Translocation of HIV TAT peptide and analogues induced by multiplexed membrane and cytoskeletal interactions

Abstract

Cell-penetrating peptides (CPPs), such as the HIV TAT peptide, are able to translocate across cellular membranes efficiently. A number of mechanisms, from direct entry to various endocytotic mechanisms (both receptor independent and receptor dependent), have been observed but how these specific amino acid sequences accomplish these effects is unknown. We show how CPP sequences can multiplex interactions with the membrane, the actin cytoskeleton, and cell-surface receptors to facilitate different translocation pathways under different conditions. Using "nunchuck" CPPs, we demonstrate that CPPs permeabilize membranes by generating topologically active saddle-splay ("negative Gaussian") membrane curvature through multidentate hydrogen bonding of lipid head groups. This requirement for negative Gaussian curvature constrains but underdetermines the amino acid content of CPPs. We observe that in most CPP sequences decreasing arginine content is offset by a simultaneous increase in lysine and hydrophobic content. Moreover, by densely organizing cationic residues while satisfying the above constraint, TAT peptide is able to combine cytoskeletal remodeling activity with membrane translocation activity. We show that the TAT peptide can induce structural changes reminiscent of macropinocytosis in actin-encapsulated giant vesicles without receptors.protein transduction domain | polyarginine | peptide-lipid interactions | pore-forming peptide | antimicrobial peptide C ell-penetrating peptides (CPPs) are effective intracellular delivery systems (1-5). These peptides are usually short (<20 amino acids) and cationic. Examples include the TAT peptide from HIV, antennapedia (ANTP) from Drosophila, and even simple polyarginines. Although unique molecular architectures incorporating CPPs have been designed for drug delivery (3, 6-8), the molecular mechanisms of cellular entry, and the relations between them, are not well understood. Different uptake mechanisms have been proposed for CPPs (9). Cell-based assays have indicated that multiple endocytotic pathways are involved (10-15). In addition to these, CPPs are also capable of direct entry mechanisms* (17-20). In general, cell-penetrating activity of CPPs has proven to be difficult to eliminate completely using a specific set of conditions (3,12,21), suggesting the existence of multiple mechanisms. A unified understanding of CPPs, which is currently lacking, must engage why the same sequence can readily activate the qualitatively distinct outcomes.How do relatively simple molecules like HIV TAT peptide facilitate mechanisms as different as direct translocation, and multiple endocytotic processes? Rather than debate priority between mechanisms, we focus on the physical chemistry of what these different mechanisms and CPPs have in common. Here, we show how the TAT peptide can multiplex different interactions with the same sequence, thus interacting with the membrane, the actin cytoskeleton, and specific receptors to produce multiple pathways of translocation under different condition...

Arginine in α-Defensins

“…If the dense presentation of the guanidine group of arginine with lipid headgroups is sufficiently diluted, its bidentate H-bonding (23) ability may diminish to the point that it behaves like lysine. Consistent with this, we have recently shown that alleviation of steric strain in polyarginine by incorporation of flexible PEG chains within polyarginine can make polyarginine behave like polylysine and thereby suppress negative Gaussian curvature generation and Pn3m phases and result in H II phases with negative mean curvature (22). Conversely, we hypothesize that a clustering of lysine residues with reduced H-bonding ability may be able to mimic the multidentate H-bonding of arginine to generate the required negative Gaussian curvature as long as the lysines do not interfere with one another.…”

“…6). Arginine generates negative Gaussian curvature in anionic membranes by inducing negative curvature along one direction via electrostatic interactions (20,44) and positive curvature along a perpendicular direction via directed lipid headgroup coordination and resultant steric "crowding" effects (22). If the dense presentation of the guanidine group of arginine with lipid headgroups is sufficiently diluted, its bidentate H-bonding (23) ability may diminish to the point that it behaves like lysine.…”

Current Understanding of the Mechanisms by which Membrane-Active Peptides Permeate and Disrupt Model Lipid Membranes

“…Thus, these findings do provide evidence that oligoarginine peptides can expand the outer leaflet surface area and generate a membrane tension gradient through the bilayer The observation of stable pores, due to adsorption of oligoarginine, has been reported in several experimental studies. Mishra et al found that hexaarginine (R6) caused rupture of a PE-rich lipid membrane such that vesicle-trapped fluorescent dyes can be released [57]. Work by Herce et al showed that R9 also induced small membrane pores.…”

“…ARCPPs and oligolysine [15,57]. They showed that ARCPPs, including TAT, penetratin and oligoarginine, all induced the bi-continuous cubic phase in PE-rich lipid membranes.…”

“…They showed that ARCPPs, including TAT, penetratin and oligoarginine, all induced the bi-continuous cubic phase in PE-rich lipid membranes. However, oligolysine instead induced an inverted hexagonal phase in similar membranes [15,57]. Wu et al carried out all-atom and coarse-grained simulations, in an attempt to determine the molecular basis for the different actions of these peptides [58].…”