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Grant
Agency: European Commission | Branch: FP7 | Program: MC-ITN | Phase: FP7-PEOPLE-2011-ITN | Award Amount: 3.34M | Year: 2012

SPHINGONET was inspired by a common vision of its partners on the training of researchers in the field of sphingolipid homeostasis and body-wide sphingolipid signaling networks using innovative technological approaches. Dysregulation of sphingolipid balances contributes to a broad range of pathological processes, spanning neurodegeneration, asthma, autoimmune disease, insuline resistance, obesity and cancer progression. Key to defining proper sites for therapeutic intervention is a comprehensive understanding of the mechanisms of sphingolipid homeostasis and how sphingolipid-mediated signaling pathways are interconnected. In spite of its many clinical implications, progress in this field is curbed by a lack of appropriate tools to monitor, quantify and manipulate sphingolipid pools in live cells. SPHINGONETS training program is designed to close these gaps in knowledge and technology by transferring the complementary expertise of its partners to a future generation of scientists who will take a leading role in decoding the full regulatory potential of the sphingolipid signaling network and maximize its therapeutic use. By merging seven academic partners working at the forefront of sphingolipid, chemical and systems biology with three (pro)drug discovery-oriented SMEs, SPHINGONET will create a challenging interdisciplinary and clinically-relevant research environment with ample opportunities for structuring industrial projects, commercial exploitation of results, entrepreneurship and complementary education adapted to the personal needs. Thus, SPHINGONET will provide its trainees with a rounded education that, besides enhancing their career perspectives, will enable them to choose a career path in Europes academia or industry, and be successful at it while retaining ties between both these bodies.


Grant
Agency: European Commission | Branch: FP7 | Program: MC-IAPP | Phase: FP7-PEOPLE-2009-IAPP | Award Amount: 643.49K | Year: 2010

The Pharmaceutical sector is a mainstay of the European economy. However, changing patterns of drug discovery and competition has greatly enhanced the need for a SME sector to produce new drug leads and targets to feed big Pharma pipelines. This application addresses the need by coalescing partners involved in the identification/validation of new targets and in drug design, synthesis and discovery. The focus is on type 2 diabetes, obesity and immune diseases which cause serious disability and reduced life expectancy globally. Dualsystems and NUIM will be involved in the identification/validation of protein interactions involved in the pathway linking retinol binding protein and insulin, through which the former, when elevated in obesity, inhibits responses to the latter thereby contributing to type 2 diabetes. A second project will identify the protein machinery involved in the failure of otherwise active natural mutants of the MC4 receptor to traffic to the cell surface. This failure produces 6-7% of cases of gross genetic obesity. The third project will identify native protein ligands for a new family of G-protein coupled receptors (LNB-GPCRs), deeply implicated in immune-related diseases and cancer. The transfer of knowledge (TOK) involve development and use of yeast 2-hybrid systems to identify putative interactors and of rapid in vitro systems for interacting protein validation. A new yeast-based biosensor system will be advanced from proof of concept to the development stage. AFChemPharm focuses on new systems of chemical synthesis. They will work with NUIM on protein modelling, drug design, in silico screening and chemical synthesis to produce selected panels of compounds to screen for hits and to optimise into leads. The TOK and skills acquisition will involve state of the art computational approaches in modelling, drug design and screening, together with their amalgamation with new synthetic processes to tailor/refine design and synthesis.


Zografou S.,Foundation for Research and Technology Hellas | Zografou S.,University of Ioannina | Basagiannis D.,Foundation for Research and Technology Hellas | Basagiannis D.,University of Ioannina | And 8 more authors.
Journal of Cell Science | Year: 2012

Weibel-Palade bodies (WPBs) are endothelial-cell-specific organelles that, upon fusion with the plasma membrane, release cargo molecules that are essential in blood vessel abnormalities, such as thrombosis and inflammation, as well as in angiogenesis. Despite the importance of WPBs, the basic mechanisms that mediate their secretion are only poorly understood. Rab GTPases play fundamental role in the trafficking of intracellular organelles. Yet, the only known WPB-associated Rabs are Rab27a and Rab3d. To determine the full spectrum of WPB-associated Rabs we performed a complete Rab screening by analysing the localisation of all Rabs in WPBs and their involvement in the secretory process in endothelial cells. Apart from Rab3 and Rab27, we identified three additional Rabs, Rab15 (a previously reported endocytic Rab), Rab33 and Rab37, on the WPB limiting membrane. A knockdown approach using siRNAs showed that among these five WPB Rabs only Rab3, Rab27 and Rab15 are required for exocytosis. Intriguingly, we found that Rab15 cooperates with Rab27a in WPB secretion. Furthermore, a specific effector of Rab27, Munc13-4, appears to be also an effector of Rab15 and is required for WPB exocytosis. These data indicate that WPB secretion requires the coordinated function of a specific group of Rabs and that, among them, Rab27a and Rab15, as well as their effector Munc13-4, cooperate to drive exocytosis. © 2012.


Hwang C.-S.,California Institute of Technology | Shemorry A.,California Institute of Technology | Auerbach D.,Dualsystems Biotech | Varshavsky A.,California Institute of Technology
Nature Cell Biology | Year: 2010

Substrates of the N-end rule pathway are recognized by the Ubr1 E3 ubiquitin ligase through their destabilizing amino-terminal residues. Our previous work showed that the Ubr1 E3 and the Ufd4 E3 together target an internal degradation signal (degron) of the Mgt1 DNA repair protein. Ufd4 is an E3 enzyme of the ubiquitin-fusion degradation (UFD) pathway that recognizes an N-terminal ubiquitin moiety. Here we show that the RING-type Ubr1 E3 and the HECT-type Ufd4 E3 interact, both physically and functionally. Although Ubr1 can recognize and polyubiquitylate an N-end rule substrate in the absence of Ufd4, the Ubr1-Ufd4 complex is more processive in that it produces a longer substrate-linked polyubiquitin chain. Conversely, Ubr1 can function as a polyubiquitylation-enhancing component of the Ubr1-Ufd4 complex in its targeting of UFD substrates. We also found that Ubr1 can recognize the N-terminal ubiquitin moiety. These and related advances unify two proteolytic systems that have been studied separately for two decades. © 2010 Macmillan Publishers Limited. All rights reserved.


Uhlmann T.,University of Oxford | Uhlmann T.,Dualsystems Biotech | Geoghegan V.L.,University of Oxford | Thomas B.,University of Oxford | And 4 more authors.
Molecular and Cellular Proteomics | Year: 2012

The lack of methods for proteome-scale detection of arginine methylation restricts our knowledge of its relevance in physiological and pathological processes. Here we show that most tryptic peptides containing methylated arginine(s) are highly basic and hydrophilic. Consequently, they could be considerably enriched from total cell extracts by simple protocols using either one of strong cation exchange chromatography, isoelectric focusing, or hydrophilic interaction liquid chromatography, the latter being by far the most effective of all. These methods, coupled with heavy methyl-stable isotope labeling by amino acids in cell culture and mass spectrometry, enabled in T cells the identification of 249 arginine methylation sites in 131 proteins, including 190 new sites and 93 proteins not previously known to be arginine methylated. By extending considerably the number of known arginine methylation sites, our data reveal a novel prolinerich consensus motif and identify for the first time arginine methylation in proteins involved in cytoskeleton rearrangement at the immunological synapse and in endosomal trafficking. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.


Veith C.,Universities of Giessen and Marburg Lung Center | Marsh L.M.,Ludwig Boltzmann Research Institute | Wygrecka M.,Justus Liebig University | Rutschmann K.,Dualsystems Biotech | And 4 more authors.
American Journal of Pathology | Year: 2012

Pulmonary hypertension (PH) is a fatal disease characterized by remodeling processes such as increased migration and proliferation of pulmonary arterial smooth muscle cells (PASMC), enhanced matrix deposition, and dysregulation of cytoskeletal proteins. However, the contribution of cytoskeletal proteins in PH is still not fully understood. In this study, we have used a yeast two-hybrid screen to identify novel binding partners of the cytoskeletal adaptor protein four-and-a-half LIM domains 1 (Fhl-1). This identified paxillin as a new Fhl-1 interacting partner, and consequently we assessed its contribution to vascular remodeling processes. Native protein-protein binding was confirmed by co-immunoprecipitation studies in murine and human PASMC. Both proteins co-localized in PASMC in vitro and in vivo. In lung samples from idiopathic pulmonary arterial hypertension patients, paxillin expression was increased on mRNA and protein levels. Laser-microdissection of murine intrapulmonary arteries revealed elevated paxillin expression in hypoxia-induced PH. Furthermore, hypoxia-dependent upregulation of paxillin was HIF-1α dependent. Silencing of paxillin expression led to decreased PASMC adhesion, proliferation, and increased apoptosis. Regulation of these processes occurred via Akt and Erk1/2 kinases. In addition, adhesion of PASMC to the extracellular matrix protein fibronectin was critically dependent on paxillin expression. To summarize, we identified paxillin as a new regulator protein of PASMC growth. © 2012 American Society for Investigative Pathology.


Schaeuble K.,University of Konstanz | Hauser M.A.,University of Konstanz | Rippl A.V.,University of Konstanz | Bruderer R.,Dualsystems Biotech | And 4 more authors.
Journal of Cell Science | Year: 2012

The chemokine receptor CCR7 is essential for lymphocyte and dendritic cell homing to secondary lymphoid organs. Owing to the ability to induce directional migration, CCR7 and its ligands CCL19 and CCL21 are pivotal for the regulation of the immune system. Here, we identify a novel function for receptor ubiquitylation in the regulation of the trafficking process of this G-protein-coupled seven transmembrane receptor. We discovered that CCR7 is ubiquitylated in a constitutive, ligand-independent manner and that receptor ubiquitylation regulates the basal trafficking of CCR7 in the absence of chemokine. Upon CCL19 binding, we show that internalized CCR7 recycles back to the plasma membrane via the trans-Golgi network. An ubiquitylation-deficient CCR7 mutant internalized normally after ligand binding, but inefficiently recycled in immune cells and was transiently retarded in the trans-Golgi network compartment of HEK293 transfectants. Finally, we demonstrate that the lack of CCR7 ubiquitylation profoundly impairs immune cell migration. Our results provide evidence for a novel function of receptor ubiquitylation in the regulation of CCR7 recycling and immune cell migration. © 2012.


Rezwan M.,Dualsystems Biotech | Auerbach D.,Dualsystems Biotech
Methods | Year: 2012

The majority of small molecule drugs act on protein targets to exert a therapeutic function. It has become apparent in recent years that many small molecule drugs act on more than one particular target and consequently, approaches which profile drugs to uncover their target binding spectrum have become increasingly important. Classical yeast two-hybrid systems have mainly been used to discover and characterize protein-protein interactions, but recent modifications and improvements have opened up new routes towards screening for small molecule-protein interactions. Such yeast "n" -hybrid systems hold great promise for the development of drugs which interfere with protein-protein interactions and for the discovery of drug-target interactions. In this review, we discuss several yeast two-hybrid based approaches with applications in drug discovery and describe a protocol for yeast three-hybrid screening of small molecules to identify their direct targets. © 2012 Elsevier Inc.


Sponder G.,Free University of Berlin | Rutschmann K.,Dualsystems Biotech | Kolisek M.,Free University of Berlin
Magnesium Research | Year: 2014

Membrane topology is an important parameter for understanding the function and regulation of any integral protein. This aspect of the NME SLC41A1 is currently under debate. The most probable model, which has been computer-predicted, exhibits ten TMh with both termini being oriented intracellularly. However, other freely accessible online prediction programs predict that SLC41A1 possesses eleven ("outside-in" configuration), nine ("outside-in" configuration), or eight ("inside-in" configuration) TMh. The consensus based on published experimental data acquired by independent research teams is that the N-terminal flanking region is located intracellularly. However, controversy remains about the orientation of the C-terminus, which has lately been proposed to be extracellular in peer-reviewed bibliography. Here, we performed splitubiquitin functional assays with transgenic SLC41A1 fused N- or C-terminally to a Cub-LexA-VP16 reporter cassette. The bait constructs were co-expressed in S. cerevisiae st. NMY51 with positive recombinant membrane markers (Ost1, Fur4, Alg5, Tom20) tagged with NubI (or NubG). Ubiquitin could only be reconstituted if the reporter moietywas exposed to the cytosol. Functional reconstitution of ubiquitin was observed when SLC41A1 C-terminally tagged with Cubwas co-expressed with NubI-tagged membrane markers, thereby, indicating a cytosolic orientation of the C-terminus of SLC41A1. Thus, our experimental data are in favor of the - the in silico analyses being strongly preferred - ten TMh model of SLC41A1 topology, with both termini being oriented intracellularly.


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