Objective The cell interprets the information conferred by ubiquitin (Ub) through Ub-receptors, proteins that harbor ubiquitin-binding domains (UBDs). Ub-receptors can be found in three mutually exclusive forms. Apo is the unbound form, whereas trans is the intermolecular form bound to ubiquitylated cargo. Intriguingly, the third form, cis, is of Ub-receptors that are themselves ubiquitylated. Monoubiquitylation of the Ub-receptors imposes an auto-inhibitory conformation, rendering them unable to bind ubiquitylated cargos, and thus providing an intrinsic regulatory mechanism. While several structures of apo and trans are available, a cis structure form of an ubiquitylated Ub-receptor has yet to be determined. Determining a cis structure is crucial for understanding the molecular mechanism of auto-inhibition. Due to the action of deubiquitylating enzymes, the ubiquitylated form of the Ub-receptors is labile and therefore it is difficult to purify them from eukaryotes. To fill this gap in knowledge, we developed a novel bacterial system for protein ubiquitylation. The system produces a high yield of ubiquitylated protein that can be easily purified and subjected to crystallization trials. Using this bacterial ubiquitylation system, we will determine cis as well as apo and trans structures of Ub-receptors. Guided by comparison of the structures, point mutations will be introduced into key residues. In vitro functional studies including the determination of oligomeric stages, and affinity measurements using isothermal titration calorimetry and analytical ultracentrifugation will be performed on all three forms. Finally, the phenotype of the wild type and mutant proteins will be studied in vivo by monitoring trafficking of GFP-cargo under confocal microscopy. The outcome of this study is predicted to provide the molecular principles of ub-receptor regulation, and serve to design drugs for diseases that involve the monoubiquitin pathways such as AIDS and cancer. Fields of science natural sciencesbiological sciencesmicrobiologybacteriologynatural sciencesbiological sciencesgeneticsmutationnatural scienceschemical sciencesanalytical chemistrycalorimetrynatural sciencesphysical sciencesopticsmicroscopyconfocal microscopynatural sciencesbiological sciencesbiochemistrybiomoleculesproteinsenzymes Keywords Biochemistry Cell biology Functional biology Structural biology Programme(s) FP7-PEOPLE - Specific programme "People" implementing the Seventh Framework Programme of the European Community for research, technological development and demonstration activities (2007 to 2013) Topic(s) PEOPLE-2007-4-3.IRG - Marie Curie Action: "International Reintegration Grants" Call for proposal FP7-PEOPLE-IRG-2008 See other projects for this call Funding Scheme MC-IRG - International Re-integration Grants (IRG) Coordinator TEL AVIV UNIVERSITY EU contribution € 75 000,00 Address RAMAT AVIV 69978 Tel Aviv Israel See on map Activity type Higher or Secondary Education Establishments Administrative Contact Lea Pais (Ms.) Links Contact the organisation Opens in new window Website Opens in new window Total cost No data
