Lipid-Specific Direct Translocation of the Cell-Penetrating Peptide NAF-1^44–67 across Bilayer Membranes

Abstract

The cell-penetrating peptide NAF-1 has recently emerged as a promising candidate for selective penetration and destruction of cancer cells. It displays numerous membraneselective behaviors including cell-specific uptake and organellespecific degradation. In this work, we explore membrane penetration and translocation of NAF-1 in model lipid bilayer vesicles as a function of lipid identity in zwitterionic phosphatidylcholine lipids mixed with anionic phosphatidylserine, phosphatidylglycerol, and phosphatidic acid lipids. By monitoring the digestion of NAF-1 using the protease trypsin located inside but not outside the vesicles, we determined that the translocation of NAF-1 was significantly enhanced by the presence of phosphatidic acid in the membrane compared to the other three anionic or zwitterionic lipids. These findings were correlated to fluorescence measurements of dansyl-labeled NAF-1, which revealed whether noncovalent interactions between NAF-1 and the bilayer were most stable either at the membrane/solution interface or within the membrane interior. Phosphatidic acid promoted interactions with fatty acid tails, while phosphatidylcholine, phosphatidylserine, and phosphatidylglycerol stabilized interactions with polar lipid headgroups. Interfacial vibrational sum frequency spectroscopy experiments revealed that the phosphate moiety on phosphatidic acid headgroups was better hydrated than on the other three lipids, which helped to shuttle NAF-1 into the hydrophobic region. Our findings demonstrate that permeation does not depend on the net charge on phospholipid lipid headgroups in these model vesicles and suggest a model wherein NAF-1 crosses membranes selectively due to lipid-specific interactions at bilayer surfaces.

Leaflet-Dependent Effect of Anionic Lipids on Membrane Insertion by Cationic Cell-Penetrating Peptides

“…In previous investigations of NAF-1 44–67 and WWWK, we determined that peptide insertion past the headgroup phosphate moieties into the core of the membrane presented the primary energy barrier for permeation followed by adoption of a membrane-immersed metastable state. − A more favorable membrane-immersed state resulted in more efficient peptide membrane insertion. Furthermore, we have previously observed that lipid compositions that enhanced insertion of NAF-1 44–67 into the membrane also enhanced transport of the peptide across the membrane into the vesicle interior . Thus, the solvatochromic measurements of NAF-1 44–67 insertion described herein may correlate to permeation and transport, although this may not be the case for all peptides. , …”

“…Furthermore, we have previously observed that lipid compositions that enhanced insertion of NAF-1 44−67 into the membrane also enhanced transport of the peptide across the membrane into the vesicle interior. 18 Thus, the solvatochromic measurements of NAF-1 44−67 insertion described herein may correlate to permeation and transport, although this may not be the case for all peptides. 29,30 When distributed symmetrically between bilayer leaflets, anionic lipids resulted in a higher-energy λ avg for all peptides with varying degrees of magnitude and lipid headgroup specificity (Figure 2a−c).…”

“…9,17 A recent study from our group using an anticancer CPP, NAF-1 44−67 , showed that phosphatidic acid (PA) lipids enhanced membrane insertion and translocation to a greater extent than phosphatidylserine (PS) and phosphatidylglycerol (PG) lipids. 18 Computational studies also suggest that this peptide permeates deeper in membranes with binary mixtures of PA or PS and zwitterionic phosphatidylcholine (PC) and locally enriches its environment with anionic lipids. 19 However, it is unclear if CPP insertion enhancement by anionic lipids is solely a result of interfacial electrostatic interactions, with outer leaflet lipids templating the insertion process, or if lipids from both leaflets play a role in stabilizing permeants as they traverse the hydrophobic region of the membrane.…”

“…We have recently shown that permeants can traverse membranes through a defect-assisted mechanism in which a cationic peptide can cross the hydrophobic region of the bilayer by increasing membrane hydration and interacting with inner and outer leaflet lipid headgroups and phosphates. − Furthermore, anionic lipids can affect CPP–membrane interactions and insertion in a headgroup-dependent manner. , A recent study from our group using an anticancer CPP, NAF-1 44–67 , showed that phosphatidic acid (PA) lipids enhanced membrane insertion and translocation to a greater extent than phosphatidylserine (PS) and phosphatidylglycerol (PG) lipids . Computational studies also suggest that this peptide permeates deeper in membranes with binary mixtures of PA or PS and zwitterionic phosphatidylcholine (PC) and locally enriches its environment with anionic lipids .…”