ATP2B4 is an essential gene for epidermal growth factor-induced macropinocytosis in A431 cells.

Macropinocytosis (MPC) is a large-scale endocytosis pathway that involves actin-dependent membrane ruffle formation and subsequent ruffle closure to generate macropinosomes for the uptake of fluid-phase cargos. MPC is categorized into two types: constitutive and stimuli-induced. Constitutive MPC in macrophages relies on extracellular Ca2+ sensing by a calcium-sensing receptor. However, the link between stimuli-induced MPC and Ca2+ remains unclear. Here, we find that both intracellular and extracellular Ca2+ are required for epidermal growth factor (EGF)-induced MPC in A431 human epidermoid carcinoma cells. Through investigation of mammalian homologs of coelomocyte uptake defective (CUP) genes, we identify ATP2B4, encoding for a Ca2+ pump called the plasma membrane calcium ATPase 4 (PMCA4), as a Ca2+-related regulator of EGF-induced MPC. Knockout (KO) of ATP2B4, as well as depletion of extracellular/intracellular Ca2+, inhibited ruffle closure and macropinosome formation, without affecting ruffle formation. We demonstrate the importance of PMCA4 activity itself, independent of interactions with other proteins via its C-terminus known as a PDZ domain-binding motif. Additionally, we show that ATP2B4-KO reduces EGF-stimulated Ca2+ oscillation during MPC. Our findings suggest that EGF-induced MPC requires ATP2B4-dependent Ca2+ dynamics.

Liquid Droplet-Mediated Formulation of Lipid Nanoparticles Encapsulating Immunoglobulin G for Cytosolic Delivery.

Antibodies are promising biopharmaceuticals that offer new therapeutic options for diseases. Since antibodies are membrane impermeable, approaches that allow immunoglobulin Gs (IgGs) to access intracellular therapeutic targets would open new horizons in antibody therapies. Lipid nanoparticles (LNPs) are among the classes of vectors that deliver biopharmaceuticals into cells. Using liquid droplets formed by IgG and polyglutamate, we report here a unique approach to forming LNPs containing IgG via liquid droplets formed in the presence of polyglutamic acid (polyE). The addition of polyE promoted the formation of smaller LNPs with cationic lipids than in its absence, and the formed LNPs were much more efficient in cytosolic IgG delivery and targeting of cellular proteins. This approach also allows for the encapsulation of intact IgG without the need for chemical or sequence modification. The intracellularly delivered IgG retained its target binding ability, as demonstrated by labeling of nuclear pore complex and HRas-GFP and inhibition of antiapoptotic cell death by phosphorylated Akt protein in live cells.

Macropinoscope: Real-Time Simultaneous Tracking of pH and Cathepsin B Activity in Individual Macropinosomes.

A fluorescent sensor that allows simultaneous analysis of environmental factors in a limited cellular space is useful for understanding precise molecular interactions in live cells and their biological responses. Macropinocytosis is a ubiquitous endocytic pathway for massive uptake of extracellular fluids, resulting in the formation of macropinosomes. Although macropinocytosis may impact intracellular delivery and cancer proliferation, information on the intracellular behaviors of macropinosomes is limited. Here, we aimed to develop a macropinoscope, a sensor that simultaneously detects pH and cathepsin B activity in individual macropinosomes. A macropinosome-specific marker, dextran (70 kDa), was employed as a platform, onto which fluorescein, Oregon Green, and tetramethylrhodamine were loaded for ratiometric pH sensing and imaging. A cathepsin-B-cleavable peptide sequence bearing sulfo-Cy5 and the quencher BHQ-3 was also mounted; cleavage of the sequence was detected as an increase in sulfo-Cy5 fluorescence. A steep decrease in pH was observed 5-10 min after macropinosome formation, which was accompanied by an immediate increase in cathepsin B activity. Our design concept will lead to the development of other macropinoscopes for the simultaneous detection of other parameters in individual macropinosomes.

Structural Dissection of Epsin-1 N-Terminal Helical Peptide: The Role of Hydrophobic Residues in Modulating Membrane Curvature.

Spatiotemporal structural alterations in cellular membranes are the hallmark of many vital processes. In these cellular events, the induction of local changes in membrane curvature often plays a pivotal role. Many amphiphilic peptides are able to modulate membrane curvature, but there is little information on specific structural factors that direct the curvature change. Epsin-1 is a representative protein thought to initiate invagination of the plasma membrane upon clathrin-coated vesicles formation. Its N-terminal helical segment (EpN18) plays a key role in inducing positive membrane curvature. This study aimed to elucidate the essential structural features of EpN18 in order to better understand general curvature-inducing mechanisms, and to design effective tools for rationally controlling membrane curvature. Structural dissection of peptides derived from EpN18 revealed the decisive contribution of hydrophobic residues to (i) enhancing membrane interactions, (ii) helix structuring, (iii) inducing positive membrane curvature, and (iv) loosening lipid packing. The strongest effect was obtained by substitution with leucine residues, as this EpN18 analog showed a marked ability to promote the influx of octa-arginine cell-penetrating peptides into living cells.

Quantitative Analysis of Extracellular Vesicle Uptake and Fusion with Recipient Cells.

In precision medicine, extracellular vesicles (EVs) are promising intracellular drug delivery vehicles. The development of a quantitative analysis approach will provide valuable information from the perspective of cell biology and system design for drug delivery. Previous studies have reported quantitative methods to analyze the relative uptake or fusion of EVs to recipient cells. However, relatively few methods have enabled the simultaneous evaluation of the "number" of EVs taken up by recipient cells and those that fuse with cellular membranes. In this study, we report a simple quantitative method based on the NanoBiT system to quantify the uptake and fusion of small and large EVs (sEVs and lEVs, respectively). We assessed the abundance of these two subtypes of EVs and determined that lEVs may be more effective vehicles for transporting cargo to recipient cells. The results also indicated that both sEVs and lEVs have very low fusogenic activity, which can be improved in the presence of a fusogenic protein.

Stearylated Macropinocytosis-Inducing Peptides Facilitating the Cellular Uptake of Small Extracellular Vesicles.

Macropinocytosis is a form of endocytosis that allows massive uptake of extracellular materials and is a promising route for intracellular delivery of biofunctional macromolecules and nanoparticles. Our laboratory developed a potent macropinocytosis-inducing peptide named P4A. However, the ability of this peptide is not apparent in the presence of serum. This study aims to endow P4A and related peptides with the ability to induce macropinocytosis in the presence of serum by N-terminal acylation with long-chain fatty acids (i.e., decanoic, myristic, and stearic acids). Stearylated P4A (stearyl-P4A) had the highest effect on stimulating macropinocytotic uptake. Moreover, the intramolecularly disulfide-bridged analogue, stearyl-oxP4A, showed an even higher ability. The effect of stearyl-oxP4A to facilitate the intracellular delivery of small extracellular vesicles (sEVs) was evaluated in terms of (i) cellular uptake using sEVs labeled with an enhanced green fluorescent protein (EGFP) and (ii) cytosolic liberation and expression of sEV-encapsulated luciferase mRNA in recipient cells. The two- to threefold uptake of both sEVs in the presence of stearyl-oxP4A suggests the potential of the peptide for sEV delivery in the presence of serum.

Grafting Hydrophobic Amino Acids Critical for Inhibition of Protein-Protein Interactions on a Cell-Penetrating Peptide Scaffold.

Stapled peptides are a promising class of conformationally restricted peptides for modulating protein-protein interactions (PPIs). However, the low membrane permeability of these peptides is an obstacle to their therapeutic applications. It is common that only a few hydrophobic amino acid residues are mandatory for stapled peptides to bind to their target proteins. Hoping to create a novel class of membrane-permeable PPI inhibitors, the phenylalanine, tryptophan, and leucine residues that play a critical role in inhibiting the p53-HDM2 interaction were grafted into the framework of CADY2─a cell-penetrating peptide (CPP) having a helical propensity. Two analogues (CADY-3FWL and CADY-10FWL) induced apoptotic cell death but lacked the intended HDM2 interaction. Pull-down experiments followed by proteomic analysis led to the elucidation of nesprin-2 as a candidate binding target. Nesprin-2 is considered to play a role in the nuclear translocation of ß-catenin upon activation of the Wnt signaling pathway, which leads to the expression of antiapoptosis proteins and cell survival. Cells treated with the two analogues showed decreased nuclear localization of ß-catenin and reduced mRNA expression of related antiapoptotic proteins. These data suggest inhibition of ß-catenin nuclear translocation as a possible mode of action of the described cell-penetrating stapled peptides.

Split luciferase-based estimation of cytosolic cargo concentration delivered intracellularly via attenuated cationic amphiphilic lytic peptides.

Intracellular delivery of biomacromolecules is challenging as these molecules are taken up by cells and encapsulated into vesicular compartments called endosomes, and the fraction of molecules that are translocated to the cytosol are particularly important to obtain desired biological responses. This study aimed to estimate the cytosolic concentrations of intracellularly delivered peptides and proteins to aid the design of novel and effective biopharmaceutical delivery systems. To this end, we employed the split NanoLuc luciferase system, using the 11-residue HiBiT peptide segment as a probe for the delivered molecules in cells expressing the complementary LgBiT protein segment. The efficacy in cytosolic HiBiT delivery was determined by measuring the resultant luciferase activity when the HiBiT segment delivered into the cytosol forms a complex with LgBiT. Mean cytosolic HiBiT concentration was calculated using cell number and cell volume analysis. L17E and HAad peptides, developed in our laboratory for intracellular protein delivery, yielded approximately 6-fold cellular HiBiT concentrations than that obtained in their absence.

Nanoscale Visualization of Morphological Alteration of Live-Cell Membranes by the Interaction with Oligoarginine Cell-Penetrating Peptides.

The interactions between the cell membrane and biomolecules remain poorly understood. For example, arginine-rich cell-penetrating peptides (CPPs), including octaarginines (R8), are internalized by interactions with cell membranes. However, during the internalization process, the exact membrane dynamics introduced by these CPPs are still unknown. Here, we visualize arginine-rich CPPs and cell-membrane interaction-induced morphological changes using a system that combines scanning ion-conductance microscopy and spinning-disk confocal microscopy, using fluorescently labeled R8. This system allows time-dependent, nanoscale visualization of structural dynamics in live-cell membranes. Various types of membrane remodeling caused by arginine-rich CPPs are thus observed. The induction of membrane ruffling and the cup closure are observed as a process of endocytic uptake of the peptide. Alternatively suggested is the concave structural formation accompanied by direct peptide translocation through cell membranes. Studies using R8 without fluorescent labeling also demonstrate a non-negligible effect of the fluorescent moiety on membrane structural alteration.

Liquid Droplet Formation and Facile Cytosolic Translocation of IgG in the Presence of Attenuated Cationic Amphiphilic Lytic Peptides.

Fc region binding peptide conjugated with attenuated cationic amphiphilic lytic peptide L17E trimer [FcB(L17E)3 ] was designed for immunoglobulin G (IgG) delivery into cells. Particle-like liquid droplets were generated by mixing Alexa Fluor 488 labeled IgG (Alexa488-IgG) with FcB(L17E)3 . Droplet contact with the cellular membrane led to spontaneous influx and distribution of Alexa488-IgG throughout cells in serum containing medium. Involvement of cellular machinery accompanied by actin polymerization and membrane ruffling was suggested for the translocation. Alexa488-IgG negative charges were crucial in liquid droplet formation with positively charged FcB(L17E)3 . Binding of IgG to FcB(L17E)3 may not be necessary. Successful intracellular delivery of Alexa Fluor 594-labeled anti-nuclear pore complex antibody and anti-mCherry-nanobody tagged with supernegatively charged green fluorescence protein allowed binding to cellular targets in the presence of FcB(L17E)3 .

Discovery of a Macropinocytosis-Inducing Peptide Potentiated by Medium-Mediated Intramolecular Disulfide Formation.

Macropinocytosis is a ubiquitous cellular uptake mechanism of peptide-based intracellular delivery. This entry pathway shows promise as a route for the intracellular uptake of biomacromolecules and nanoparticles. In this work, we obtained the 8-residue analogue P4A bearing higher macropinocytosis induction ability. P4A contains vital cysteine residues in its sequence, which immediately reacts with cystine in culture medium to convert into its oxidized forms, including the intramolecularly oxidized form (oxP4A) as the dominant and active species. The conjugate of oxP4A and the membrane lytic peptide LK15 delivered bioactive proteins into cells; notably, this peptide delivered functional proteins fused with a negatively charged protein tag at a significantly reduced amount (up to nanomolar range) without compromising the delivery efficiency and the cellular activities of delivered proteins.

An Artificial Amphiphilic Peptide Promotes Endocytic Uptake by Inducing Membrane Curvature.

Membrane curvature plays a pivotal role in cellular life, including cellular uptake and membrane trafficking. The modulation of membrane curvature provides a novel means of manipulating cellular events. In this report, we show that a nine-residue amphiphilic peptide (R6W3) stimulates endocytic uptake by inducing membrane curvature. Curvature formation on cell membranes was confirmed by observing the cellular distribution of the curvature-sensing protein amphiphysin fused with a yellow fluorescent protein (Amp-YFP). Dot-like signals of Amp-YFP were visible following the addition of R6W3, suggesting curvature formation in cell membranes, leading to endocytic cup and vesicle formation. The promotion of endocytic uptake was confirmed using the endocytosis marker polydextran. Treatment of cells with R6W3 yielded a 4-fold dextran uptake compared with untreated cells. The amphiphilic helical structure of R6W3 was also crucial for R6W3-stimulated endocytic uptake.

Stimulating Macropinocytosis for Intracellular Nucleic Acid and Protein Delivery: A Combined Strategy with Membrane-Lytic Peptides To Facilitate Endosomal Escape.

Delivery of biomacromolecules via endocytic pathways requires the efficient accumulation of cargo molecules into endosomes, followed by their release to the cytosol. We propose a unique intracellular delivery strategy for bioactive molecules using a new potent macropinocytosis-inducing peptide derived from stromal-derived factor 1α (SN21). This peptide allowed extracellular materials to enter cells through the activation of macropinocytosis. To provide the ability to release internalized cargoes from endosomes, we conjugated SN21 with membrane-lytic peptides. The combination of a macropinocytosis-inducing peptide and a membrane-lytic peptide successfully delivered functional siRNA and proteins, which include antibodies, Cre recombinase, and an artificial transcription regulator protein having a transcription activator-like effector (TALE) motif. This study shows the feasibility of combining the physiological stimulation of macropinocytosis with the physicochemical disruption of endosomes as a strategy for intracellular delivery.

Improved cytosolic delivery of macromolecules through dimerization of attenuated lytic peptides.

Intracellular delivery of biomacromolecules is a challenging research field in chemical biology and drug delivery. We previously reported a peptide named L17E, which successfully delivered functional proteins, including antibodies, into cells. However, relatively high concentrations of L17E and proteins are needed. In this study, we prepared dimers of L17E and its analog L17E/Q21E. Dimerization of L17E increased cytotoxicity leading to reduced intracellular delivery compared with L17E. On the other hand, the dimers of the L17E analog, L17E/Q21E, especially when tethered at the N-termini, yielded a comparable level of intracellular delivery with L17E at decreased amounts of delivery peptides and cargoes.

Enhancing the activity of membrane remodeling epsin-peptide by trimerization.

Modulating the structural dynamics of biomembranes by inducing bilayer curvature and lipid packing defects has been highlighted as a practical tool to modify membrane-dependent cellular processes. Previously, we have reported on an amphipathic helical peptide derived from the N-terminal segment (residues 1-18, EpN18) of epsin-1, which can promote membrane remodeling including lipid packing defects in cell membranes. However, a high concentration is required to exhibit a pronounced effect. In this study, we demonstrate a significant increase in the membrane-remodeling effect of EpN18 by constructing a branched EpN18 homotrimer. Both monomer and trimer could enhance cell internalization of octaarginine (R8), a cell-penetrating peptide. The EpN18 trimer, however, promoted the uptake of R8 at an 80-fold lower concentration than the monomer. Analysis of the generalized polarization of a polarity-sensitive dye (di-4-ANEPPDHQ) revealed a higher efficacy of trimeric EpN18 in loosening the lipid packing in the cell membrane. Circular dichroism measurements in the presence of lipid vesicles showed that the EpN18 trimer has a higher α-helix content compared with the monomer. The stronger ability of the EpN18 trimer to impede negative bilayer curvature is also corroborated by solid-state 31P NMR spectroscopy. Hence, trimerizing peptides can be considered a promising approach for an exponential enhancement of their membrane-remodeling performance.

A Case Study on the Keap1 Interaction with Peptide Sequence Epitopes Selected by the Peptidomic mRNA Display.

Many protein-protein and peptide-protein interactions (PPIs) play key roles in the regulation of biological functions, and therefore, the modulation of PPIs has become an attractive target of new drug development. Although a number of PPIs have already been identified, over 100 000 unknown PPIs are predicted to exist. To uncover such unknown PPIs, it is important to devise a conceptually distinct method from that of currently available methods. Herein, an mRNA display by using a total RNA library derived from various human tissues, which serves as a unique method to physically isolate peptide epitopes that potentially bind to a target protein of interest, is reported. In this study, selection was performed against Kelch-like ECH-associated protein (Keap1) as a model target protein, leading to a peptide epitope originating from astrotactin-1 (ASTN1). It turned out that this ASTN1 peptide was able to interact with Keap1 more strongly than that with a known peptide derived from Nrf2; a well-known, naturally occurring Keap1 binder. This case study demonstrates the applicability of peptidomic mRNA display for the rapid exploration of consensus binding peptide motifs and the potential for the discovery of unknown PPIs with other proteins of interest.

Oligoarginine-Bearing Tandem Repeat Penetration-Accelerating Sequence Delivers Protein to Cytosol via Caveolae-Mediated Endocytosis.

To facilitate the cytosolic delivery of larger molecules such as proteins, we developed a new cell-penetrating peptide sequence, named Pas2r12, consisting of a repeated Pas sequence (FFLIG-FFLIG) and d-dodeca-arginine (r12). This peptide significantly enhanced the cellular uptake and cytosolic release of enhanced green fluorescent protein and immunoglobulin G as cargos. We found that simply mixing Pas2r12 with cargos could generate cytosolic introducible forms. The cytosolic delivery of cargos by Pas2r12 was found to be an energy-requiring process, to rely on actin polymerization, and to be suppressed by caveolae-mediated endocytosis inhibitors (genistein and methyl-ß-cyclodextrin) and small interfering RNA against caveolin-1. These results suggest that Pas2r12 enhances membrane penetration of cargos without the need for cross-linking and that caveolae-mediated endocytosis may be the route by which cytosolic delivery is enhanced.

Targeting surface voids to counter membrane disorders in lipointoxication-related diseases.

Saturated fatty acids (SFA), which are abundant in the so-called western diet, have been shown to efficiently incorporate within membrane phospholipids and therefore impact on organelle integrity and function in many cell types. In the present study, we have developed a yeast-based two-step assay and a virtual screening strategy to identify new drugs able to counter SFA-mediated lipointoxication. The compounds identified here were effective in relieving lipointoxication in mammalian ß-cells, one of the main targets of SFA toxicity in humans. In vitro reconstitutions and molecular dynamics simulations on bilayers revealed that these molecules, albeit according to different mechanisms, can generate voids at the membrane surface. The resulting surface defects correlate with the recruitment of loose lipid packing or void-sensing proteins required for vesicular budding, a central cellular process that is precluded under SFA accumulation. Taken together, the results presented here point at modulation of surface voids as a central parameter to consider in order to counter the impacts of SFA on cell function.

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. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 430-439, 2016.

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.