Multiple faces of cell-penetrating peptides – their intracellular trafficking, stability and endosomal escape during protein transduction
Date
2011-05-13
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Paljude ravimite ja bioaktiivsete ainete (nagu DNA, valgud jms) rakkudesse sisenemine on pärsitud nende ebasobilike mõõtmete, laengu või muude omaduste tõttu, tuues sageli kaasa antud ühendite vähese või puuduliku toime organismis. Selleks, et nende sisenemist soodustada, on võimalik kasutada spetsiaalseid transportivaid molekule, näiteks rakku sisenevaid peptiide (ehk RSP-sid), mis on lühikesed ja enamasti positiivselt laetud valgulised järjestused. RSP-d vahendavad nende külge seotud lastmolekulide sisenemist rakkudesse, tõstes mitmekordselt viimaste rakusisest toimet. Seetõttu omavad nad kõrget potentsiaali ka biomeditsiini valdkonna edasiarendamisel.
RSP-lastmolekuli rakkudesse sisenemine toimub eelkõige membraaniga ümbritsetud kandjate ehk endosoomide kaudu, mis punguvad raku sisemusse ja haaravad raku väliskeskkonnast endasse ka RSP koos lastmolekuliga. Sedasorti sisenemine ehk endotsütoos on ühelt poolt küll rakkudele kahjutu, kuid teisalt takistab lastmolekuli sisenemist raku tsütoplasmasse ja/või tuuma, kus tavaliselt paikneb bioaktiivse lastmolekuli sihtmärk. Lisaks sellele suunatakse suur osa endosoomide sisust lagundamisele, mistõttu endosoomidest vabanemine on lastmolekulile tema toime säilimiseks absoluutselt vajalik. Kahjuks on mõistetud, et just endosoomidest vabanemine ning ühendite vähene vastupanuvõime lagundamisele on hetkel ühed olulisemad tagasilöögid RSP-vahendatud transpordis.
Seega keskenduski antud töö RSP-valk-komplekside rakusisese suunamise, stabiilsuse ning endosoomidest vabanemise uurimisele. Selgus, et antud kompleksid suunatakse rakkudes järk-järgult happelise keskkonnaga lagundavatesse endosoomidesse. Siiski paikneb vähemalt osa komplekse ka neutraalsema pH-ga endosoomides, kus nad püsivad lagundamata vähemalt kuni 12 tundi pärast sisenemist. Uurides komplekside terviklikkust lähemalt, leidsime, et peptiidi hulga suurendamine kompleksis tagab nii lastmolekuli parema sisenemise kui ka vastupidavuse lagundamisele. Lisaks sellele vallandas peptiidi kontsentratsiooni tõstmine rakkudes valguse kaastoimel endosoomide membraani „katkemise“, mille tagajärjel lekkisid nii peptiid kui lastmolekul raku tsütoplasmasse. Seega avastasime mooduse, mille abil on võimalik tõsta lastmolekuli(de) rakusisese toime efektiivsust.
Kuna suur osa kompleksidest suunatakse raku sees siiski lagundamisele, uurisime lagundamise suhtes keemiliselt stabiilsemaks muudetud peptiidide vastupanuvõimet antud protsessile. Vastupidiselt ootustele täheldasime, et teatud peptiidid võivad sellisel kujul olla rakkudele kahjulikud – ühendite tekitatud plasma membraani häiritus ning rakkude energiasünteesi katkemine viis järk-järgulise raku surmani. Seetõttu on ääretult oluline alati kontrollida erinevate muutuste/modifikatsioonide mõju rakkudele.
Viimaks uurisime RSP-motiivi sisaldava uudse inhibiitori ARC toimet rakkudes. ARC-i peamine sihtmärkvalk on oluline rakkude kokkutõmbumises, mistõttu antud valgu liigne aktiivsus võib põhjustada näiteks südamehaigusi. Töös esitatud katsetulemused näitasid, et ARC on suuteline edukalt rakkudesse sisenema, seal oma sihtmärgiga seonduma ning takistama temast lähtuvate signaalide levikut. Seega on RSP-sid võimalik tõhusalt ära kasutada ka enamakski kui ainult transpordi vahendajatena.
Several potential drugs and bioactive molecules (for instance DNA, proteins, etc.) cannot carry out their effect(s) inside the body due to their limited entrance to cells. This is primarily caused by the unfavorable characteristics of the (bio)active molecule (like its charge or size) that hinders its binding to the cell membrane and/or its delivery across the membrane barrier. To overcome this hurdle, the bioactive cargo can be attached to special transport vectors, for example cell-penetrating peptides (CPPs), that can facilitate an enhanced entrance of the cargo into the target cells elevating drastically the cellular effects elicited by the cargo. Because of this, CPPs carry great promise in the development of the field of biomedicine. As CPPs mainly use endocytosis to gain entry to the cells, the complexes containing the peptide and the cargo are intracellularly found inside membrane-bordered vesicles called endosomes. Even though endocytosis is a natural and therefore also a harmless way to acquire material from the surrounding environment, the entrapment of the complexes inside vesicles hampers their release into the cytoplasm, where the cargo could be directed to its intracellular target(s). Furthermore, most of the endocytosed material is targeted to low pH structures called lysosomes, where it faces the destiny of being degraded by the cellular enzymes. Over the past years, it has become increasingly apparent that the limited endosomal escape is one of the major drawbacks in the CPP-mediated delivery today. The major goals of this work were to establish the route by which the CPP-protein complexes are targeted inside the cells, where they eventually end up and how stable the complexes remain in a cellular environment. Additionally, the endosomal escape of the complexes and also the subsequent cellular effects of the bioactive molecule were addressed. First, it was established that the CPP-protein complexes follow the endo-lysosomal, rather than the recycling pathway inside the cells and a large amount of the complexes are in time targeted to low pH lysosomes. Nevertheless, a portion of complexes were found to reside in endosomes that did not experience a pH shift, suggesting that not all of the complexes were targeted to degradation. Indeed, the complexes remained stable inside cell for at least 12 hours. Furthermore, elevation of the peptide to cargo concentration revealed a photo-inducible endosomal escape process, which could be a simple technique for biomedical application to increase the cellular effects of the (bio)active cargo molecule. As the degradation still remains an issue and the cleavage of the peptide diminishes its potential as the delivery vector, the cellular effects of degradation-resistant forms of CPPs were evaluated. It was discovered that some CPPs in this form can be cytotoxic to cells hampering the use of some degradative-resistant CPPs as delivery vectors. Finally, the cellular effects of a novel protein kinase inhibitor ARC carrying a CPP-like moiety were assessed. The results imply that the CPP-like compound can indeed enter cells, bring about its endosomal escape, bind to its target kinase inside the cytoplasm and inhibit the signal cascade emanating from the target. Thus, CPPs can be efficiently used in the delivery of therapeutics and (bio)active molecules. Hopefully, the future studies will reveal additional possibilities for conquering the setbacks we face today.
Several potential drugs and bioactive molecules (for instance DNA, proteins, etc.) cannot carry out their effect(s) inside the body due to their limited entrance to cells. This is primarily caused by the unfavorable characteristics of the (bio)active molecule (like its charge or size) that hinders its binding to the cell membrane and/or its delivery across the membrane barrier. To overcome this hurdle, the bioactive cargo can be attached to special transport vectors, for example cell-penetrating peptides (CPPs), that can facilitate an enhanced entrance of the cargo into the target cells elevating drastically the cellular effects elicited by the cargo. Because of this, CPPs carry great promise in the development of the field of biomedicine. As CPPs mainly use endocytosis to gain entry to the cells, the complexes containing the peptide and the cargo are intracellularly found inside membrane-bordered vesicles called endosomes. Even though endocytosis is a natural and therefore also a harmless way to acquire material from the surrounding environment, the entrapment of the complexes inside vesicles hampers their release into the cytoplasm, where the cargo could be directed to its intracellular target(s). Furthermore, most of the endocytosed material is targeted to low pH structures called lysosomes, where it faces the destiny of being degraded by the cellular enzymes. Over the past years, it has become increasingly apparent that the limited endosomal escape is one of the major drawbacks in the CPP-mediated delivery today. The major goals of this work were to establish the route by which the CPP-protein complexes are targeted inside the cells, where they eventually end up and how stable the complexes remain in a cellular environment. Additionally, the endosomal escape of the complexes and also the subsequent cellular effects of the bioactive molecule were addressed. First, it was established that the CPP-protein complexes follow the endo-lysosomal, rather than the recycling pathway inside the cells and a large amount of the complexes are in time targeted to low pH lysosomes. Nevertheless, a portion of complexes were found to reside in endosomes that did not experience a pH shift, suggesting that not all of the complexes were targeted to degradation. Indeed, the complexes remained stable inside cell for at least 12 hours. Furthermore, elevation of the peptide to cargo concentration revealed a photo-inducible endosomal escape process, which could be a simple technique for biomedical application to increase the cellular effects of the (bio)active cargo molecule. As the degradation still remains an issue and the cleavage of the peptide diminishes its potential as the delivery vector, the cellular effects of degradation-resistant forms of CPPs were evaluated. It was discovered that some CPPs in this form can be cytotoxic to cells hampering the use of some degradative-resistant CPPs as delivery vectors. Finally, the cellular effects of a novel protein kinase inhibitor ARC carrying a CPP-like moiety were assessed. The results imply that the CPP-like compound can indeed enter cells, bring about its endosomal escape, bind to its target kinase inside the cytoplasm and inhibit the signal cascade emanating from the target. Thus, CPPs can be efficiently used in the delivery of therapeutics and (bio)active molecules. Hopefully, the future studies will reveal additional possibilities for conquering the setbacks we face today.
Description
Väitekirja elektrooniline versioon ei sisalda publikatsioone.
Keywords
dissertatsioonid, bioloogia, rakufüsioloogia, bioloogiline transport, peptiidid, cell physiology, peptides