Amphiphatic peptide CT2

The influenza virus M2 amphipathic helix (M2AH) alters membrane curvature in a cholesterol-dependent manner, mediating viral membrane scission during influenza virus budding. Here, we have investigated the biophysical effects of cholesterol on the ability of an M2AH peptide to manipulate membrane properties. We see that the ability of the M2AH to interact with membranes and form an α-helix is independent of membrane cholesterol concentration; however, cholesterol affects the angle of the M2AH peptide within the membrane. This change in membrane orientation affects the ability of the M2AH to alter lipid order. In low-cholesterol membranes, the M2AH is inserted near the level of the lipid head groups, increasing lipid order, which may contribute to generation of the membrane curvature. As the cholesterol content increases, the M2AH insertion becomes flatter and slightly deeper in the membrane below the lipid headgroups, where the polar face can continue to interact with the headgroups while the hydrophobic face binds cholesterol. This changed orientation minimizes lipid packing defects and lipid order changes, likely reducing the generation of membrane curvature. Thus, cholesterol regulates M2 membrane scission by precisely modulating M2AH positioning within the membrane. This has implications for the understanding of many of amphipathic-helix-driven cellular budding processes that occur in specific lipid environments.

Previous cell-penetrating peptides (CPPs) generally have low cytosolic delivery efficiencies, because of inefficient endosomal escape. In this study, a family of small, amphipathic cyclic peptides was found to be highly efficient CPPs, with cytosolic delivery efficiencies of up to 120% (compared to 2.0% for Tat). These cyclic CPPs bind directly to the plasma membrane phospholipids and enter mammalian cells via endocytosis, followed by efficient release from the endosome. Their total cellular uptake efficiency correlates positively with the binding affinity for the plasma membrane, whereas their endosomal escape efficiency increases with the endosomal membrane-binding affinity. The cyclic CPPs induce membrane curvature on giant unilamellar vesicles and budding of small vesicles, which subsequently collapse into amorphous lipid/peptide aggregates. These data suggest that cyclic CPPs exit the endosome by binding to the endosomal membrane and inducing CPP-enriched lipid domains to bud off as small vesicles. Together with their high proteolytic stability, low cytotoxicity, and oral bioavailability, these cyclic CPPs should provide a powerful system for intracellular delivery of therapeutic agents and chemical probes.

Membrane scission is a crucial step in all budding processes, from endocytosis to viral budding. Many proteins have been associated with scission, though the underlying molecular details of how scission is accomplished often remain unknown. Here, we investigate the process of M2-mediated membrane scission during the budding of influenza viruses. Residues 50-61 of the viral M2 protein bind membrane and form an amphipathic α-helix (AH). Membrane binding requires hydrophobic interactions with the lipid tails but not charged interactions with the lipid headgroups. Upon binding, the M2AH induces membrane curvature and lipid ordering, constricting and destabilizing the membrane neck, causing scission. We further show that AHs in the cellular proteins Arf1 and Epsin1 behave in a similar manner. Together, they represent a class of membrane-induced AH domains that alter membrane curvature and fluidity, mediating the scission of constricted membrane necks in multiple biological pathways.