All-peptide-based polyion complex vesicles: Facile preparation and encapsulation of the protein in active form

The polyion complex vesicle (PICsome) is a promising platform for bioactive molecule delivery as well as nanoreactor systems. In addition to anionic and cationic charged blocks, a hydrophilic poly(ethylene glycol) (PEG) block is mostly employed for PICsome formation; however, the long-term safety of the PEG component in vivo is yet to be clarified. In this study, we developed novel PEG-free PICsome comprising all peptide components. Instead of the PEG block, we selected the sarcosine (Sar) oligomer as a hydrophilic block and fused it with anionic oligo(l-glutamic acid). Mixing the Sar-containing anionic peptide with cationic oligo(l-lysine) resulted in the formation of stable vesicles. The peptide-based PICsome was able to encapsulate a model protein in its hollow structure. After modification of the surface with a cell-penetrating peptide, the protein-encapsulated PICsome was successfully delivered into plant cells, indicating its promised for application as a biocompatible carrier for protein delivery

Cell wall‐denaturing molecules for plant gene modification

Zwitterionic molecules, such as zwitterionic liquids (ZILs) and polypeptides (ZIPs), are attracting attention for application in new methods that can be used to loosen tight cell wall networks in a biocompatible manner. These novel methods can enhance the cell wall permeability of nanocarriers and increase their transfection efficiency into targeted subcellular organelles in plants. Herein, we provide an overview of the recent progress and future perspectives of such molecules that function as boosters for cell wall-penetrating nanocarriers

Molecular dynamics simulation of complexation between plasmid DNA and cationic peptides

The elucidation of the process by which cationic peptides condense plasmid DNA (pDNA) is important for unraveling the mechanism of peptide/pDNA complex formation, which plays a vital role in gene delivery for the genetic transformation of living cells. We performed atomic MD simulations of the complexation of pDNA in the presence of two cationic peptides, KH9 (with an alternating sequence of lysine and histidine) and Cytcox (functioning as a mitochondria-targeting signal), to investigate the mechanism of pDNA condensation. The simulations revealed that the cationic peptides bound to the pDNA and that defects in pDNA formed in response to the densely packed cationic peptides, presumably initiating the folding of the double-stranded pDNA into a globule morphology. The decrease in the radius of gyration and the number of hydrogen bonds and the increase in the writhe structure, with a slightly higher tendency for the Cytcox/pDNA system, strongly support the formation of pDNA defects leading to the bending of the double helix. The results provided insight into the mechanism of pDNA complexation with cationic peptides, which should contribute to the future design of highly efficient gene delivery systems using peptide-mediated nanocarriers.This work was financially supported by Japan Science and Technology Agency Exploratory Research for Advanced Technology (JST ERATO; Grant No. JPMJER1602). We acknowledge the RIKEN Advanced Center for Computing and Communication (ACCC) for providing access to HOKUSAI BigWaterfall Supercomputer resources.Peer ReviewedPostprint (published version

Plant Mitochondrial-Targeted Gene Delivery by Peptide/DNA Micelles Quantitatively Surface-Modified with Mitochondrial Targeting and Membrane-Penetrating Peptides

Plant mitochondria play essential roles in metabolism and respiration. Recently, there has been growing interest in mitochondrial transformation for developing crops with commercially valuable traits, such as resistance to environmental stress and shorter fallow periods. Mitochondrial targeting and cell membrane penetration functions are crucial for improving the gene delivery efficiency of mitochondrial transformation. Here, we developed a peptide-based carrier, referred to as Cytcox/KAibA-Mic, that contains multifunctional peptides for efficient transfection into plant mitochondria. We quantified the mitochondrial targeting and cell membrane-penetrating peptide modification rates to control their functions. The modification rates were easily determined from high-performance liquid chromatography chromatograms. Additionally, the gene carrier size remained constant even when the mitochondrial targeting peptide modification rate was altered. Using this gene carrier, we can quantitatively investigate the relationships between various peptide modifications and transfection efficiency and optimize the gene carrier conditions for mitochondrial transfection

A silk composite fiber reinforced by telechelic-type polyalanine and its strengthening mechanism

Antiparallel β-sheets play a key role in determining the physical properties of fibroins, e.g., degradation and mechanical properties, and are typically formed by poly(A) motifs from spider dragline silks and GAGAGS motifs from Bombyx mori silkworm silks. To explore the interaction between these two motifs within the same system, a telechelic-type polyalanine (TPA) was prepared through chemoenzymatic synthesis and doped in silkworm silk fibroins to fabricate silk composite fibers. Interestingly, when TPA was added at suitable ratios of 1 and 3 wt%, the mechanical properties of the composite fibers were largely improved by approximately 42% and 51% compared with those of silk-only fibers in terms of tensile strength and toughness, respectively. As revealed by wide-angle X-ray diffraction (WAXD), silk composite fibers achieved the highest crystallinity at a TPA ratio of 1 wt%, largely contributing to their tensile strength. Evidenced by simultaneous stretching during WAXD measurement, TPA did not compete with the silk matrix by forming its own crystallization. Ultimately, a strengthening mechanism of nucleus-dependent crystallization was discussed to show the favorable heterogeneous nucleation created from TPA molecules for the promotion of crystallization in silk fibroins. Interestingly, regularly packed and aligned granules within composite fibers were detected by AFM to further support the enhanced mechanical performance. This work envisions sophisticated control of β-sheet crystals to better understand the structure–property relationship

Relaxation of the Plant Cell Wall Barrier via Zwitterionic Liquid Pretreatment for Micelle‐Complex‐Mediated DNA Delivery to Specific Plant Organelles

Targeted delivery of genes to specific plant organelles is a key challenge for fundamental plant science, plant bioengineering, and agronomic applications. Nanoscale carriers have attracted interest as a promising tool for organelle-targeted DNA delivery in plants. However, nanocarrier-mediated DNA delivery in plants is severely hampered by the barrier of the plant cell wall, resulting in insufficient delivery efficiency. Herein, we propose a unique strategy that synergistically combines a cell wall-loosening zwitterionic liquid (ZIL) with a peptide-displaying micelle complex for organelle-specific DNA delivery in plants. We demonstrated that ZIL pretreatment can enhance cell wall permeability without cytotoxicity, allowing micelle complexes to translocate across the cell wall and carry DNA cargo into specific plant organelles, such as nuclei and chloroplasts, with significantly augmented efficiency. Our work offers a novel concept to overcome the plant cell wall barrier for nanocarrier-mediated cargo delivery to specific organelles in living plants

Papain-Catalyzed, Sequence-Dependent Polymerization Yields Polypeptides Containing Periodic Histidine Residues

His-containing polypeptides, including polyHis, are attractive materials due to the unique characteristics of the imidazole ring of the His residue. In particular, His-containing polypeptides with repetitive sequences have a variety of distinctive features based on their periodic structure. In this study, chemoenzymatic polymerization of ethyl ester monomers with sequences His, GlyHis, HisGly, and GlyHisGly with hydrophobic side chains on the imidazole ring was performed using papain as a catalyst. Sequence dependence in chemoenzymatic polymerization was observed for GlyHis- and HisGly-based monomers: GlyHis-based monomers did not undergo polymerization, whereas polymerization of HisGly-based monomers afforded polypeptides with a degree of polymerization from 6 to 38 and from 5 to 31 and a number-average degree of polymerization of 16.4 and 12.4 for poly(HisGly) and poly[His(Bu)Gly], respectively. The difference in polymerizability of these dipeptide monomers was supported by a docking simulation between these monomers and papain, where the ester group of the HisGly-based monomer was closer to the catalytic center of papain than that of the GlyHis-based monomer. Infrared spectroscopy and synchrotron wide-angle X-ray diffraction measurements indicated that poly(HisGly) formed a β-sheet structure whose crystallinity was 41.6%, whereas the other tripeptide-based polypeptides were more amorphous showing 19.6–30.7% of crystallinity. Poly(HisGly) exhibited the highest thermal stability among all of the polypeptides in the thermogravimetric analysis, reflecting the difference in the secondary structures

Chemoenzymatic Polymerization of l-Serine Ethyl Ester in Aqueous Media without Side-Group Protection

Poly(l-serine) (polySer) has tremendous potential as a polypeptide-based functional material due to the utility of the hydroxyl group on its side chain; however, tedious protection/deprotection of the hydroxyl groups is required for its synthesis. In this study, polySer was synthesized by the chemoenzymatic polymerization (CEP) of l-serine ethyl ester (Ser-OEt) or l-serine methyl ester (Ser-OMe) using papain as a catalyst in an aqueous medium. The CEP of Ser-OEt proceeded at basic pH ranging from 7.5 to 9.5 and resulted in the maximum precipitate yield of polySer at an optimized pH of 8.5. A series of peaks detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry revealed that the formed precipitate consisted of polySer with a degree of polymerization ranging from 5 to 22. Moreover, infrared spectroscopy, circular dichroism spectroscopy, and synchrotron wide-angle X-ray diffraction measurements indicated that the obtained polySer formed a β-sheet/strand structure. This is the first time the synthesis of polySer was realized by CEP in aqueous solution without protecting the hydroxyl group of the Ser monomer

Synthetic Mitochondria-Targeting Peptides Incorporating α-Aminoisobutyric Acid with a Stable Amphiphilic Helix Conformation in Plant Cells

In the genetic modification of plant cells, the mitochondrion is an important target in addition to the nucleus and plastid. However, gene delivery into the mitochondria of plant cells has yet to be established by conventional methods, such as particle bombardment, because of the small size and high mobility of mitochondria. To develop an efficient mitochondria-targeting signal (MTS) that functions in plant cells, we designed the artificial peptide (LURL)₃ and its analogues, which periodically feature hydrophobic α-aminoisobutyric acid (Aib, U) and cationic arginine (R), considering the consensus motif recognized by the mitochondrial import receptor Tom20. Circular dichroism measurements and molecular dynamics simulation studies revealed that (LURL)₃ had a propensity to form a stable α-helix in 0.1 M phosphate buffer solution containing 1.0 wt % sodium dodecyl sulfate. After internalization into plant cells via particle bombardment, (LURL)₃ revealed highly selective accumulation in the mitochondria, whereas its analogue (LARL)₃ was predominantly located in the vacuoles in addition to mitochondria. The high selectivity of (LURL)₃ can be attributed to the incorporation of Aib, which promotes the hydrophobic interaction between the MTS and Tom20 by increasing the hydrophobicity and helicity of (LURL)₃. The present study provided a prospective mitochondrial targeting system using the simple design of artificial peptides

Synchronization of Circadian Per2 Rhythms and HSF1-BMAL1:CLOCK Interaction in Mouse Fibroblasts after Short-Term Heat Shock Pulse

Circadian rhythms are the general physiological processes of adaptation to daily environmental changes, such as the temperature cycle. A change in temperature is a resetting cue for mammalian circadian oscillators, which are possibly regulated by the heat shock (HS) pathway. The HS response (HSR) is a universal process that provides protection against stressful conditions, which promote protein-denaturation. Heat shock factor 1 (HSF1) is essential for HSR. In the study presented here, we investigated whether a short-term HS pulse can reset circadian rhythms. Circadian Per2 rhythm and HSF1-mediated gene expression were monitored by a real-time bioluminescence assay for mPer2 promoter-driven luciferase and HS element (HSE; HSF1-binding site)-driven luciferase activity, respectively. By an optimal duration HS pulse (43°C for approximately 30 minutes), circadian Per2 rhythm was observed in the whole mouse fibroblast culture, probably indicating the synchronization of the phases of each cell. This rhythm was preceded by an acute elevation in mPer2 and HSF1-mediated gene expression. Mutations in the two predicted HSE sites adjacent (one of them proximally) to the E-box in the mPer2 promoter dramatically abolished circadian mPer2 rhythm. Circadian Per2 gene/protein expression was not observed in HSF1-deficient cells. These findings demonstrate that HSF1 is essential to the synchronization of circadian rhythms by the HS pulse. Importantly, the interaction between HSF1 and BMAL1:CLOCK heterodimer, a central circadian transcription factor, was observed after the HS pulse. These findings reveal that even a short-term HS pulse can reset circadian rhythms and cause the HSF1-BMAL1:CLOCK interaction, suggesting the pivotal role of crosstalk between the mammalian circadian and HSR systems