Overcoming the plasma membrane barrier: uptake of amphipathic cell-penetrating peptides induces influx of calcium ions and downstream responses
Kuupäev
2012-08-09
Autorid
Ajakirja pealkiri
Ajakirja ISSN
Köite pealkiri
Kirjastaja
Abstrakt
Mitmed potentsiaalsed ravimid ning bioaktiivsed ühendid, mis on lahuses efektiivsed, ei ole kahjuks võimelised läbima rakumembraani ja ei ole seetõttu kasutatavad elusrakkude korral, kus need peaksid oma toimet avaldama. Samas on rakumembraani barjääri ületamiseks välja töötatud mitmeid meetoteid. Näiteks peptiidsete järjestuste avastamine, mis on võimelised iseseisvalt rakkudesse tungima, on kaasa aidanud efektiivsete kandjamolekulide arendusse. Neid spetsiifilisi peptiide hakati nimetama rakkudesse sisenevateks peptiidideks (RSP-d) ning need on võimelised efektiivselt rakkudesse transportima ka nendega liidetud lastmolekule. Samas on enne RSP-de laiaulatuslikku kasutuselevõttu vaja täpselt välja selgitada nende sisenemise mehhanismid. Nimelt arvatakse, et osad RSP-d põhjustavad rakkude plasmamembraani häiritusi, mis võivad osutuda rakkudele kahjulikuks. Seetõttu on oluline välja selgitada, kas ja kuidas on rakud võimelised toime tulema stressiga, mida võib põhjustada RSP-de rakkudesse sisenemine. Antud töö põhieesmärgiks oli uurida, kas teatud RSP-d muudavad rakumembraani piisavalt lekkivaks, et põhjustada kaltsiumioonide sissevoolu rakkudesse ning kas see omakorda aktiveerib teisi rakulisi vastuseid.
Kasutades elektronmikroskoopiat tegime me kindlaks, et osad RSP-d on tõepoolest võimelised mõjutama rakumembraani lipiidset kaksikkihti, muutes selle raskesti detekteeritavaks. Lisaks sellele olid nende RSP-de poolt tekitatud membraani häiritused piisavalt ulatuslikud, et põhjustada kaltsiumi sissevoolu rakkudesse. Tuginedes saadud tulemustele on RSP-d võimalik jaotada kahte suurde gruppi: a) amfipaatsed RSP, mis mõjutavad plasmamembaani ja seetõttu põhjustavad rakumembraani häiritusi ning b) mitte-amfipaatsed RSP, mis ei mõjuta raku pinda. Samas aktiveerib kaltsiumi sissevool efektiivse plasmamembraani parandamise vastuse, mis omakorda sulgeb läbilaskvad rakupinna regioonid ja aitab rakkudel toime tulla RSP-de põhjustatud stressiga. Käesoleva töö tulemused näitavad, et tulevikus on võimalik kasutada RSP-sid kui efektiivseid kandjamolekule ning kahjulikud kõrvalmõjud, mida võivad RSP-d rakkudesse sisenemisel tekitada, on välditavad.
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.
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.
Kirjeldus
Väitekirja elektrooniline versioon ei sisalda publikatsioone.
Märksõnad
peptiidid, penetratsioon, rakumembraan, bioloogiline transport, rakufüsioloogia, peptides, penetration, cell membrane, biological transport, cell physiology