. "0"^^ . " proteomics" . . "3"^^ . " intramembrane protease" . . "0"^^ . . "http://www.isvav.cz/projectDetail.do?rowId=LK11206"^^ . "3"^^ . "1"^^ . "Rhomboids are intramembrane proteases highly conserved in evolution. In insects, they mediate secretion of bioactive forms of growth factors, thus regulating developmental signaling, while their fuctions within the secretory pathway of mammals are unclear. The key to the understanding of their biological functions are their substrates, but these are mostly unknown in mammals. Progress in this field is also limited by our poor understanding of rhomboid substrate recognition and by the lack of effective rhomboid inhibitors. These compounds would be important tools for basic reseach, but they also have an emerging medical potential. We propose to define substrate repertoires of mouse rhomboids RHBDL1-4 using quantitative proteomics and mass spectrometry, which will enable us to characterise their substrate specificity and identify their endogenous substrates. Based on the analysis of the substrates in cell culture assays we will generate hypotheses about the physiological functions of mammalian rhomboids, which will be then tested in cell lines derived from rhomboid knock out animals. We will initially focus on RHBDL4, where preliminary data suggest that it affects the susceptibility of cells to endoplasmic reticulum (ER) stress induced apoptosis. ER stress response is an important cellular adaptation mechanism, whose dysregulation can have pathological consequences. Importantly, the connection between ER stress and apoptosis is poorly understood. Finally, specific inhibitors are important tools to regulate rhomboid function, and we will thus be employing medicinal chemistry to develop new generation of these compounds. In summary, this integrated effort will yield new biological knowledge, provide a new methodological platform for the discovery of intramembrane protease substrates, contribute to the understanding of rhomboid specificity and mechanism, and lead to a new generation of rhomboid inhibitors applicable both in basic and translational research."@en . " serine protease" . "Rhomboidy jsou intramembr\u00E1nov\u00E9 proteasy \u0161iroce konservovan\u00E9 v evoluci. U hmyzu umo\u017E\u0148uj\u00ED sekreci bioaktivn\u00EDch forem r\u016Fstov\u00FDch faktor\u016F, \u010D\u00EDm\u017E reguluj\u00ED v\u00FDvojovou signalizaci, zat\u00EDmco u savc\u016F jsou jejich funkce v sekre\u010Dn\u00ED dr\u00E1ze zat\u00EDm nepoznan\u00E9. Kl\u00ED\u010Dem k pln\u00E9mu pochopen\u00ED biologick\u00FDch funkc\u00ED rhomboid\u016F jsou jejich p\u0159irozen\u00E9 substr\u00E1ty. Ty jsou v\u0161ak u savc\u016F v\u011Bt\u0161inou nezn\u00E1m\u00E9. Brzdami v\u00FDvoje tohoto oboru nav\u00EDc je, \u017Ee mechanismus rozpozn\u00E1v\u00E1n\u00ED substr\u00E1t\u016F rhomboidy je nejasn\u00FD a nejsou dostupn\u00E9 jejich specifick\u00E9 a \u00FA\u010Dinn\u00E9 inhibitory. Tyto l\u00E1tky by byly d\u016Fle\u017Eit\u00FDmi n\u00E1stroji pro z\u00E1kladn\u00ED v\u00FDzkum, ale maj\u00ED tak\u00E9 potenci\u00E1ln\u00ED medic\u00EDnsk\u00FD v\u00FDznam. Na\u0161\u00EDm c\u00EDlem je nejprve pomoc\u00ED kvantitativn\u00ED hmotov\u00E9 spektrometrie definovat reperto\u00E1r substr\u00E1t\u016F my\u0161\u00EDch rhomboid\u016F RHBDL1-4. To n\u00E1m umo\u017En\u00ED charakterisovat jejich substr\u00E1tovou specifitu a identifikovat p\u0159irozen\u00E9 substr\u00E1ty. Analysou funkce p\u0159irozen\u00FDch substr\u00E1t\u016F v bun\u011B\u010Dn\u00FDch kultur\u00E1ch v p\u0159\u00EDtomnosti a absenci dan\u00FDch rhomboid\u016F pak vytvo\u0159\u00EDme hypot\u00E9zy o molekul\u00E1rn\u00EDch a fyziologick\u00FDch funkc\u00EDch. Ty budeme n\u00E1sledn\u011B testovat v bun\u011B\u010Dn\u00FDch lini\u00EDch odvozen\u00FDch z rhomboid knock-out my\u0161\u00ED. Zpo\u010D\u00E1tku se soust\u0159ed\u00EDme na rhomboid RHBDL4, u n\u011Bho\u017E p\u0159edb\u011B\u017En\u00E9 v\u00FDsledky nazna\u010Duj\u00ED, \u017Ee ovliv\u0148uje n\u00E1chylnost bun\u011Bk k apoptose p\u0159i nadm\u011Brn\u00E9m stresu v endoplasmatick\u00E9m retikulu (ER). Odpov\u011B\u010F na ER stres je d\u016Fle\u017Eit\u00FD bun\u011B\u010Dn\u00FD adapta\u010Dn\u00ED mechanismus, jeho\u017E dysfunkce m\u016F\u017Ee m\u00EDt pathologick\u00E9 d\u016Fsledky a zejm\u00E9na propojen\u00ED ER stresu a apoptosy nen\u00ED dnes pln\u011B pochopeno. Kone\u010Dn\u011B d\u016Fle\u017Eit\u00FDm n\u00E1strojem k regulaci funkce rhomboid\u016F a jejich v\u00FDzkumu jsou specifick\u00E9 inhibitory, kter\u00E9 budeme vyv\u00EDjet postupy medicin\u00E1ln\u00ED chemie. Tento integrovan\u00FD p\u0159\u00EDstup poskytne nov\u00E9 biologick\u00E9 poznatky d\u00EDky objasn\u011Bn\u00ED funkc\u00ED rhomboid\u016F v sav\u010D\u00EDch bu\u0148k\u00E1ch, vytvo\u0159\u00ED platformu pro identifikaci substr\u00E1t\u016F intramembr\u00E1nov\u00FDch proteas vyu\u017Eitelnou v jin\u00FDch biologick\u00FDch kontextech v\u010Detn\u011B pathogen\u016F, p\u0159isp\u011Bje k pochopen\u00ED specifity a mechanismu rhomboid\u016F, a poskytne novou generaci inhibitor\u016F rhomboid vyu\u017Eiteln\u00FDch v z\u00E1kladn\u00EDm i aplikovan\u00E9m v\u00FDzkumu." . . "2014-02-03+01:00"^^ . "membrane protein; intramembrane protease; serine protease; signalling, secretory pathway, endoplasmic reticulum, proteomics, SILAC, inhibitor"@en . . "2016-12-31+01:00"^^ . "Intramembrane proteases of the rhomboid family in the secretory pathway of mammalian cells: substrate repertoire, specificity, biological roles and their inhibition"@en . " endoplasmic reticulum" . . . . . . . "Intramembr\u00E1nov\u00E9 proteasy rodiny rhomboid\u016F v sekre\u010Dn\u00ED dr\u00E1ze sav\u010D\u00EDch bun\u011Bk: reperto\u00E1r substr\u00E1t\u016F, specifita, biologick\u00E9 \u00FAlohy a jejich inhibice" . . " secretory pathway" . . " SILAC" . . . "2015-02-16+01:00"^^ . . "membrane protein" . "2012-04-01+02:00"^^ . . "LK11206" . " signalling" .