Publications by authors named "Shiroh Futaki"

193 Publications

A facile combinatorial approach to construct a ratiometric fluorescent sensor: application for the real-time sensing of cellular pH changes.

Chem Sci 2021 May 8;12(23):8231-8240. Epub 2021 May 8.

Institute of Advanced Energy, Kyoto University Kyoto Japan

Realtime monitoring of the cellular environment, such as the intracellular pH, in a defined cellular space provides a comprehensive understanding of the dynamics processes in a living cell. Considering the limitation of spatial resolution in conventional microscopy measurements, multiple types of fluorophores assembled within that space would behave as a single fluorescent probe molecule. Such a character of microscopic measurements enables a much more flexible combinatorial design strategy in developing fluorescent probes for given targets. Nanomaterials with sizes smaller than the microscopy spatial resolution provide a scaffold to assemble several types of fluorophores with a variety of optical characteristics, therefore providing a convenient strategy for designing fluorescent pH sensors. In this study, fluorescein (CF) and tetramethylrhodamine (CR) were assembled on a DNA nanostructure with controlling the number of each type of fluorophore. By taking advantage of the different responses of CF and CR emissions to the pH environment, an appropriate assembly of both CF and CR on DNA origami enabled a controlled intensity of fluorescence emission and ratiometric pH monitoring within the space defined by DNA origami. The CF and CR-assembled DNA origami was successfully applied for monitoring the intracellular pH changes.
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http://dx.doi.org/10.1039/d1sc01575cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8208317PMC
May 2021

Functional Peptides That Target Biomembranes: Design and Modes of Action.

Authors:
Shiroh Futaki

Chem Pharm Bull (Tokyo) 2021 ;69(7):601-607

Institute for Chemical Research, Kyoto University.

Biomembranes are important targets in molecular design. Our laboratory has been exploring the design of functional peptides that modulate membrane barrier function, lipid packing, and structure. Evaluation of the results obtained and analyses of cellular mechanisms have yielded peptides with more refined designs and functions. This review highlights the progress made in our laboratory towards the development of unique peptides that modulate membrane properties.
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http://dx.doi.org/10.1248/cpb.c21-00140DOI Listing
January 2021

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

Angew Chem Int Ed Engl 2021 Jun 11. Epub 2021 Jun 11.

Institute for Chemical Research, Kyoto University, Uji, Kyoto, 611-0011, Japan.

Fc region binding peptide conjugated with attenuated cationic amphiphilic lytic peptide L17E trimer [FcB(L17E) ] 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) . 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) . Binding of IgG to FcB(L17E) 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) .
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http://dx.doi.org/10.1002/anie.202105527DOI Listing
June 2021

Chemical passports to cross biological borders.

Nat Chem 2021 Jun;13(6):517-519

Institute for Chemical Research, Kyoto University, Uji, Kyoto, Japan.

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http://dx.doi.org/10.1038/s41557-021-00694-2DOI Listing
June 2021

Influence of the Dabcyl group on the cellular uptake of cationic peptides: short oligoarginines as efficient cell-penetrating peptides.

Amino Acids 2021 Jul 25;53(7):1033-1049. Epub 2021 May 25.

Department of Organic Chemistry, Eötvös L. University, Pázmány P. Setany 1/A, Budapest, 1117, Hungary.

Cell-penetrating peptides (CPPs) are promising delivery vehicles. These short peptides can transport wide range of cargos into cells, although their usage has often limitations. One of them is the endosomatic internalisation and thus the vesicular entrapment. Modifications which increases the direct delivery into the cytosol is highly researched area. Among the oligoarginines the longer ones (n > 6) show efficient internalisation and they are well-known members of CPPs. Herein, we describe the modification of tetra- and hexaarginine with (4-((4-(dimethylamino)phenyl)azo)benzoyl) (Dabcyl) group. This chromophore, which is often used in FRET system increased the internalisation of both peptides, and its effect was more outstanding in case of hexaarginine. The modified hexaarginine may enter into cells more effectively than octaarginine, and showed diffuse distribution besides vesicular transport already at low concentration. The attachment of Dabcyl group not only increases the cellular uptake of the cell-penetrating peptides but it may affect the mechanism of their internalisation. Their conjugates with antitumor drugs were studied on different cells and showed antitumor activity.
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http://dx.doi.org/10.1007/s00726-021-03003-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8241751PMC
July 2021

Membrane anchoring of a curvature-inducing peptide, EpN18, promotes membrane translocation of octaarginine.

Bioorg Med Chem Lett 2021 Jul 10;43:128103. Epub 2021 May 10.

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan. Electronic address:

EpN18 is a curvature-inducing peptide, which loosens lipid packing upon interaction with the cell membrane, and facilitates cell-membrane penetration by arginine-rich cell-penetrating peptides, including octaarginine (R8). In the present study, we conjugated the N-terminal of EpN18 with a pyrenebutyryl (pBu) moiety, which acts as an anchoring unit that increases membrane interactions. Enhanced lipid-packing loosening and cytosolic translocation of R8 were observed by the pBu anchoring of EpN18.
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http://dx.doi.org/10.1016/j.bmcl.2021.128103DOI Listing
July 2021

Potentiating the Membrane Interaction of an Attenuated Cationic Amphiphilic Lytic Peptide for Intracellular Protein Delivery by Anchoring with Pyrene Moiety.

Bioconjug Chem 2021 05 16;32(5):950-957. Epub 2021 Apr 16.

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.

We previously reported an approach for intracellular protein delivery by attenuating membrane-lytic activity of cationic amphiphilic peptides on cell surfaces. HAad is one such peptides that cytosolically delivers proteins of interest, including antibodies, by stimulating their endosomal escape. Additionally, HAad elicits ruffling of cell membrane, accompanied by transient membrane permeabilization, allowing for the efficient cytosolic translocation of proteins. In this study, we prepared a conjugate of HAad with pyrenebutyric acid as a membrane-anchoring unit (pBu-HAad). pBu-HAad demonstrated protein delivery into cells with only 1/20 concentration of HAad. However, the conjugates with cholesteryl hemisuccinate and aliphatic fatty acids (C = 3, 6, and 10) did not yield such marked effects. The results of time-course and inhibitor studies suggest that the membrane anchoring of HAad by a pyrene moiety leads to enhanced peptide-membrane interaction and to loosen lipid packing, thus facilitating cytosolic translocation through membranes.
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http://dx.doi.org/10.1021/acs.bioconjchem.1c00101DOI Listing
May 2021

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

Anal Chem 2021 04 26;93(13):5383-5393. Epub 2021 Mar 26.

Graduate School of Environmental Studies, Tohoku University, Sendai, Miyagi 980-8579, Japan.

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.
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http://dx.doi.org/10.1021/acs.analchem.0c04097DOI Listing
April 2021

Use of homoarginine to obtain attenuated cationic membrane lytic peptides.

Bioorg Med Chem Lett 2021 05 9;40:127925. Epub 2021 Mar 9.

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan. Electronic address:

Our research group has been studying the design of intracellular delivery peptides based on cationic lytic peptides. By placing negatively charged amino acids on potentially hydrophobic faces of the peptides, membrane lytic activity is attenuated on the cell surface, whereas it recovers in endosomes, enabling cytosolic delivery of proteins including antibodies. These lytic peptides generally contain multiple lysines, facilitating cell surface interaction and membrane perturbation. This study evaluated the effect of lysine-to-homoarginine substitution using HAad as a model delivery peptide. The resulting peptide had a comparable or better delivery efficacy for Cre recombinase, antibodies, and the Cas9/sgRNA complex with one-quarter of the concentration of HAad, implying that a subtle structural difference can affect delivery activity.
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http://dx.doi.org/10.1016/j.bmcl.2021.127925DOI Listing
May 2021

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

Angew Chem Int Ed Engl 2021 05 14;60(21):11928-11936. Epub 2021 Apr 14.

Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto, 611-0011, Japan.

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.
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http://dx.doi.org/10.1002/anie.202016754DOI Listing
May 2021

Environmental pH stress influences cellular secretion and uptake of extracellular vesicles.

FEBS Open Bio 2021 03 18;11(3):753-767. Epub 2021 Feb 18.

Keio University School of Medicine, Tsukuba, Japan.

Exosomes (extracellular vesicles/EVs) participate in cell-cell communication and contain bioactive molecules, such as microRNAs. However, the detailed characteristics of secreted EVs produced by cells grown under low pH conditions are still unknown. Here, we report that low pH in the cell culture medium significantly affected the secretion of EVs with increased protein content and zeta potential. The intracellular expression level and location of stably expressed GFP-fused CD63 (an EV tetraspanin) in HeLa cells were also significantly affected by environmental pH. In addition, increased cellular uptake of EVs was observed. Moreover, the uptake rate was influenced by the presence of serum in the cell culture medium. Our findings contribute to our understanding of the effect of environmental conditions on EV-based cell-cell communication.
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http://dx.doi.org/10.1002/2211-5463.13107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7931216PMC
March 2021

Direct entry of cell-penetrating peptide can be controlled by maneuvering the membrane curvature.

Sci Rep 2021 01 8;11(1):31. Epub 2021 Jan 8.

Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan.

A biomembrane's role is to be a barrier for interior cytosol from an exterior environment to execute the cell's normal biological functions. However, a water-soluble peptide called cell-penetrating peptide (CPP) has been known for its ability to directly penetrate through the biomembranes into cells (cytolysis) without perturbating cell viability and expected to be a promising drug delivery vector. Examples of CPP include peptides with multiple arginine units with strong cationic properties, which is the key to cytolysis. Here we show the conclusive evidence to support the mechanism of CPP's cytolysis and way to control it. The mechanism we proposed is attributed to biomembrane's physicochemical nature as lamellar liquid crystal (Lα). Cytolysis occurs as the temporal and local dynamic phase transitions from Lα to an undulated lamellar with pores called Mesh. We have shown this phase transfer of Lα composed of dioleoyl-phosphatidylcholine (DOPC) with water by adding oligo-arginine (Rx) as CPP at the equilibrium. Using giant unilamellar vesicle composed of DOPC as a single cell model, we could control the level of cytolysis of CPP (FITC-R8) by changing the curvature of the membrane through osmotic pressure modulation. The cytolysis of CPP utilizes biomembrane's inherent topological and functional flexibility corresponding to the stimuli.
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http://dx.doi.org/10.1038/s41598-020-79518-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7794472PMC
January 2021

Effect of Surface Modifications on Cellular Uptake of Gold Nanorods in Human Primary Cells and Established Cell Lines.

ACS Omega 2020 Dec 10;5(50):32744-32752. Epub 2020 Dec 10.

Faculty of Advanced Science and Technology, Kumamoto University, Kumamoto 860-8555, Japan.

Endocytosis is a cellular process in which substances are engulfed by the cellular membrane and budded off inside the cells to form vesicles. It plays key roles in controlling nutritional component uptake, immune responses, and other biological functions. A comprehensive understanding of endocytosis gives insights into such physiological functions and informs the design of medical nanodevices that need to enter cells. So far, endocytosis has been studied mostly using established cell lines. However, the established cell lines generally originate from cancer cells or are transformed from normal cells into immortalized cells. Therefore, primary cells may give us more reliable information about the endocytosis process of nanoparticles into cells. In this research, we studied the uptake of gold nanorods (AuNRs) with four different surface modifications (anionic/cationic polymers and anionic/cationic silica) by two kinds of primary cells (human monocyte-derived macrophages and human umbilical vein endothelial cells) and two kinds of established cell lines (HeLa cells and RAW 264.7 cells). We found that the surface properties of AuNRs affected their cellular uptake, and the cationic surface tended was advantageous for uptake, but it depended on the cell types. Control experiments using inhibitors of representative endocytosis pathways (macropinocytosis, clathrin-mediated endocytosis, and caveolae-mediated endocytosis) indicated that primary cells had a dominant uptake pathway for internalization of the AuNRs, whereas the established cell lines had multiple pathways. Our results provide us with novel insights into cellular uptake of AuNRs in that they depend not only on surface characters of the nanoparticles but also cell types, such as primary cells and established cell lines.
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http://dx.doi.org/10.1021/acsomega.0c05162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7758961PMC
December 2020

Effect of Vesicle Size on the Cytolysis of Cell-Penetrating Peptides (CPPs).

Int J Mol Sci 2020 Oct 7;21(19). Epub 2020 Oct 7.

Chiba Institute of Science, Choshi, Chiba 288-0025, Japan.

A specific series of peptides, called a cell-penetrating peptide (CPP), is known to be free to directly permeate through cell membranes into the cytosol (cytolysis); hence, this CPP would be a potent carrier for a drug delivery system (DDS). Previously, we proposed the mechanism of cytolysis as a temporal and local phase transfer of membrane lipid caused by positive membrane curvature generation. Moreover, we showed how to control the CPP cytolysis. Here, we investigate the phospholipid vesicle's size effect on CPP cytolysis because this is the most straightforward way to control membrane curvature. Contrary to our expectation, we found that the smaller the vesicle diameter (meaning a higher membrane curvature), the more cytolysis was suppressed. Such controversial findings led us to seek the reason for the unexpected results, and we ended up finding out that the mobility of membrane lipids as a liquid crystal is the key to cytolysis. As a result, we could explain the cause of cytolysis suppression by reducing the vesicle size (because of the restriction of lipid mobility); osmotic pressure reduction to enhance positive curvature generation works as long as the membrane is mobile enough to modulate the local structure. Taking all the revealed vital factors and their effects as a tool, we will further explore how to control CPP cytolysis for developing a DDS system combined with appropriate cargo selection to be tagged with CPPs.
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http://dx.doi.org/10.3390/ijms21197405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582850PMC
October 2020

Development of a Simple and Rapid Method for In Situ Vesicle Detection in Cultured Media.

J Mol Biol 2020 11 12;432(22):5876-5888. Epub 2020 Sep 12.

Laboratory of Biofunctional Design Chemistry, Institute for Chemical Research, Kyoto University, Gokasho, Uji 611-0011, Japan.

Extracellular membrane vesicles (EMVs) are biogenic secretory lipidic vesicles that play significant roles in intercellular communication related to human diseases and bacterial pathogenesis. They are being investigated for their possible use in diagnosis, vaccines, and biotechnology. However, the existing methods suffer from a number of issues. High-speed centrifugation, a widely used method to collect EMVs, may cause structural artifacts. Immunostaining methods require several steps and thus the separation and detection of EMVs from the secretory cells is time-consuming. Furthermore, detection of EMVs using these methods requires specific and costly antibodies. To tackle these problems, development of a simple and rapid detection method for the EMVs in the cultured medium without separation from the secretory cells is a pressing task. In this study, we focused on the Gram-negative bacterium Shewanella vesiculosa HM13, which produces a large amount of EMVs including a cargo protein with high purity, as a model. Curvature-sensing peptides were used for EMV-detection tools. FAAV, a peptide derived from sorting nexin protein 1, selectively binds to the EMVs even in the presence of the secretory cells in the complex cultured medium. FAAV can fully detect the EMVs within a few minutes, and the resistance of FAAV to proteases enables it to withstand prolonged use in the cultured medium. Fluorescence/Förster resonance energy transfer was used to develop a method to detect changes in the amount of the EMVs with high sensitivity. Overall, our results indicate the potential applicability of FAAV for in situ EMV detection in cultured media.
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http://dx.doi.org/10.1016/j.jmb.2020.09.009DOI Listing
November 2020

Key Process and Factors Controlling the Direct Translocation of Cell-Penetrating Peptide through Bio-Membrane.

Int J Mol Sci 2020 Jul 30;21(15). Epub 2020 Jul 30.

Department of Pure and Applied Chemistry, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.

Cell-penetrating peptide (CPP) can directly penetrate the cytosol (cytolysis) and is expected to be a potent vector for a drug delivery system (DDS). Although there is general agreement that CPP cytolysis is related to dynamic membrane deformation, a distinctive process has yet to be established. Here, we report the key process and factors controlling CPP cytolysis. To elucidate the task, we have introduced trypsin digestion of adsorbed CPP onto giant unilamellar vesicle (GUV) to quantify the adsorption and internalization (cytolysis) separately. Also, the time-course analysis was introduced for the geometric calculation of adsorption and internalization amount per lipid molecule consisting of GUV. As a result, we found that adsorption and internalization assumed to occur successively by CPP molecule come into contact with membrane lipid. Adsorption is quick to saturate within 10 min, while cytolysis of each CPP on the membrane follows successively. After adsorption is saturated, cytolysis proceeds further linearly by time with a different rate constant that is dependent on the osmotic pressure. We also found that temperature and lipid composition influence cytolysis by modulating lipid mobility. The electrolyte in the outer media is also affected as a chemical mediator to control CPP cytolysis by following the Hoffmeister effect for membrane hydration. These results confirmed the mechanism of cytolysis as temporal and local phase transfer of membrane lipid from Lα to Mesh, which has punctured bilayer morphologies.
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http://dx.doi.org/10.3390/ijms21155466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432884PMC
July 2020

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

Bioorg Med Chem Lett 2020 09 20;30(17):127362. Epub 2020 Jun 20.

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bmcl.2020.127362DOI Listing
September 2020

Pseudo-Membrane Jackets: Two-Dimensional Coordination Polymers Achieving Visible Phase Separation in Cell Membrane.

Angew Chem Int Ed Engl 2020 10 13;59(41):17931-17937. Epub 2020 Aug 13.

Department of Chemistry, Graduate School of Science and Technology and Institute of Pulsed Power Science, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto, 860-8555, Japan.

Cell membranes contain lateral systems that consist of various lipid compositions and actin cytoskeleton, providing two-dimensional (2D) platforms for chemical reactions. However, such complex 2D environments have not yet been used as a synthetic platform for artificial 2D nanomaterials. Herein, we demonstrate the direct synthesis of 2D coordination polymers (CPs) at the liquid-cell interface of the plasma membrane of living cells. The coordination-driven self-assembly of networking metal complex lipids produces cyanide-bridged CP layers with metal ions, enabling "pseudo-membrane jackets" that produce long-lived micro-domains with a size of 1-5 μm. The resultant artificial and visible phase separation systems remain stable even in the absence of actin skeletons in cells. Moreover, we show the cell application of the jackets by demonstrating the enhancement of cellular calcium response to ATP.
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http://dx.doi.org/10.1002/anie.202006600DOI Listing
October 2020

Optimizing Charge Switching in Membrane Lytic Peptides for Endosomal Release of Biomacromolecules.

Angew Chem Int Ed Engl 2020 11 20;59(45):19990-19998. Epub 2020 Aug 20.

Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto, 611-0011, Japan.

Endocytic pathways are practical routes for the intracellular delivery of biomacromolecules. Along with this, effective strategies for endosomal cargo release into the cytosol are desired to achieve successful delivery. Focusing on compositional differences between the cell and endosomal membranes and the pH decrease within endosomes, we designed the lipid-sensitive and pH-responsive endosome-lytic peptide HAad. This peptide contains aminoadipic acid (Aad) residues, which serve as a safety catch for preferential permeabilization of endosomal membranes over cell membranes, and His-to-Ala substitutions enhance the endosomolytic activity. The ability of HAad to destabilize endosomal membranes was supported by model studies using large unilamellar vesicles (LUVs) and by increased intracellular delivery of biomacromolecules (including antibodies) into live cells. Cerebral ventricle injection of Cre recombinase with HAad led to Cre/loxP recombination in a mouse model, thus demonstrating potential applicability of HAad in vivo.
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http://dx.doi.org/10.1002/anie.202005887DOI Listing
November 2020

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

Bioconjug Chem 2020 06 13;31(6):1611-1615. Epub 2020 May 13.

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.

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.
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http://dx.doi.org/10.1021/acs.bioconjchem.0c00239DOI Listing
June 2020

Rational Design Principles of Attenuated Cationic Lytic Peptides for Intracellular Delivery of Biomacromolecules.

Mol Pharm 2020 06 12;17(6):2175-2185. Epub 2020 May 12.

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.

Intracellular delivery of bioactive macromolecules via endocytic pathways has utility in biotechnological and medicinal applications. Various endosomolytic peptides bearing glutamic acid (Glu) residues have been developed with the aim to achieve selective lysis of endosomal membranes without damaging cell membranes (plasma membranes) to release endosome-entrapped macromolecules and obtain their bioactivity. Glu residues on peptides are negatively charged in the extracellular medium, and substitution of this residue onto membrane-lytic peptides prevents its peptide-membrane interaction and its lytic activity. On the other hand, within endosomes, which have a reduced pH of ∼5, Glu is protonated, resulting in the reduction of the hydrophilicity of the peptide, unmasking its lytic activity. Despite this, a limited number of studies have elucidated the optimum positions for Glu substitution. This report investigated the positioning of Glu and the endosomolytic activities of cationic lytic peptides, ponericin-W3, and melittin. By cell-based assays, biophysical analyses, and molecular dynamics simulations, we found that analogues with Glu positioned on the borders between the hydrophobic and hydrophilic faces of the helical structures showed better performance than placing Glu within said faces.
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http://dx.doi.org/10.1021/acs.molpharmaceut.0c00312DOI Listing
June 2020

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

Bioorg Med Chem Lett 2020 06 15;30(12):127190. Epub 2020 Apr 15.

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan. Electronic address:

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 P NMR spectroscopy. Hence, trimerizing peptides can be considered a promising approach for an exponential enhancement of their membrane-remodeling performance.
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http://dx.doi.org/10.1016/j.bmcl.2020.127190DOI Listing
June 2020

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

Bioconjug Chem 2020 03 7;31(3):547-553. Epub 2020 Feb 7.

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.

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.
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http://dx.doi.org/10.1021/acs.bioconjchem.0c00064DOI Listing
March 2020

Programmable RNA methylation and demethylation using PUF RNA binding proteins.

Chem Commun (Camb) 2020 Jan 7;56(9):1365-1368. Epub 2020 Jan 7.

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.

New methods to control local RNA methylation are needed to elucidate the function of individual mA. Here, fusion proteins between the programmable RNA binding protein PUF and the mA demethylase FTO or METTL14 methyltransferase were designed. FTO-PUFs and METTL14-PUFs showed sequence-specific RNA demethylation and methylation activities, respectively.
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http://dx.doi.org/10.1039/c9cc09298fDOI Listing
January 2020

Intracellular target delivery of cell-penetrating peptide-conjugated dodecaborate for boron neutron capture therapy (BNCT).

Chem Commun (Camb) 2019 Nov;55(93):13955-13958

Graduate School of Science, Osaka Prefecture University, 1-1, Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.

In this study, we designed and synthesized organelle-targeted cell-penetrating peptide (CPP)-conjugated boron compounds to increase their cellular uptake and to control the intracellular locations for the induction of sophisticated anticancer biological activity in boron neutron capture therapy (BNCT), leading to anticancer effects with ATP reduction and apoptosis when irradiated with neutrons in an in vitro BNCT assay.
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http://dx.doi.org/10.1039/c9cc03924dDOI Listing
November 2019

Meeting Peptides in Kyoto.

Chembiochem 2019 08 2;20(16):2015-2016. Epub 2019 Aug 2.

Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1, Hongo, Bunkyo, Tokyo, 113-0033, Japan.

The 10th International Peptide Symposium was held in Kyoto last December in conjunction with the 55th Japanese Peptide Symposium. Around 800 peptide scientists from 31 different countries and regions enjoyed sessions covering various aspects of state-of-the-art peptide science, such as synthetic methodology, chemical biology, cell biology, biophysics, and medicinal/ medical applications.
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http://dx.doi.org/10.1002/cbic.201900456DOI Listing
August 2019

Development of a Membrane Curvature-Sensing Peptide Based on a Structure-Activity Correlation Study.

Chem Pharm Bull (Tokyo) 2019 Oct 17;67(10):1131-1138. Epub 2019 Jul 17.

Institute for Chemical Research, Kyoto University.

Membrane curvature formation is important for various biological processes such as cell motility, intracellular signal transmission, and cellular uptake of foreign substances. However, it remains still a challenging topic to visualize the membrane curvature formation on the cell membranes in real-time imaging. To develop and design membrane curvature-sensors, we focused on amphipathic helical peptides of proteins belonging to the Bin/Amphiphysin/Rvs (BAR) family as the starting point. BAR proteins individually have various characteristic structures that recognize different curvatures, and the derived peptides possess the potential to function as curvature sensors with a variety of recognition abilities. Peptide-based curvature sensors can have wide applications in biological research fields due to their small size, easy modification, and large production capability in comparison to protein-based sensors. In the present study, we found that an amphipathic peptide derived from sorting nexin1 (SNX1) has a curvature-recognition ability. The mutation studies of the initial peptide revealed a close correlation between the α-helicity and lipid binding ability of the peptides. In particular, the amino acids located on the hydrophobic face played a vital role in curvature recognition. The α-helix formation of the peptides was thought to serve to accommodate lipid-packing defects on the membrane surface and to maintain their binding to lipid vesicles. The structure-activity correlation found in this study have the potential to contribute to the design of peptide-based curvature sensors that will enable the capture of various life phenomena in cells.
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http://dx.doi.org/10.1248/cpb.c19-00465DOI Listing
October 2019

An influenza-derived membrane tension-modulating peptide regulates cell movement and morphology via actin remodeling.

Commun Biol 2019 26;2:243. Epub 2019 Jun 26.

1Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 Japan.

Tension in cell membranes is closely related to various cellular events, including cell movement and morphogenesis. Therefore, modulation of membrane tension can be a new approach for manipulating cellular events. Here, we show that an amphipathic peptide derived from the influenza M2 protein (M2[45-62]) yields lamellipodia at multiple sites in the cell. Effect of M2[45-62] on cell membrane tension was evaluated by optical tweezer. The membrane tension sensor protein FBP17 was involved in M2[45-62]-driven lamellipodium formation. Lysine-to-arginine substitution in M2[45-62] further enhanced its activity of lamellipodium formation. M2[45-62] had an ability to reduce cell motility, evaluated by scratch wound migration and transwell migration assays. An increase in neurite outgrowth was also observed after treatment with M2[45-62]. The above results suggest the potential of M2[45-62] to modulate cell movement and morphology by modulating cell membrane tension.
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http://dx.doi.org/10.1038/s42003-019-0486-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594980PMC
May 2020

Loosening of Lipid Packing by Cell-Surface Recruitment of Amphiphilic Peptides by Coiled-Coil Tethering.

Chembiochem 2019 08 9;20(16):2151-2159. Epub 2019 Aug 9.

Institute for Chemical Research, Kyoto University, Uji, Kyoto, 611-0011, Japan.

Lipid packing has a strong influence on the formation and structural dynamics of cell membranes. Techniques to modulate lipid packing may thus enable modification of cellular functions and events. An 18-residue amphiphilic helical peptide derived from the N-terminal segment of epsin-1 (EpN18) is reported to induce positive membrane curvature and to loosen lipid packing in the cell membrane. In this study, it is shown that EpN18, crosslinked to a leucine-zipper peptide K4, is recruited to the cell surface by interacting with a cell-surface-expressed E3 leucine-zipper segment. Cell-surface tethering markedly enhanced loosening of lipid packing, which led to the promotion of membrane translocation of octaarginine. The loosening of lipid packing by EpN18 was also confirmed by analyzing the generalized polarization value with a membrane-environment-sensitive dye, 2-hydroxy-3-{2-[(2-hydroxyethyl)dimethylamino]ethyl}-4-{2-[6-(dibutylamino)-2-naphthyl]ethenyl}pyridiniumdibromide (di-4-ANEPPDHQ). This approach thus shows promise for the control of lipid packing and related cellular events.
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http://dx.doi.org/10.1002/cbic.201900347DOI Listing
August 2019

Cell-penetrating mechanism of intracellular targeting albumin: Contribution of macropinocytosis induction and endosomal escape.

J Control Release 2019 06 10;304:156-163. Epub 2019 May 10.

Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan; Center for Clinical Pharmaceutical Sciences, School of Pharmacy, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan. Electronic address:

We recently developed a cell-penetrating drug carrier composed of albumin (HSA) combined with palmitoyl-cyclic-(D-Arg). While it is possible that the palmitoyl-cyclic-(D-Arg)/HSA enters the cell mainly via macropinocytosis, the mechanism responsible for the induction of macropinocytosis and endosomal escape remain unknown. We report herein that palmitoyl-cyclic-(D-Arg)/HSA might interact with heparan sulfate proteoglycan and the chemokine receptor CXCR4 followed by multiple activations of the PKC/PI3K/JNK/mTOR signaling pathways to induce macropinocytosis. This result was further confirmed by a co-treatment with 70 kDa dextran, a macropinocytosis marker. Using liposomes that mimic endosomes, the leakage of 5,6-carboxyfluorescein from liposome was observed in the presence of palmitoyl-cyclic-(D-Arg)/HSA only in the case of the anionic late endosome-like liposomes but not the neutral early endosome-like liposomes. Heparin largely inhibited this leakage, suggesting the importance of electrostatic interactions between palmitoyl-cyclic-(D-Arg)/HSA and the late-endosomal membrane. Immunofluorescence staining and Western blotting data indicated that the intact HSA could be transferred from endosomes to the cytosol. These collective data suggest that the palmitoyl-cyclic-(D-Arg)/HSA is internalized via macropinocytosis and intact HSA is released from the late endosomes to the cytoplasm before the endosomes fuse with lysosomes. Palmitoyl-cyclic-(D-Arg)/HSA not only functions as an intracellular drug delivery carrier but also as an inducer of macropinocytosis.
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http://dx.doi.org/10.1016/j.jconrel.2019.05.015DOI Listing
June 2019
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