Proximity labeling and identification of endogenous client proteins recruited to Y15-based artificial granules tethering a bait protein.

Protein clustering is a ubiquitous event in diverse cellular processes. Self-association of proteins triggers recruitment of downstream proteins to regulate cellular signaling. To investigate the interactions in detail, chemical biology tools to identify proteins recruited to defined assemblies are required. Here, we exploit an identification of proteins recruited in artificial granules (IPRAG) platform that combines intracellular Y15-based supramolecule construction with a proximity labeling method. We validated the IPRAG tool using Nck1 as a target bait protein. We constructed Nck1-tethering granules, labeled the recruited proteins with biotin, and analyzed them by LC-MS/MS. As a result, we successfully identified proteins that directly or indirectly interact with Nck1.

Selection of fluorescent biosensors against galectin-3 from an NBD-modified phage library displaying designed α-helical peptides.

Fluorescent biosensors are indispensable tools for molecular imaging, detection, and drug screening. Conventionally, fluorescent biosensors were constructed by incorporating fluorophores into ligands. Here, to develop ligand-independent biosensors, we demonstrated biosensor selection from a fluorophore-modified peptide phage library. In this library, the peptides were designed to form α-helical structures, and one cysteine, the probe modification site, was located at the center of four randomized residues on the same face of the helix. By conjugation with 4-nitrobenzoxadiazole (NBD), we constructed an NBD-modified phage library. We conducted selection against galectin-3 (Gal-3), a galactose-specific lectin associated with various biological events such as tumor metastasis and insulin resistance. After biopanning, we obtained NBD-modified peptides that selectively bind to Gal-3 from the library. The fluorescence intensity of the hit biosensors increased with the concentration of Gal-3, and this fluorescent response was visually observed.

Biofunctional supramolecular hydrogels fabricated from a short self-assembling peptide modified with bioactive sequences for the 3D culture of breast cancer MCF-7 cells.

Self-assembling peptides are a type of molecule with promise as scaffold materials for cancer cell engineering. We have reported a short self-assembling peptide, (FFiK)2, that had a symmetric structure connected via a urea bond. In this study, we functionalized (FFiK)2 by conjugation with various bioactive sequences for the 3D culture of cancer cells. Four sequences, RGDS and PHSRN derived from fibronectin and AG73 and C16 derived from laminin, were selected as bioactive sequences to promote cell adhesion, proliferation or migration. (FFiK)2, and its derivatives could co-assemble into supramolecular nanofibers displaying bioactive sequences and form hydrogels. MCF-7 cells were encapsulated in functionalized peptide hydrogels without significant cytotoxicity. Encapsulated MCF-7 cells proliferated under 3D culture conditions. MCF-7 cells proliferated with spheroid formation in hydrogels that displayed RGDS or PHSRN sequences, which will be able to be applied to drug screening targeting cancer stem cells. On the other hand, since MCF-7 cells migrated in a 3D hydrogel that displayed AG73, we could construct the metastatic model of breast cancer cells, which is helpful for the elucidation of breast cancer cells and drug screening against cancer cells under metastatic state. Therefore, functionalized (FFiK)2 hydrogels with various bioactive sequences can be used to regulate cancer cell function for tumor engineering and drug screening.

Affinity Control of Monosaccharide Conjugated Peptides against Lectins with a Set of Amino Acid Substitutions on α-Helical Structures.

Saccharides are well-known to play important roles in various biological events through specific interactions with target molecules such as carbohydrate-binding proteins (so-called lectins). Although characterization and identification of lectin molecules with saccharides are essential to understand biological events, they are still difficult due to weak interactions of saccharides, especially with monosaccharides. Herein, we demonstrate enhancement and control of monosaccharide affinity toward lectin proteins using chemical conjugation of monosaccharides with structurally regulated peptide and amino acid substitution. Thermodynamic analyses of the interactions by isothermal calorimetry measurements were performed to characterize the interactions between monosaccharide-conjugated peptide and the lectin molecules in detail. Conjugation with α-helical 16-mer short peptides drastically enhanced the affinity to lectins as compared with peptides with random coil structures, indicating that the α-helical peptide-based scaffold cooperatively interacted with lectins through additional interactions by suitable amino acids. Furthermore, suitable arrangement of the amino acids surrounding the monosaccharides on the α-helix afforded the conjugated peptides with varied affinities for two types of lectins. Our results indicate that the affinity of monosaccharide-conjugated peptides toward lectins is generally designable by appropriate conjugation of a simple monosaccharide with designed peptides, leading to the construction of a monosaccharide-modified peptide microarray toward high-throughput identification and/or screening of lectins in various biological events.

hDM2 protein-binding peptides screened from stapled α-helical peptide phage display libraries with different types of staple linkers.

Chemically modified peptide ligands were identified from α-helix peptide phage libraries with different types of staple linkers. The hDM2-protein was used as a representative target of protein-protein interactions to screen ligands for p53 binding sites in hDM2. Two types of staple linkers were used for the chemical modification of the peptide phage display libraries before affinity selection. The identified stapled peptides could bind to hDM2 competitively with the p53 peptide. The stapled peptide phage libraries developed in this study will improve the discovery of protein-protein interaction inhibitors through the synergistic effect of peptide units and staple linkers.

Osteoblastic differentiation on hydrogels fabricated from Ca2+-responsive self-assembling peptides functionalized with bioactive peptides.

We recently developed an amphiphilic peptide, E1Y9 (Ac-E-YEYKYEYKY-NH2), that self-assembles into nanofibers and forms a hydrogel in the presence of Ca2+ ion. Four E1Y9-derivatives (E1Y9-ALK, E1Y9-DGR, E1Y9-PRG and E1Y9-RGD) were designed as conjugates of E1Y9 with bioactive peptide sequences named as ALK (ALKRQGRTLYGF), DGR (DGRDSVAYG), PRG (PRGDSGYRGDS) and RGD (RGDS), respectively, and stimulated osteoblast cells growth as well as differentiation. In this study, E1Y9/E1Y9-derivative mixed hydrogels were constructed to serve as scaffolds for osteoblastic differentiation of MC3T3-E1 cells. E1Y9 and E1Y9-derivatives co-assembled into networked nanofibers and formed hydrogels in response to Ca2+ ion. The pre-osteoblast cell line MC3T3-E1 was cultured and differentiated on mixed hydrogels. An E1Y9/E1Y9-ALK mixed hydrogel exhibited the highest cell proliferation and differentiation activity among the peptide hydrogels. The peptide sequence ALK promoted expression of RUNX2 and osteopontin, a key transcription factor and bone tissue matrix protein, respectively, during the differentiation stage. During the later stage, localization of RUNX2 and osteopontin was regulated in the cytosol and extracellularly, respectively, indicating that the E1Y9/E1Y9-ALK mixed hydrogel controlled the differentiation of MC3T3-E1 cells. Thus, the E1Y9/E1Y9-ALK mixed hydrogel developed in this study showed potential for the culture and regulation of differentiation of osteoblast cells for bone regeneration.

Fluorescent and luminescent fusion proteins for analyses of amyloid beta peptide aggregation.

The amyloid beta (Aß) peptide is regarded as a causative agent of Alzheimer's disease. In this study, fluorescent and luminescent fusion proteins were constructed to analyze Aß aggregation. A system was developed to monitor changes in luminescence that provides information about Aß aggregation. In the presence of monomeric Aß, the fusion protein exhibits higher luminescence intensity, and the luminescence intensity is diminished after aggregation of the fusion protein and Aß. In contrast, the fluorescence is sustained in the presence of Aß. In the absence of Aß, the fusion protein self-aggregates, and its luminescence and fluorescence are quenched, thus decreasing the background fluorescence and enhancing the detection of Aß inside and outside the cells. The ratio of the luminescence intensity to the fluorescence intensity would allow the aggregation degrees of Aß to be distinguished. This study would be a promising method for analyzing the aggregation state of a particular amyloid protein/peptide (monomer, oligomer, or fibril), as well as the distribution of the amyloid protein/peptide within and at the cell surface, by using a single fusion protein. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Cell differentiation on disk- and string-shaped hydrogels fabricated from Ca(2+) -responsive self-assembling peptides.

We recently developed a self-assembling peptide, E1Y9, that self-assembles into nanofibers and forms a hydrogel in the presence of Ca(2+) . E1Y9 derivatives conjugated with functional peptide sequences derived from extracellular matrices (ECMs) reportedly self-assemble into peptide nanofibers that enhance cell adhesion and differentiation. In this study, E1Y9/E1Y9-IKVAV-mixed hydrogels were constructed to serve as artificial ECMs that promote cell differentiation. E1Y9 and E1Y9-IKVAV co-assembled into networked nanofibers, and hydrogels with disk and string shapes were formed in response to Ca(2+) treatment. The neuronal differentiation of PC12 cells was facilitated on hydrogels of both shapes that contained the IKVAV motifs. Moreover, long neurites extended along the long axis of the string-shaped gel, suggesting that the structure of hydrogels of this shape can affect cellular orientation. Thus, E1Y9 hydrogels can potentially be used as artificial ECMs with desirable bioactivities and shapes that could be useful in tissue engineering applications. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 476-483, 2016.

Cellular differentiation assessments by measuring the degree of cellular internalization and membrane adsorption using designed peptides.

We demonstrate examples of cellular differentiation assessments, including cellular neurite outgrowth and fat cell maturation, by measuring the degree of membrane adsorption or cellular internalization using designed peptides. Because changes in the cellular membrane and cytosol during differentiation were shown to influence membrane adsorption and cellular internalization, we could successfully evaluate the extent of differentiation simply like stain indicators.

Interaction of amphiphilic α-helical cell-penetrating peptides with heparan sulfate.

Cell-penetrating peptides (CPPs) are able to be taken up by cells and can deliver macromolecular cargos. However, the mechanism of this internalization is not yet fully understood. Recent theories suggest that the binding of cationic CPPs to negatively charged extracellular glycosaminoglycans, such as heparan sulfate (HS), is a possible mechanism of cellular uptake (CU). Our group has screened the CU activities of 54 systematically designed amphiphilic α-helical peptides in HeLa cells. Notably, a mutation in even a single residue significantly alters the CU ability of a peptide. To determine the structure-CU activity relationship of CPPs, four peptides, which contain a difference in one or two amino acids (i.e., Arg/Glu and Ala/Phe), were chosen from our CPP library to examine their interactions with HS. Fluorescence spectroscopy, isothermal titration calorimetry (ITC) and dynamic light scattering analysis indicated that the HS-binding affinities and HS-clustering abilities of the four CPPs correlated well with their CU activities in HeLa and A549 cells. The heat capacities of the CPPs, determined using ITC and binding free energy decomposition analyses in molecular dynamics simulations, revealed that electrostatic interactions were more dominant in the HS-binding processes of Arg-containing peptides in comparison to Glu-containing peptides, whereas hydrophobic contributions were the primary mode of interaction of Phe-containing peptides in comparison to Ala-containing peptides. Furthermore, it was implied that hydrophobic interactions may be more favourable than electrostatic interactions during the CU process.

Effects of Group 3 LEA protein model peptides on desiccation-induced protein aggregation.

Group 3 late embryogenesis abundant (G3LEA) proteins have amino acid sequences with characteristic 11-mer motifs and are known to reduce aggregation of proteins during dehydration. Previously, we clarified the structural and thermodynamic properties of the 11-mer repeating units in G3LEA proteins using synthetic peptides composed of two or four tandem repeats originating from an insect (Polypedilum vanderplanki), nematodes and plants. The purpose of the present study is to test the utility of such 22-mer peptides as protective reagents for aggregation-prone proteins. For lysozyme, desiccation-induced aggregation was abrogated by low molar ratios of a 22-mer peptide, PvLEA-22, derived from a P. vanderplanki G3LEA protein sequence. However, an unexpected behavior was noted for the milk protein, α-casein. On drying, the resultant aggregation was significantly suppressed in the presence of PvLEA-22 with its molar ratios>25 relative to α-casein. However, when the molar ratio was <10, aggregation occurred on addition of PvLEA-22 to aqueous solutions of α-casein. Other peptides derived from nematode, plant and randomized G3LEA protein sequences gave similar results. Such an anomalous solubility change in α-casein was shown to be due to a pH shift to ca. 4, a value nearly equal to the isoelectric point (pI) of α-casein, when any of the 22-mer peptides was mixed. These results demonstrate that synthetic peptides derived from G3LEA protein sequences can reduce protein aggregation caused both by desiccation and, at high molar ratios, also by pH effects, and therefore have potential as stabilization reagents.

Modification of a small ß-barrel protein, to give pseudo-amyloid structures, inhibits amyloid ß-peptide aggregation.

Aggregation of amyloid ß-peptide (Aß) is closely related to the pathogenesis of Alzheimer's disease (AD). Although much effort has been devoted to the construction of molecules that inhibit the aggregation of Aß1-42, high doses are needed for the inhibition of Aß aggregation in many cases. Previously, we reported that designed green fluorescent protein (GFP) analogues that gives pseudo-Aß ß-sheet structures can work as an aggregation inhibitor against Aß. To further test this design strategy, we constructed protein analogues that mimic Aß ß-sheet structures of amyloids by using insulin-like growth factor 2 receptor domain 11 (IGF2R-d11) as a scaffold. A designed protein, named IG11KK, which has a parallel configuration of Aß-like ß sheets, can bind more preferentially to oligomeric Aß1-42 than the monomer. Moreover, IG11KK suppressed the aggregation of Aß1-42 efficiently, even though lower concentrations of IG11KK than Aß were used. The aggregation kinetics of Aß in the presence of the designed proteins revealed that IG11KK can work as an inhibitor not only for the early to middle stages, but also in the latter stage of Aß aggregation owing to its favorable binding to oligomeric structures of Aß. The design strategy using ß-barrel proteins such as IGF2R-d11 and GFP is useful in generating excellent inhibitors of protein misfolding and amyloid formation.

Self-assembling peptide nanofibers promoting cell adhesion and differentiation.

There is an increasing need for the development of functional artificial extracellular matrices (ECMs) for tissue engineering. Recently, we have successfully designed a self-assembling peptide, named E1Y9, to construct functional ECMs. We describe here an enhancement of abilities of E1Y9 materials to promote cell adhesion and differentiation, using functional peptide sequences derived from natural extracellular matrix proteins. We designed functionalized self-assembling peptides, RGDS-conjugated E1Y9 (E1Y9-RGDS) and IKVAV-conjugated E1Y9 (E1Y9-IKVAV). E1Y9-RGDS and E1Y9-IKVAV formed peptide nanofibers in a similar manner to E1Y9, with ß-sheet secondary structures. Surfaces coated with peptide nanofibers displayed the higher bioactivities of E1Y9-RGDS for cell adhesion and E1Y9-IKVAV for cell differentiation than those of E1Y9, with the activities being dependent on the concentrations of the functional peptides. These functionalized peptides will be useful for the construction of functional ECMs in cell and tissue engineering.

Peptides as new smart bionanomaterials: molecular-recognition and self-assembly capabilities.

Biomolecules express exquisite properties that are required for molecular recognition and self-assembly on the nanoscale. These smart capabilities have developed through evolution and such biomolecules operate based on smart functions in natural systems. Recently, these remarkable smart capabilities have been utilized in not only biologically related fields, but also in materials science and engineering. A peptide-screening technology that uses phage-display systems has been developed based on this natural smart evolution for the generation of new functional peptide bionanomaterials. We focused on peptides that specifically bound to synthetic polymers. These polymer-binding peptides were screened by using a phage-display peptide library to recognize nanostructures that were derived from polymeric structural features and were utilized for possible applications as new bionanomaterials. We also focused on self-assembling peptides with ß-sheet structures that formed nanoscale, fibrous structures for applications in new bottom-up nanomaterials. Moreover, nanofiber-binding peptides were also screened to introduce the desired functionalities into nanofibers without the need for additional molecular design. Our approach to construct new bionanomaterials that employ peptides will open up excellent opportunities for the next generation of materials science and technology.

A monosaccharide-modified peptide phage library for screening of ligands to carbohydrate-binding proteins.

A monosaccharide-modified ß-loop peptide library displayed on phage has been constructed and used for the screening of glycopeptide ligands against a carbohydrate-binding protein. The ß-loop peptide library was designed and modified with a mannose derivative on phage. The glycopeptide ligands to concanavalin A (ConA), a mannose-binding protein, were obtained from the mannose-modified peptide phage library. The amino acids neighboring the mannose unit of glycopeptides not only reinforced the binding affinity but also gave diverse binding characteristics.

Systematic screening of the cellular uptake of designed alpha-helix peptides.

The cellular penetration (CP) activity of functional molecules has attracted significant attention as one of the most promising new approaches for drug delivery. In particular, cell-penetrating peptides (CPPs) have been studied extensively in cellular engineering. Because there have been few large-scale systematic studies to identify peptide sequences with optimal CP activity or that are suitable for further applications in cell engineering, such as cell-specific penetration and cell-selective culture, we screened and compared the cellular uptake (CU) activity of 54 systematically designed α-helical peptides in HeLa cells. Furthermore, the CU activity of 24 designed peptides was examined in four cell lines using a cell fingerprinting technique and statistical approaches. The CU activities in various cells depended on amino acid residues of peptide sequences as well as charge, α-helical content and hydrophobicity of the peptides. Notably, the mutation of a single residue significantly altered the CU ability of a peptide, highlighting the variability of cell uptake mechanisms. Moreover, these results demonstrated the feasibility of cell-selective culture by conducting cell-selective permeation and death in cultures containing two cell types. These studies may lead to further peptide library design and screening for new classes of CPPs with useful functions.

Gold nanoparticles conjugated with monosaccharide-modified peptide for lectin detection.

Gold nanoparticles (GNPs) conjugated with monosaccharide-modified peptides have been developed as optical probes for lectin detection. Mannose-modified peptides were designed and conjugated with GNPs. The GNPs with mannose-modified peptide showed remarkable red shift of absorption maximum due to the aggregation with concanavalin A (ConA), a mannose-binding lectin. The aggregation activity of glycopeptide-modified GNPs with ConA depended on the amino acid sequence around the mannose unit of glycopeptides.

Effects of Hydrophobic Residues on the Intracellular Self-Assembly of De Novo Designed Peptide Tags and Their Orthogonality.

Protein assemblies forming nano- to micro-sized structures underlie versatile biological events in living systems. For mimicking and engineering these protein assemblies through a bottom-up approach, self-assembling peptides (SAPs) that form nanofibril structures via ß-sheets serve as potential practical tags. Nevertheless, the development of SAP tags is still in its infancy, and insight into the relationship between peptide sequences and intracellular self-assembly is limited. In this study, we focused on hydrophobic residues in SAPs and examined the self-assembly of SAP-tagged superfolder GFPs (green fluorescent proteins) in COS-7 cells. Based on XEXK (X; hydrophobic amino acids: F, L, I, V, W, or Y) sequence units, we designed a panel of Xn peptides with different hydrophobic residues (X) and chain lengths (n). We observed that the self-assembly propensity, the size of the assemblies, the influence on protein denaturation, and the subcellular localization differed significantly depending on the hydrophobic amino acid. F9, L9, I7, and V13 peptides formed µm-scaled granules, W13 formed small oligomeric clusters in the cytoplasm, and Y15 formed assemblies in the nucleus. In addition, we investigated the orthogonality of their interaction. Strikingly, W13- and Y15-tagged proteins interacted independently and formed two distinct assemblies in cells. Herein, we have demonstrated the great opportunities for rationalizing artificial protein assemblies and orthogonal structures in an intracellular context using the designed SAPs.

Pattern enrichment analysis for phage selection of stapled peptide ligands.

Phage display is the most widely used technique to discover de novo peptides that bind to target proteins. However, it is associated with some challenges such as compositional bias. In this study, to overcome these difficulties, we devised a 'pattern enrichment analysis.' In this method, two samples (one obtained by affinity selection, the other simply amplified without selection) are prepared, and the two sequence datasets read on next-generation sequencer are compared to find the three-residue pattern most enriched in the selected sample. This allows us to compare two sequence datasets with high coverage and facilitates the identification of peptide sequences and the key residues for binding. We also demonstrated that this approach in the combination with structured peptide libraries allowed spatial mapping of the enriched sequence patterns. Here, we prepared a phage library displaying chemically stapled helical peptides with the X1C2X3X4X5X6X7X8C9X10 sequence, where X is any amino acid. To validate our method, we performed screening against the HDM2 protein. The results showed that the hydrophobic residues (Phe, Tyr, Trp and Leu) that are key to interactions with HDM2 were clearly identified by the pattern enrichment analysis. We also performed selection targeting the SARS-CoV-2 spike RBD in the same manner. The results showed that similar patterns were enriched among the hit peptides that inhibited the protein-protein interaction.

Cell fingerprint patterns using designed α-helical peptides to screen for cell-specific toxicity.

We conducted cell-based cytotoxicity screening of a 101-membered α-helical peptide library using cell fingerprints (CFPs). The CFP data suggested that there is a relationship between cytotoxicity and peptide characteristics, such as hydrophobicity, charge, and amino acid composition. In spite of the small size of the library used in this study, several peptides demonstrated cell-specific toxicity. The strategy of combining a designed peptide library with CFP thus shows real promise for peptide-based screening with cells.