteins (GFPs) have been found in more than 100 species of animals, with some species containing six or more copies producing a variety of colours. Thus far, however, these species have all been within three phyla: Cnidaria, Arthropoda and Chordata. We have discovered GFP-type fluorescent proteins in the phylum Ctenophora, the comb jellies. The ctenophore proteins share the xYG chromophore motif of all other characterized GFP-type proteins. These proteins exhibit the uncommon property of reversible photoactivation, in which fluorescent emission becomes brighter upon exposure to light, then gradually decays to a non-fluorescent state. In addition to providing potentially useful optical probes with novel properties, finding a fluorescent protein in one of the earliest diverging metazoans adds further support to the possibility that these genes are likely to occur throughout animals. © 2009 The Royal Society.

Gavina M.,University of Milan | Za L.,University of Milan | Za L.,Axxam SpA | Molteni R.,University of Milan | And 2 more authors.
Biology of the Cell | Year: 2010

Background information. Cell motility entails the reorganization of the cytoskeleton and membrane trafficking for effective protrusion. The GIT-PIX protein complexes are involved in the regulation of cell motility and adhesion and in the endocytic traffic of members of the family of G-protein-coupled receptors. We have investigated the function of the endogenous GIT complexes in the regulation of cell motility stimulated by fMLP (formyl-Met-Leu-Phe) peptide, in a rat basophilic leukaemia RBL-2H3 cell line stably expressing an HA (haemagglutinin)-tagged receptor for the fMLP peptide. Results. Our analysis shows that RBL cells stably transfected with the chemoattractant receptor expressed both GIT1-PIX and GIT2-PIX endogenous complexes. We have used silencing of the different members of the complex by small interfering RNAs to study the effects on a number of events linked to agonist-induced cell migration. We found that cell adhesion was not affected by depletion of any of the proteins of the GIT complex, whereas agonist-enhanced cell spreading was inhibited. Analysis of agonist-stimulated haptotactic cell migration indicated a specific positive effect of GIT1 depletion on trans-well migration. The internalization of the formyl-peptide receptor was also inhibited by depletion of GIT1 and GIT2. The effects of the GIT complexes on trafficking of the receptors was confirmed by an antibody-enhanced agonist-induced internalization assay, showing that depletion of PIX, GIT1 or GIT2 protein caused decreased perinuclear accumulation of internalized receptors. Conclusions. Our results show that endogenous GIT complexes are involved in the regulation of chemoattractantinduced cell motility and receptor trafficking, and support previous findings indicating an important function of the GIT complexes in the regulation of different G-protein-coupled receptors. Our results also indicate that endogenous GIT1 and GIT2 regulate distinct subsets of agonist-induced responses and suggest a possible functional link between the control of receptor trafficking and the regulation of cell motility by GIT proteins.

Saluste G.,Spanish National Cancer Research Center | Albarran M.I.,Spanish National Cancer Research Center | Alvarez R.M.,Spanish National Cancer Research Center | Rabal O.,Spanish National Cancer Research Center | And 12 more authors.
PLoS ONE | Year: 2012

A new chemical series, triazolo[4,5-b]pyridines, has been identified as an inhibitor of PIM-1 by a chemotype hopping strategy based on a chemically feasible fragment database. In this case, structure-based virtual screening and in silico chemogenomics provide added value to the previously reported strategy of prioritizing among proposed novel scaffolds. Pairwise comparison between compound 3, recently discontinued from Phase I clinical trials, and molecule 8, bearing the selected novel scaffold, shows that the primary activities are similar (IC50 in the 20 to 150 nM range). At the same time, some ADME properties (for example, an increase of more than 45% in metabolic stability in human liver microsomes) and the off-target selectivity (for example, an increase of more than 2 log units in IC50 vs. FLT3) are improved, and the intellectual property (IP) position is enhanced. The discovery of a reliable starting point that fulfills critical criteria for a plausible medicinal chemistry project is demonstrated in this prospective study. © 2012 Saluste et al.

Veitinger S.,RWTH Aachen | Veitinger S.,Ruhr University Bochum | Veitinger T.,RWTH Aachen | Cainarca S.,Axxam SpA | And 9 more authors.
Journal of Physiology | Year: 2011

Intimate bidirectional communication between Sertoli cells and developing germ cells ensures the integrity and efficiency of spermatogenesis. Yet, a conceptual mechanistic understanding of the physiological principles that underlie Sertoli cell autocrine and paracrine signalling is lacking. Here, we characterize a purinergic Ca 2+ signalling network in immature mouse Sertoli cells that consists of both P2X2 and P2Y2 purinoceptor subtypes, the endoplasmic reticulum and, notably, mitochondria. By combining a transgenic mouse model with a dedicated bioluminescence imaging device, we describe a novel method to monitor mitochondrial Ca 2+ mobilization in Sertoli cells at subcellular spatial and millisecond temporal resolution. Our data identify mitochondria as essential components of the Sertoli cell signalling 'toolkit' that control the shape of purinergic Ca 2+ responses, and probably several other paracrine Ca 2+-dependent signals. © 2011 The Authors. Journal compilation © 2011 The Physiological Society.

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