The permeability barrier imposed by cellular membranes limits the access of exogenous compounds to the interior of cells. Researchers and patients alike would benefit from efficient methods for intracellular delivery of a wide range of membrane-impermeant molecules, including biochemically active small molecules, imaging agents, peptides, peptide nucleic acids, proteins, RNA, DNA, and nanoparticles. There has been a sustained effort to exploit cell penetrating peptides (CPPs) for the delivery of such useful cargoes in vitro and in vivo because of their biocompatibility, ease of synthesis, and controllable physical chemistry. Here, we discuss the many mechanisms by which CPPs can function, and describe a taxonomy of mechanisms that could be help organize future efforts in the field.

Peptides that self-assemble, at low concentration, into bilayer-spanning pores which allow the passage of macromolecules would be beneficial in multiple areas of biotechnology. However, there are few, if any, natural or designed peptides that have this property. Here we show that the 26-residue peptide “MelP5”, a synthetically evolved gain-of-function variant of the bee venom lytic peptide melittin identified in a high-throughput screen for small molecule leakage, enables the passage of macromolecules across bilayers under conditions where melittin and other pore-forming peptides do not. In surface-supported bilayers, MelP5 forms unusually high conductance, equilibrium pores at peptide:lipid ratios as low as 1:25000. The increase in bilayer conductance due to MelP5 is dramatically higher, per peptide, than the increase due to the parent sequence of melittin or other peptide pore formers. Here we also develop two novel assays for macromolecule leakage from vesicles, and we use them to characterize MelP5 pores in bilayers. We show that MelP5 allows the passage of macromolecules across vesicle membranes at peptide:lipid ratios as low as 1:500, and under conditions where neither osmotic lysis nor gross vesicle destabilization occur. The macromolecule-sized, equilibrium pores formed by MelP5 are unique as neither melittin nor other pore-forming peptides release macromolecules significantly under the same conditions. MelP5 thus appears to belong to a novel functional class of peptide that could form the foundation of multiple potential biotechnological applications.

Root hairs are fast-growing tubular protrusions on root epidermal cells that play important roles in water and nutrient uptake in plants. The tip-focused polarized growth of root hairs is accomplished by the secretion of newly synthesized materials to the tip via the polarized membrane trafficking mechanism. Here, we report the function of two different types of plasma membrane (PM) Qa-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), SYP123 and SYP132, in the growth of root hair in Arabidopsis. We found that SYP123, but not SYP132, localizes in the tip region of root hairs by recycling between the brefeldin A (BFA)-sensitive endosomes and the PM of the expanding tip in an F-actin-dependent manner. The vesicle-associated membrane proteins VAMP721/722/724 also exhibited tip-focused localization in root hairs and formed ternary SNARE complexes with both SYP123 and SYP132. These results demonstrate that SYP123 and SYP132 act in a coordinated fashion to mediate tip-focused membrane trafficking for root hair tip growth.

To better understand the sequence–structure–function relationships that control the activity and selectivity of membrane-permeabilizing peptides, we screened a peptide library, based on the archetypal pore-former melittin, for loss-of-function variants. This was accomplished by assaying library members for failure to cause leakage of entrapped contents from synthetic lipid vesicles at a peptide-to-lipid ratio of 1:20, 10-fold higher than the concentration at which melittin efficiently permeabilizes the same vesicles. Surprisingly, about one-third of the library members are inactive under these conditions. In the negative peptides, two changes of hydrophobic residues to glycine were especially abundant. We show that loss-of-function activity can be completely recapitulated by a single-residue change of the leucine at position 16 to glycine. Unlike the potently cytolytic melittin, the loss-of-function peptides, including the single-site variant, are essentially inactive against phosphatidylcholine vesicles and multiple types of eukaryotic cells. Loss of function is shown to result from a shift in the binding–folding equilibrium away from the active, bound, α-helical state toward the inactive, unbound, random-coil state. Accordingly, the addition of anionic lipids to synthetic lipid vesicles restored binding, α-helical secondary structure, and potent activity of the “negative” peptides. While nontoxic to mammalian cells, the single-site variant has potent bactericidal activity, consistent with the anionic nature of bacterial membranes. The results show that conformational fine-tuning of helical pore-forming peptides is a powerful way to modulate their activity and selectivity.

Background: Spontaneous membrane-translocating peptides were discovered by screening in synthetic lipid vesicles. Results: The translocating peptides carry membrane-impermeant cargos directly across cell membranes and drive systemic biodistribution in small animals. Conclusion: These peptides constitute a new class of delivery vehicle for membrane-impermeant cargos. Significance: Spontaneous membrane-translocating peptides could expand the universe of useful drugs.