Hisaaki Hirose

A sub-nanometre view of how membrane curvature and composition modulate lipid packing and protein recruitment

Two parameters of biological membranes, curvature and lipid composition, direct the recruitment of many peripheral proteins to cellular organelles. Although these traits are often studied independently, it is their combination that generates the unique interfacial properties of cellular membranes. Here, we use a combination of in vivo, in vitro and in silico approaches to provide a comprehensive map of how these parameters modulate membrane adhesive properties. The correlation between the membrane partitioning of model amphipathic helices and the distribution of lipid-packing defects in membranes of different shape and composition explains how macroscopic membrane properties modulate protein recruitment by changing the molecular topography of the membrane interfacial region. Furthermore, our results suggest that the range of conditions that can be obtained in a cellular context is remarkably large because lipid composition and curvature have, under most circumstances, cumulative effects.

Cell-surface Accumulation of Flock House Virus-derived Peptide Leads to Efficient Internalization via Macropinocytosis

Arginine-rich cell-penetrating peptides (CPPs), including human immunodeficiency virus type 1 (HIV-1) Tat (48-60) and oligoarginines, have been applied as carriers for delivery of cargo molecules, because of their capacity to internalize into cells and penetrate biological membranes. Despite the fact that they have been extensively studied, the factors required for the efficient internalization of CPPs are still unclear. In this report, we evaluated the internalization efficiencies of seven CPPs derived from DNA/RNA-binding peptides, and discovered that a peptide derived from the flock house virus (FHV) coat protein was internalized most efficiently into Chinese hamster ovary (CHO-K1), HeLa, and Jurkat cells. Comparison of the factors facilitating the internalization with those of the Tat peptide revealed that the FHV peptide induces macropinocytosis much more efficiently than the Tat peptide, which leads to its high cellular uptake efficiency. Additionally, the strong adsorption of the FHV peptide on cell membranes via glycosaminoglycans (GAGs) was shown to be a key factor for induction of macropinocytosis, and these steps were successfully monitored by live imaging of the peptide internalization into cells in relation to the actin organization. The remarkable methods of FHV peptide internalization thus highlighted the critical factors for internalizations of the arginine-rich CPPs.

Acylation of octaarginine: Implication to the use of intracellular delivery vectors

Cell-penetrating peptides (CPPs) have the ability to efficiently internalize into cells and thus have been used as a vector for the intracellular delivery of various bioactive molecules. The introduction of a hydrophobic core to CPPs may increase their interaction with membranes and facilitate their translocation into cells. While the usefulness of acylated oligoarginine to gene and siRNA delivery has been largely reported, little information is available about their use for the delivery of small molecular-weight compounds, peptides and proteins. In this report, we employed octaarginine (R8) as a typical arginine-rich CPP and evaluated the effect of acylation using butanoic, hexanoic and decanoic acids on its capacity as a delivery vector. Hexanoyl octaarginine (C6R8-Alexa) showed the highest efficiency of cellular uptake of the studied variants, ten times higher than R8-Alexa. C6R8-Alexa also produced a diffuse cytosolic distribution. On the other hand, a less significant effect of C6R8 over R8 was observed for the delivery of proteins, suggesting that the advantage of C6R8 may be obtained during the delivery of relatively small molecular-weight compounds. Although less prominent than at 37°C, a significant cytosolic distribution of C6R8-Alexa was observed at 4°C, and this suggested the potential ability of the C6R8 peptide for direct penetration through plasma membranes.

Arginine-rich Peptides: Methods of Translocation Through Biological Membranes

Intracellular delivery using cell-penetrating peptides (CPPs) has received considerable attention as a promising method for introducing exogenous molecules into cells. The mechanisms that enable efficient internalization of CPPs together with cargo molecules are also of interest. In this review, we describe our current views of membrane translocation of CPPs, especially those rich in arginine, as these peptides represent one of the most frequently employed classes of CPPs.

Effect of the attachment of a penetration accelerating sequence and the influence of hydrophobicity on octaarginine-mediated intracellular delivery.

Arginine-rich cell-penetrating peptides (CPPs), including oligoarginine peptides, have been widely used as a tool for intracellular delivery of various molecules with low membrane permeability. We previously reported the enhanced cytosolic entry of arginine-rich CPPs by the attachment of a short peptide segment, the penetration accelerating sequence (Pas). In this study, the importance of hydrophobic sequences, especially phenylalanine residues, in the Pas segment was demonstrated for this enhanced translocation through cell membranes. The advantage of using Pas for intracellular delivery was particularly marked for delivering cargoes with a relatively small molecular weight, such as bioactive peptides. In addition, the results of this study indicate the important roles that the total hydrophobicity of the PasR8 conjugates play in cytosolic translocation and the eventual bioactivity thus attained.

Modeling the endosomal escape of cell-penetrating peptides using a transmembrane pH gradient.

Cell-penetrating peptides (CPPs) can internalize into cells with covalently or non-covalently bound biologically active cargo molecules, which by themselves are not able to pass the cell membrane. Direct penetration and endocytosis are two main pathways suggested for the cellular uptake of CPPs. Cargo molecules which have entered the cell via an endocytotic pathway must be released from the endosome before degradation by enzymatic processes and endosomal acidification. Endosomal entrapment seems to be a major limitation in delivery of these molecules into the cytoplasm. Bacteriorhodopsin (BR) asymmetrically introduced into large unilamellar vesicles (LUVs) was used to induce a pH gradient across the lipid bilayer. By measuring pH outside the LUVs, we observed light-induced proton pumping mediated by BR from the outside to the inside of the LUVs, creating an acidic pH inside the LUVs, similar to the late endosomes in vivo. Here we studied the background mechanism(s) of endosomal escape. 20% negatively charged LUVs were used as model endosomes with incorporated BR into the membrane and fluorescein-labeled CPPs entrapped inside the LUVs, together with a fluorescence quencher. The translocation of different CPPs in the presence of a pH gradient across the membrane was studied. The results show that the light-induced pH gradient induced by BR facilitates vesicle membrane translocation, particularly for the intermediately hydrophobic CPPs, and much less for hydrophilic CPPs. The presence of chloroquine inside the LUVs or addition of pyrenebutyrate outside the LUVs destabilizes the vesicle membrane, resulting in significant changes of the pH gradient across the membrane.

Curvature Engineering: Positive Membrane Curvature Induced by Epsin N-Terminal Peptide Boosts Internalization of Octaarginine

Epsin-1 is a representative protein for inducing the positive curvature necessary for the formation of clathrin-coated pits. Here we demonstrate that the N-terminus 18-residue peptide of epsin-1 (EpN18) has this ability per se, as proved by differential scanning calorimetry (DSC) and solid-state NMR. Moreover, it is shown how this positive curvature promotion can be exploited for promoting the direct penetration of a representative cell-penetrating peptide (CPP), octaarginine (R8), through artificial and plasma membranes. This synergistic effect has been used for the efficient delivery of a proapoptotic domain peptide (PAD), which induced high level of apoptosis only when coadministered with R8 and EpN18, thus emphasizing the importance of positive curvature induction for achieving the desired ultimate cargo bioavailability.

Expressed protein ligation for the preparation of fusion proteins with cell penetrating peptides for endotoxin removal and intracellular delivery

Expressed protein ligation (EPL) is a useful method for the native chemical ligation of proteins with other proteins or peptides. This study assessed the practicability of EPL in the preparation of fusion proteins of enhanced green fluorescent protein (EGFP) with chemically synthesized cell-penetrating peptides (CPPs) for intracellular delivery. Using intein-mediated purification with an affinity chitin-binding tag (IMPACT) system, the thioester of EGFP (EGFP-SR) was prepared. Optimization of the ligation of EGFP-SR with arginine 12-mer (R12) produced the fusion protein in high yield. The EPL procedure also allows the preparation of EGFP-R12 containing a low level of endotoxin (ET), via the satisfactory ET removal of EGFP-SR prior to ligation with the R12 peptide. Fusion proteins of EGFP with R12 and the d-isomer of R12 prepared by EPL showed similar levels of cellular uptake compared to the fusion protein directly expressed in Escherichiacoli.

Effect of amino acid substitution in the hydrophobic face of amphiphilic peptides on membrane curvature and perturbation: N‐terminal helix derived from adenovirus internal protein VI as a model

The N‐terminal amphipathic helical segment of adenovirus internal protein VI (AdVpVI) plays a critical role in viral infection. Here, we report that the peptide segment corresponding to AdVpVI (positions 33–55) can induce positive membrane curvature together with membrane perturbation. The enhanced perturbation ability of the peptide was observed for membranes containing negatively charged phospholipids. Based on the liposome leakage assay, substitution of leucine at position 40 to other aliphatic (isoleucine) and aromatic (phenylalanine and tryptophan) residues yielded a similar degree of membrane perturbation by the peptides, which was considerably diminished by the substitution to glutamine. Further studies using the wild‐type AdVpVI (33–55) (WT) and phenylalanine‐substituted peptides (L40F) demonstrated that both peptides have positive membrane‐curvature‐inducing ability. These peptides showed higher binding affinity to 50‐nm large unilamellar vesicles (LUVs) than to 200‐nm LUVs. However, no enhanced perturbation by these peptides was observed for 50‐nm LUVs compared to 200‐nm LUVs, suggesting that both the original membrane curvature and the additional strain due to peptide insertion affect the membrane perturbation ability of these peptides. In the case of L40F, this peptide rather had a lower membrane perturbation ability for 50‐nm LUVs than for 200‐nm LUVs, which can be attributed to possible shallower binding of L40F on membranes.