Functional evaluation of an electrophilic focused library to identify a covalent inhibitor against intrinsically disordered circadian clock transcription factors.

In vitro screening of a focused library of compounds containing an electrophilic warhead identified N-chloroacetyl-bis(trifluoromethyl)aniline derivative 15 as a potent inhibitor of BMAL1-CLOCK heterodimer binding to an E-box DNA fragment. Kinetic analysis of thiol-reactivity demonstrated that iodoacetamide and structurally related 20 are significantly more reactive than or equally reactive as 15, respectively, whereas none inhibited BMAL1-CLOCK interaction with the E-box DNA fragment. These results suggest that 15 binds and reacts with a specific nucleophilic residue. This low-molecular-weight compound may serve as a useful lead for further development of BMAL1-CLOCK inhibitors.

Recognition of G-quadruplex RNA by a crucial RNA methyltransferase component, METTL14.

N6-methyladenosine (m6A) is an important epitranscriptomic chemical modification that is mainly catalyzed by the METTL3/METTL14 RNA methyltransferase heterodimer. Although m6A is found at the consensus sequence of 5'-DRACH-3' in various transcripts, the mechanism by which METTL3/METTL14 determines its target is unclear. This study aimed to clarify the RNA binding property of METTL3/METTL14. We found that the methyltransferase heterodimer itself has a binding preference for RNA G-quadruplex (rG4) structures, which are non-canonical four-stranded structures formed by G-rich sequences, via the METTL14 RGG repeats. Additionally, the methyltransferase heterodimer selectively methylated adenosines close to the rG4 sequences. These results suggest a possible process for direct recruitment of METTL3/METTL14 to specific methylation sites, especially near the G4-forming regions. This study is the first to report the RNA binding preference of the m6A writer complex for the rG4 structure and provides insights into the role of rG4 in epitranscriptomic regulation.

The past, present, and future of artificial zinc finger proteins: design strategies and chemical and biological applications.

Zinc finger proteins are abundant in the human proteome and are responsible for a variety of functions. The domains that constitute zinc finger proteins are compact spherical structures, each comprising approximately 30 amino acid residues, but they also have precise molecular factor functions: zinc binding and DNA recognition. Due to the biological importance of zinc finger proteins and their unique structural and functional properties, many artificial zinc finger proteins have been created and are expected to improve their functions and biological applications. In this study, we review previous studies on the redesign and application of artificial zinc finger proteins, focusing on the experimental results obtained by our research group. In addition, we systematically review various design strategies used to construct artificial zinc finger proteins and discuss in detail their potential biological applications, including gene editing. This review will provide relevant information to researchers involved or interested in the field of artificial zinc finger proteins as a potential new treatment for various diseases.

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.

Artificial Nanocage Formed via Self-Assembly of ß-Annulus Peptide for Delivering Biofunctional Proteins into Cell Interiors.

Nanocarriers that deliver functional proteins to cell interiors are an attractive platform for the intracellular delivery of intact proteins without further modification, with in vivo compatibility. Development of efficient methods for cargo protein encapsulation and release in recipient cell cytosol is needed. Herein, we assess the feasibility of the abovementioned requirements using a protein nanocage (artificial nanocage) without compromising the structure and functions of the original protein and allowing for design flexibility of the surfaces and interiors. The protein nanocage formed via the self-assembly of the ß-annulus peptide (24-amino acid peptide) in water was used as a model framework. The nitrilotriacetic acid moiety was displayed on the nanocage lumen for effective encapsulation of hexahistidine-tagged proteins in the presence of Ni2+, and the amphiphilic cationic lytic peptide HAad was displayed on a nanocage surface to attain cell permeability. Successful intracellular delivery of cargo proteins and targeting of cytosolic proteins by a nanobody were achieved, indicating the validity of the approach employed in this study.

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.

L17ER4: A cell-permeable attenuated cationic amphiphilic lytic peptide.

We have developed a series of attenuated cationic amphiphilic lytic (ACAL) peptides that can efficiently bring immunoglobulin G (IgG) and other functional proteins into cells. Delivery is generally achieved through the coadministration of ACAL peptides with cargo proteins. However, conjugation of ACAL peptides with cargos may be a promising approach for in vivo application to link in vivo outcomes of ACAL peptides and cargos. This study describes the creation of a new cell-permeable ACAL peptide, L17ER4. L17E is an optimized prototype of ACAL peptides previously developed in our laboratory for efficient delivery of IgGs into cells. Delivery was improved by functionalizing L17E with a tetra-arginine (R4) tag. Compared to the use of R8, a representative cell-penetrating peptide with high intracellular delivery efficacy, conjugation with L17ER4 afforded approximately four-fold higher cellular uptake of model small-molecule cargos (fluorescein isothiocyanate and HiBiT peptide). L17ER4 was also able to deliver proteins to cells. Fused with L17ER4, Cre recombinase was delivered into cells. Intracerebroventricular injection of Cre-L17ER4 into green red reporter mice, R26GRR, led to significant in vivo gene recombination in ependymal cells, suggesting that L17ER4 may be used as a cell-penetrating peptide for delivering protein therapeutics into cells in vivo.

Use of homoarginine to obtain attenuated cationic membrane lytic peptides.

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.

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.

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.

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.

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

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.

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

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.

Nested PUF Proteins: Extending Target RNA Elements for Gene Regulation.

RNA-binding proteins recognizing unique sequences within large transcriptomes serve as a powerful tool to control RNA metabolism. Pumilio and fem-3 mRNA-binding factor (PUF) proteins are considered good candidates for such tools, because they are typically composed of eight highly homologous repeat segments and can be designed to recognize arbitrary 8 nt RNA sequences. However, a specific 8 nt RNA sequence is found at multiple sites in various RNAs in the transcriptome, making it difficult to specifically target a single RNA. Designer PUF proteins recognizing longer RNA sequences should achieve more selective binding. Here, we propose an approach for creating 16-repeat PUFs capable of targeting a single, unique mRNA in the transcriptome. Our design is simple and involves either the tandem alignment of two PUF segments or the nesting of one PUF segment within another. Designed 16-repeat PUFs bound to the target RNA sequence without partial recognition derived from the original 8-repeat PUF. Furthermore, based on our strategy, expression of an endogenous mRNA was selectively and effectively modulated, demonstrating the applicability of 16-repeat PUF proteins for regulating endogenous RNA metabolism.

Sequence-specific 5mC detection in live cells based on the TALE-split luciferase complementation system.

We established a method for converting TALE-DNA binding to luminescence, by combining a TALE and a split luciferase system. Furthermore, using a methylation-sensitive TALE, sequence-specific 5mC detection of genomic DNA was achieved in live cells. This study provides a new strategy for exploring the biological functions of 5mC.

Calmodulin EF-hand peptides as Ca2+ -switchable recognition tags.

Calmodulin is a representative calcium-binding protein comprised of four Ca2+ -binding motifs with a helix-loop-helix structure (EF-hands). In this study, we clarified the potential of peptide segments derived from the third and fourth EF-hands (EF3 and EF4) to act as recognition tags. Through an analysis of the mode of disulfide formation among cysteines inserted at the N- or C-terminus of these peptide segments, EF3 and EF4 peptides were suggested to form a heterodimer with a topology similar to that in the wild-type protein. Heterodimer formation was shown to be a function of the Ca2+ concentration, suggesting that these structures may be used as Ca2+ -switchable recognition tags. An example of an "EF-tag" system involving the membrane fusion of liposomes decorated with EF3 and EF4 peptides is presented. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2016.

Preparation of peptide thioesters from naturally occurring sequences using reaction sequence consisting of regioselective S-cyanylation and hydrazinolysis.

The vital roles of peptide/protein thioesters in protein chemistry, including chemical or semi-synthesis of proteins, have encouraged studies on the development of methods for the preparation of such chemical units. Biochemical protocols using intein or sortase have proved to be useful in protein chemistry as methods suitable for naturally occurring sequences, including recombinant proteins. Although chemical protocols are potential options for thioester preparation, only a few are applicable to naturally occurring sequences, because standard chemical protocols require an artificial chemical device for producing thioesters. In this context, the chemical preparation of thioesters based on a reaction sequence consisting of regioselective S-cyanylation and hydrazinolysis was investigated. Regioselective S-cyanylation, which is required for cysteine-containing thioesters, was achieved with the aid of a zinc-complex formation of a CCHH-type zinc-finger sequence. Free cysteine residues that are not involved in complex formation were selectively protected with a 6-nitroveratryl group followed by S-cyanylation of the zinc-binding cysteine. Hydrazinolysis of the resulting S-cyanopeptide and subsequent photo-removal of the 6-nitroveratryl group yielded the desired peptide hydrazide, which was then converted to the corresponding thioester. The generated thioester was successfully used in N-to-C-directed one-pot/sequential native chemical ligation using an N-sulfanylethylanilide peptide to give a 64-residue peptide toxin. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 531-546, 2016.

Creating a TALE protein with unbiased 5'-T binding.

Transcription activator-like effectors (TALEs) are convenient tools for genome engineering at specific genomic sites. However, their use is constrained because most TALE binding sites are preceded by a highly conserved 5' terminal T nucleotide (5'-T). To remove the 5'-T constraint, we substituted tryptophan 232 in the repeat-1 loop region of the dHax3 N-terminal domain for other amino acids. Furthermore, we randomized four amino acid residues of the hairpin loop region of repeat-1. Although point mutation was insufficient to remove the 5'-T constraint, directed evolution from the randomized library yielded repeat-1 mutants with unbiased targeting sites for 5'-bases. Our result indicates that the repeat-1 loop region of dHax3 is important for 5'-base accommodation, and that molecular evolution of repeat-1 of TALEs is an efficient strategy to remove the 5'-T constraint and thus allow targeting of any DNA sequences.