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.

Fig. 1.

Mechanism of membrane permeation depends on peptide sequence. (A and B) R₆ (unlabeled) added to 20/60/20 PS/PE/PC GUVs (red) with encapsulated Alexa Fluor 633 dye (blue) induced permeation fast enough to rupture vesicles. (C and D) Addition of a single tryptophan hydrophobic residue drastically changed the dominant mode of translocation/escape: R₆W (unlabeled) induced slow leakage of intact vesicles, similar to antimicrobial peptides. (E and F) Labeling R₆ with the hydrophobic aromatic rings of fluorescein isothiocyanate (FITC, green) also gave rise to slow leakage of encapsulated dye, followed by eventual equilibrium distribution of the R₆-FITC peptide (G). White scale bars are 20 μm.

Fig. 2.

Membrane activity of CPPs controlled by lipid crowding effects and amino acid content. (A) SAXS data for R₉, incubated with SUVs (DOPS∶DOPE = 20∶80) at peptide-to-lipid molar ratio (P/L) = 1/40 show diffraction peak positions well described by , characteristic of a bicontinuous cubic Pn3m phase (h, k, l are Miller indices; a is the lattice parameter). Tetraarginine blocks connected with a short PEG spacer (R₄-(PEG)₅-R₄) induces a Pn3m phase (P/L = 1/40) with a larger lattice constant. SAXS data for R₄-(PEG)₂₇-R₄, with a longer PEG spacer, show that the Pn3m phase is suppressed, replaced by H_II and L_α phases with no saddle-splay curvature (P/L = 1/40). (B) Multidentate coordination of arginine’s guanidinium side chain induces positive curvature strain along the peptide. (C) Monodentate coordination of lysine’s amino side chain does not induce positive curvature. (D) The cubic Pn3m phase (the zero-mean-curvature surface at the midplane between the two membrane leaflets) (E) The average induced Gaussian curvature ; χ = -2 and A_o = 1.919 for Pn3m) is maximized near N_R = 9, where observed membrane transduction activity is empirically highest. (F) ANTP, TAT peptide, R₉ all induce the cubic Pn3m phase in membranes (DOPS∶DOPE = 20∶80 at P/L = 1/40). (G) The ratio of the number of arginines to the number of arginines + number of lysines (N_R/(N_R + N_K)) plotted against hydrophobicity (Eisenberg hydrophobicity scale) using the amino acid sequences of 39 cell-penetrating peptides and 1080 cationic antimicrobial peptides shows that a reduction in arginine content can be compensated for by an increase in both lysine and hydrophobic content.

Fig. 3.

The induction of saddle-splay curvature alone is not a sufficient condition for full strength CPP activity. (A) TAMRA-labeled TAT peptide (TAMRA-TAT; red) readily crossed the membrane of PS∶PC∶PE = 20∶40∶40 GUVs. (B) TAT peptide covalently coupled to Cy5-tagged PLA nanoparticles (TAT-NP, approximately 30-nm diameter, blue) did not cross the membrane but were localized at the periphery of GUVs. (C) PS∶PC∶PE = 20∶40∶40 GUVs loaded with G-actin and rhodamine phalloidin (red). (D) TAT-NPs (blue), added to these GUVs and localized at the periphery, were able to induce negative Gaussian membrane curvature and membrane permeation, enabling Mg²⁺ ions to cross GUV membrane and polymerize the encapsulated G-actin into F-actin bundles (red). White scale bars are 10 μm.

Fig. 4.

TAT peptide can penetrate membranes and actively induce cytoskeletal actin response. (A) Confocal image of a GUV comprising 40/40/20 PE/PC/PS (labeled green with DiO) with 5% calcium ionophore (Calcimycin) and encapsulating 7 μM globular actin (G-actin). Exposure to 8 mM Mg²⁺, diffusing into the GUV via the ionophores, induced polymerization into filamentous actin (F-actin, labeled red with rhodamine phallodin) network without any accompanying deformation of vesicle. (B and C) Exposure to approximately 4 μM TAT peptide-induced dimple instabilities on the membrane, and promoted growth of F-actin bundles encapsulated within the GUV. (D) In certain cases, the F-actin bundles distorted originally spherical vesicles to form sharp filopodium-like protusions, reminiscent of membrane ruffling and macropinocytosis. (E) Schematic showing a proposed autonomous pathway for TAT cellular transduction. TAT peptide can generate saddle-splay membrane curvature and enter through an induced pore, but large conjugated cargos cannot. The TAT peptide interacts strongly with cytoplasmic actin to promote cellular uptake of anchored cargo via endocytotic pathways. White scale bars are 10 μm.