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Late endosomes and its unique anionic lipid bis(monoacylglycero)phosphate act as doorways for the endosomal escape and cytosolic entry of the fluorescently‐labeled dimeric cell‐penetrating peptide dfTAT and bioactive cargos
Author(s) -
ErazoOliveras Alfredo,
Najjar Kristina,
Truong Dat,
Wang TingYi,
Pellois JeanPhilippe
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.842.9
Subject(s) - endosome , endocytic cycle , microbiology and biotechnology , cytosol , organelle , chemistry , cell penetrating peptide , endocytosis , biophysics , cell , biochemistry , biology , enzyme
One of the key challenges and a multibillion dollar issue in the field of delivery of biologics/therapeutics inside live cells is endosomal entrapment. Currently, there is an urgent need to define the mechanisms of cell penetration and endosomal escape at a cellular and molecular level. A better understanding of how cell penetration and endosomal escape occurs will allow the improvement of existing delivery strategies. Our laboratory has shown that dfTAT, a recently identified delivery tool, can circumvent this giant hurdle. In this work we describe the use of dfTAT, a dimeric fluorescently‐labeled peptide found to promote endosomal leakage extremely efficiently and without deleterious effects to cells 1 , as a model cell‐penetrating peptide that can reveal the cellular organelles and factors involved in endosomal escape. By controlling/fine‐tuning the trafficking of dfTAT within the endocytic pathway, we have identified late endosomes as the unique organelles rendered leaky by dfTAT and as the exit point for dfTAT endosomal escape. Following endocytic uptake and trafficking, dfTAT binds bis(monoacylglycero)phosphate (BMP), a negatively‐charged phospholipid enriched in late endosomes and, consequently, causes endosomal leakage in cellulo. Moreover, dfTAT binds and causes leakage of bilayers containing BMP but not bilayers containing two other negatively‐charged lipids, phosphatidic acid (PA) and phosphatydilglycerol (PG), in vitro (PG being a structural isomer of BMP). Finally, we establish that an anti‐BMP antibody is capable of blocking leakage in vitro and endosomal escape in cellulo. Together, these data not only identify late endosomes as the cellular gateways utilizable for efficient delivery protocols, but also sheds light on a mechanism that may provide a fundamental basis for the rational design of future cell‐permeable molecules that are highly active yet display low cytotoxicity. Support or Funding Information RO1GM087227, R01GM087981 and RO1GM110137