Studying in vivo effect of cell-penetrating inhibitor peptide conjugate on protein-protein interaction
Mitogen-activated protein kinases (MAPK) are among the most ancient and ubiquitous signaling proteins of eukaryotic organisms. They play crucial roles in diverse cellular functions such as proliferation, differentiation, apoptosis, cell survival, inflammatory and neurodegenerative processes. Their dysfunction or hyperfunctioning can lead to several diseases and pathological disorders. Much effort has been devoted so far to develop potent inhibitors of these signaling molecules, since mortality caused by malignant tumor is the second leading cause of death. Moreover, resistance and toxic side-effects evolved during treatment pose a serious problems, so development of newer inhibitors is continuously needed. One family of these inhibitors binds to the ATP-binding pocket and inhibits the ATP-ase activity of the kinases. These ATP-competitive inhibitors are less specific and influence the function of other enzymes in the cell as well. Our goal is to develop a more specific inhibitor which is selective to the MAPK-pathway and targets the interactions of the MAPK protein. Specific peptides consisting of 12-10 amino acids-so called linear motifs-are able to compete with MAPK-interaction partners and inhibit the kinase action. We have successfully tested the effect of these competitive inhibitor peptides in vitro by means of purified recombinant proteins, and now would like to test their in vivo effect. However the transfer of the peptides into the cell must be solved, therefore we use cell-penetrating peptide conjugate, which are efficient candidate for delivering peptide into the cell. The main goal of our cooperation is to develop effective cell-penetrating peptide conjugate inhibitors against the different MAPK-pathways and test their biological effect. We follow the fate of the cell-penetrating conjugate by fluorescence microscopy and their effect on the MAPKs' interaction and activity of the signaling pathway by Protein Fragment Complementation Assay and Western-blot or transcription reporter system, respectively.
Anita Alexa - Zoltán Bánóczi