Penetratin translocation mechanism through asymmetric droplet interface bilayers

Abstract

Penetratin is a cell penetrating peptide (CPP) that can enter cells by direct translocation through the plasma membrane. The molecular mechanism of this translocation still remains poorly understood. Here we provide insights on this mechanism by studying the direct translocation of the peptide across model membranes based on Droplet Interface Bilayers (DIBs), which are bilayers at the interface between two adhering aqueous-in-oil droplets. We first showed with symmetric bilayers made of a mix of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-racglycerol) (POPG) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (POPC) that the translocation of penetratin required the presence of at least 40% of POPG on both leaflets. Interestingly when replacing POPG with another anionic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-Lserine (POPS), translocation was inefficient. To elucidate the lipid partners required at each step of the CPP translocation process, we then investigated the crossing of asymmetric bilayers.We found that POPG on the proximal leaflet and POPS on the distal leaflet allowed penetratin translocation. Translocation was not observed when POPS was on the proximal leaflet and POPG on the distal leaflet or if POPS on the distal leaflet was replaced with POPC. These observations led us to propose a three-step translocation mechanism: (i) peptide recruitment by anionic lipids, (ii) formation of a transient peptide-lipid structure leading to the initiation of translocation which required specifically POPG on the proximal leaflet, (iii) termination of the translocation process favored by a driving force provided by anionic lipids in the distal leaflet.

Impact of Cyclization and Methylation on Peptide Penetration through Droplet Interface Bilayers

“…For instance, such a membrane recruitment-based mechanism promotes the translocation of amphipathic peptides. 15,54 However, C2/C2m showed inconsistent behaviors (Figure 6d), as their permeation rates were not reflected by the irinterfacial recruitment (except if C2m was considered alone). This observation indicates that the capacity of a peptide to insert into the bilayer membrane represents only one parameter controlling permeability.…”

“…Their membrane recruitment was likely mediated by the hydrophobic residues in the peptides (L, Y) , and the methylation at the leucine could improve their orientation and interaction with the membrane. For instance, such a membrane recruitment-based mechanism promotes the translocation of amphipathic peptides. , …”

Structural Bases for the Involvement of Phosphatidylinositol-4,5-bisphosphate in the Internalization of the Cell-Penetrating Peptide Penetratin

“…Penetratin also tends to form peptide-rich regions at the surface of PG liposomes . Finally, PG appears as a permissive lipid for direct translocation across pure lipid membranes. ,,, As opposed to PG, PS is a biologically relevant lipid when investigating internalization mechanisms of CPPs, but it has been scarcely used. Penetratin has a higher affinity for PS-containing membranes than just PC and adopts an α-helical structure in its presence .…”

“…Penetratin has a higher affinity for PS-containing membranes than just PC and adopts an α-helical structure in its presence . Regarding direct translocation, the role of PS is not clear, as it was shown to allow direct translocation or not, depending on the experimental setup. Interactions of Penetratin with model membranes incorporating PI­(4,5)­P 2 have never been studied, though it was shown that direct translocation could occur in liposomes composed of PC, PI, and PI-phosphates …”

“…Many studies have investigated the interaction of Penetratin with negatively charged model membranes through various methods. It is now clearly admitted that Penetratin strongly interacts with anionic lipids − and that they are involved in Penetratin translocation through membranes. − …”