EB-SxIP · Modulating EB protein interactions through small molecules

Modulating EB protein interactions through small molecules

Plus-end tracking proteins (+TIPs) are specialized microtubule associated proteins distinguished by their preferential accumulation at microtubule growing ends. +TIPs are known to form protein networks that play an important role in many fundamental cellular processes, including mitosis, intracellular transport, cell polarity and migration. Within +TIP networks, End Binding (EB) proteins are the master organizers and central recruitment points. Most +TIPs contain a short linear sequence motif, denoted SxIP, that is specifically recognized by EB proteins and constitutes an evolutionarily conserved mechanism for microtubule tip localization. This research project deals with the characterization of this recognition mechanism and the search for small molecule inhibitors able to disrupt it and, consequently, disrupt the vast majority of +TIP networks. Such molecules would allow the functional dissection of the diverse cellular functions that has been described to depend on these +TIP networks. Furthermore, given the involvement of EB proteins in some malignancies, these molecules might have a potential therapeutic interest, which needs to be evaluated. The project has involved a multi-disciplinary research combining methodologies from different fields including computational, structural, cell and chemical biology and it has been performed through the establishment of several collaborations with scientists from research institutes all over Europe. Among others, the results obtained include the development and validation of a fluorescence polarization quantitative assay, which is at present being used in diverse high-throughput screening campaigns to identify inhibitors of the EB-SxIP interaction. We have also identified the sequence determinants of the SxIP short linear motif and quantitatively correlated the EB binding and microtubule tip tracking activities of different human +TIPs, as well as its regulation by phosphorylation. Altogether, these results provide essential experimental data for bioinformatics approaches to carry out genome-wide predictions of novel +TIPs in multiple organisms and pave the way for finding small molecules able to disrupt +TIP networks. We have also exhaustively explored the effects of abrogating the EB-SxIP interaction in in vitro reconstitution systems as well as in mammalian and fission yeast cells using point mutations in EB proteins that disrupt SxIP binding while keeping intact the ability of EBs to track microtubule growing tips. Interestingly, in contrast to the results obtained in vitro, the EB-SxIP interaction is isoform-specific in mammalian cells. Moreover, the disruption of the EB1-SxIP interaction shows significant differences in mitotic timing and defects in spindle positioning, although cells could progress along the cell cycle with relative normality. In fission yeast, the disruption of EB-SxIP did not alter neither microtubule dynamics nor cell morphology but significantly compromised spore viability. Despite the significant effort invested, we have not been able to identify small molecule efficient inhibitors of the EB-SxIP interaction so far, but we continue at present exploring new possibilities to accomplish this objective. It is well known that inhibiting a protein-peptide interaction represents an enormous challenge but, worth to tackle in any case, given that this achievement would represent a very useful and convenient biological tool that would greatly benefit the scientific community studying the cytoskeleton.

Data: CORDIS, © European Union