The Homeodomain Derived Peptide Penetratin Induces Curvature of Fluid Membrane Domains

BACKGROUND:Protein membrane transduction domains that are able to cross the plasma membrane are present in several transcription factors, such as the homeodomain proteins and the viral proteins such as Tat of HIV-1. Their discovery resulted in both new concepts on the cell communication during development, and the conception of cell penetrating peptide vectors for internalisation of active molecules into cells. A promising cell penetrating peptide is Penetratin, which crosses the cell membranes by a receptor and metabolic energy-independent mechanism. Recent works have claimed that Penetratin and similar peptides are internalized by endocytosis, but other endocytosis-independent mechanisms have been proposed. Endosomes or plasma membranes crossing mechanisms are not well understood. Previously, we have shown that basic peptides induce membrane invaginations suggesting a new mechanism for uptake, "physical endocytosis". METHODOLOGY/PRINCIPAL FINDINGS:Herein, we investigate the role of membrane lipid phases on Penetratin induced membrane deformations (liquid ordered such as in "raft" microdomains versus disordered fluid "non-raft" domains) in membrane models. Experimental data show that zwitterionic lipid headgroups take part in the interaction with Penetratin suggesting that the external leaflet lipids of cells plasma membrane are competent for peptide interaction in the absence of net negative charges. NMR and X-ray diffraction data show that the membrane perturbations (tubulation and vesiculation) are associated with an increase in membrane negative curvature. These effects on curvature were observed in the liquid disordered but not in the liquid ordered (raft-like) membrane domains. CONCLUSIONS/SIGNIFICANCE:The better understanding of the internalisation mechanisms of protein transduction domains will help both the understanding of the mechanisms of cell communication and the development of potential therapeutic molecular vectors. Here we showed that the membrane targets for these molecules are preferentially the fluid membrane domains and that the mechanism involves the induction of membrane negative curvature. Consequences on cellular uptake are discussed

Distinct Behaviour of the Homeodomain Derived Cell Penetrating Peptide Penetratin in Interaction with Different Phospholipids

Penetratin is a protein transduction domain derived from the homeoprotein Antennapedia. Thereby it is currently used as a cell penetrating peptide to introduce diverse molecules into eukaryotic cells, and it could also be involved in the cellular export of transcription factors. Moreover, it has been shown that it is able to act as an antimicrobial agent. The mechanisms involved in all these processes are quite controversial.In this article, we report spectroscopic, calorimetric and biochemical data on the penetratin interaction with three different phospholipids: phosphatidylcholine (PC) and phosphatidylethanolamine (PE) to mimic respectively the outer and the inner leaflets of the eukaryotic plasma membrane and phosphatidylglycerol (PG) to mimic the bacterial membrane. We demonstrate that with PC, penetratin is able to form vesicle aggregates with no major change in membrane fluidity and presents no well defined secondary structure organization. With PE, penetratin aggregates vesicles, increases membrane rigidity and acquires an α-helical structure. With PG membranes, penetratin does not aggregate vesicles but decreases membrane fluidity and acquires a structure with both α-helical and β–sheet contributions.These data from membrane models suggest that the different penetratin actions in eukaryotic cells (membrane translocation during export and import) and on prokaryotes may result from different peptide and lipid structural arrangements. The data suggest that, for eukaryotic cell penetration, penetratin does not acquire classical secondary structure but requires a different conformation compared to that in solution

Sequence Defined Disulfide-Linked Shuttle for Strongly Enhanced Intracellular Protein Delivery

Intracellular protein transduction technology is opening the door for a promising alternative to gene therapy. Techniques have to address all critical steps, like efficient cell uptake, endolysosomal escape, low toxicity, while maintaining full functional activity of the delivered protein. Here, we present the use of a chemically precise, structure defined three-arm cationic oligomer carrier molecule for protein delivery. This carrier of exact and low molecular weight combines good cellular uptake with efficient endosomal escape and low toxicity. The protein cargo is covalently attached by a bioreversible disulfide linkage. Murine 3T3 fibroblasts could be transduced very efficiently with cargo nlsEGFP, which was tagged with a nuclear localization signal. We could show subcellular delivery of the nlsEGFP to the nucleus, confirming cytosolic delivery and expected subsequent subcellular trafficking. Transfection efficiency was concentration-dependent in a directly linear mode and 20-fold higher in comparison with HIV-TAT-nlsEGFP containing a functional TAT transduction domain. Furthermore, β-galactosidase as a model enzyme cargo, modified with the carrier oligomer, was transduced into neuroblastoma cells in enzymatically active form