Abstract
Cystine-knot peptides are attractive templates in drug discovery due to a number of features they possess including their 3D conformation, physicochemical stability and synthetic tractability. Yet, their cellular uptake mechanisms remain largely unexplored. Recently, we demonstrated that the cystine-knot peptide EETI-II is internalized into cells and that its cellular uptake could be modulated by using a protein transfection reagent Xfect. However, the mechanism of Xfect-mediated cellular internalization of EETI-II remained unclear. Here, by using high resolution electron microscopy, we observe the formation of EETI-II-positive macropinosomes and clathrin-coated pits at early time points after treatment of cells with EETI-II/Xfect complexes. Internalized EETI-II subsequently accumulates in intracellular Xfect-induced detergent-resistant membrane compartments which appear to lack characteristic endosomal or lysosomal markers. Notably, Xfect enables the uptake of cell impermeable nuclear dyes into similar intracellular compartments that do not seem to deliver the cargo to the cytosol or nucleus. Altogether, our findings reveal mechanistic insights into the cellular uptake route of Xfect, and underscore the need for the development of effective tools to enhance the cytosolic delivery of cystine-knot peptides. Finally, our data illustrate that electron microscopy is a powerful approach for studying endocytic mechanisms of cell-penetrating peptides and their effects on cellular membranes.
Highlights
Cystine-knot peptides (CKP) have a highly constrained structure, comprising a knotted arrangement of three disulfide bonds[1], which confers remarkable thermal, chemical and proteolytic stability[2]
Typical macropinocytic events were observed on the plasma membrane of cells that were treated with rEETI-II-Alexa488/Xfect
Parts of the observed invaginated plasma membrane were decorated with a characteristic clathrin coat, suggesting a role for clathrin-mediated endocytosis in rEETI-II-Alexa488/Xfect internalization (Fig. 1d)
Summary
Cystine-knot peptides (CKP) have a highly constrained structure, comprising a knotted arrangement of three disulfide bonds[1], which confers remarkable thermal, chemical and proteolytic stability[2]. MCoTI is internalized into mammalian cells primarily via macropinocytosis and, to a lesser extent, cholesterol-dependent and clathrin-mediated endocytosis, eventually accumulating in lysosomes[15,16] Another CKP which belongs to the kalata B1 family enters cells through a combination of endocytic mechanisms and direct interaction with phosphatidylethanolamine on the plasma membrane[17,18,19]. We have used the acyclic cystine-knot peptide EETI-II (peptide sequence, GCPRILMRCKQDSDCLAGC VCGPNGFCG) as a model system to study the feasibility of re-targeting cystine-knot peptides out of the endosomal/lysosomal pathways with protein transfection reagents One such example of a protein transfection reagent is Xfect[20], which is a cell-penetrating peptide of unknown composition that has been reported to bind to protein cargos through ionic pairing and deliver proteins into cells. Studies performed in reconstituted liposomes revealed that Xfect influences membrane thickness and integrity
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