Supervisor: Professor Margus Pooga, Institute of Molecular and Cell Biology
Opponent: Professor Roland Brock, Radboud University Nijmegen, The Netherlands
Many potential drugs or other bioactive compounds that are efficient in solutions are not able to traverse cellular membranes and, therefore, can't be utilized in living organisms. To confer the barrier function of the cell surface, different delivery methods have been developed. For example, discovery of sequences that possess cell-pene¬trating properties has paved the way for the development of effi¬cient carriers for cell-impermeable molecules. These so called cell-penetrating peptides (CPPs) are able to gain access into cells and, more importantly, transport various payloads attached to them into intracellular compartments in a non-invasive manner. However, before these promising transport vectors can be used in therapeutics, their mechanism of entry needs to be carefully elucidated, since it appears that some CPPs are able to induce disturbances in the plasma membrane. This might become harmful for cells and, thus, it is important to understand if and how cells can deal with the potential stress induced by CPPs. Therefore, the main focus of this study was to elucidate whether CPPs are able to make the plasma membrane leaky enough to induce the influx of calcium ions into cells, and activate downstream responses.
By using electron microscopy we demonstrated that, indeed, some CPPs interfere with the tight packing of membrane lipids, and make the cell surface hardly detectable. Furthermore, the disturbances caused by CPPs were sufficient enough to induce the influx of calcium ions into cells. According to membrane destabilizing capacity we were able to categorize CPPs into two groups: (i) amphipathic CPPs that are membrane-active and, thus, hamper the integrity of the cell surface and (ii) non-amphipathic CPPs that do not affect the plasma membrane of cells. However, the influx of calcium activated an efficient repair mechanism that helped cells to reseal the damaged plasma membrane and overcome the stress caused by CPPs. Thus, the present study demonstrates that CPPs can be efficiently used as transport vectors, and there are means of overcoming seemingly harmful side-effects induced by some CPPs.