Peptide array screening with anti-GLP-1 monoclonal antibody: Discovery of cysteine-containing DPP-IV inhibitory peptides.

Inhibition of dipeptidyl peptidase IV (DPP-IV) is an effective pharmacotherapy for the management of type 2 diabetes. Recent findings have suggested that various dietary proteins can serve as precursors to peptides that inhibit DPP-IV. Although several DPP-IV inhibitory peptides derived from food materials have been reported, more effective inhibitory peptides remain to be discovered. This study aimed to identify potent DPP-IV inhibitory peptides that earlier approaches had overlooked by employing a screening method that combined peptide arrays and neutralizing antibodies. Octa-peptides covering the complete amino acid sequences of four casein proteins and two whey proteins were synthesized on arrays via a solid-phase method. These peptides were then reacted with a monoclonal antibody specifically engineered to recognize glucagon-like peptide 1 (GLP-1), a substrate of DPP-IV. The variable region of the anti-GLP-1 monoclonal antibody is utilized to mimic the substrate-binding region of DPP-IV, enabling the antibody to bind to peptides that interact with DPP-IV. Based on this feature, 26 peptides were selected as DPP-IV inhibitory peptide candidates, 11 of which showed strong DPP-IV inhibitory activity. Five of these peptides consistently contained cysteines positioned two to four residues from the N-terminus. Treatment with disulfide formation decreased the DPP-IV inhibitory activity of these cysteine-containing peptides, while the inhibitory activity of α-lactalbumin hydrolysates increased with reducing treatment. These results revealed that the thiol group is important for DPP-IV inhibitory activity. This study provides a useful screen for DPP-IV inhibitory peptides and indicates the importance of reductive cysteine residues within DPP-IV inhibitory peptides.

Overexpression of TSPAN8 in consensus molecular subtype 3 colorectal cancer.

BACKGROUND: Recently, consensus molecular subtypes (CMSs) have been proposed as a robust transcriptome-based classification system for colorectal cancer (CRC). Tetraspanins (TSPANs) are transmembrane proteins. They have been associated with the development of numerous malignancies, including CRC, through their role as "master organizers" for multi-molecular membrane complexes. No previous study has investigated the correlation between TSPANs and CMS classification. Herein, we investigated the expression of TSPANs in patient-derived primary CRC tissues and their CMS classifications. METHODS: RNA samples were derived from primary CRC tissues (n = 100 patients diagnosed with colorectal adenocarcinoma) and subjected to RNA sequencing for transcriptome-based CMS classification and TSPAN-relevant analyses. Immunohistochemistry (IHC) and immunofluorescence (IF) stains were conducted to observe the protein expression level. To evaluate the relative biological pathways, gene-set enrichment analysis was performed. RESULTS: Of the highly expressed TSPAN genes in CRC tissues (TSPAN8, TSPAN29, and TSPAN30), TSPAN8 was notably overexpressed in CMS3-classified primary tissues. The overexpression of TSPAN8 protein in CMS3 CRC was also observed by IHC and IF staining. As a result of gene-set enrichment analysis, TSPAN8 may potentially play a role in organizing signaling complexes for kinase-based metabolic deregulation in CMS3 CRC. CONCLUSIONS: The present study reports the overexpression of TSPAN8 in CMS3 CRC. This study proposes TSPAN8 as a subtype-specific biomarker for CMS3 CRC. This finding provides a foundation for future CMS-based studies of CRC, a complex disease and the second leading cause of cancer mortality worldwide.

Enantioselective Detection of Gaseous Odorants with Peptide-Graphene Sensors Operating in Humid Environments.

Replicating the sense of smell presents an ongoing challenge in the development of biomimetic devices. Olfactory receptors exhibit remarkable discriminatory abilities, including the enantioselective detection of individual odorant molecules. Graphene has emerged as a promising material for biomimetic electronic devices due to its unique electrical properties and exceptional sensitivity. However, the efficient detection of nonpolar odor molecules using transistor-based graphene sensors in a gas phase in environmental conditions remains challenging due to high sensitivity to water vapor. This limitation has impeded the practical development of gas-phase graphene odor sensors capable of selective detection, particularly in humid environments. In this study, we address this challenge by introducing peptide-functionalized graphene sensors that effectively mitigate undesired responses to changes in humidity. Additionally, we demonstrate the significant role of humidity in facilitating the selective detection of odorant molecules by the peptides. These peptides, designed to mimic a fruit fly olfactory receptor, spontaneously assemble into a monomolecular layer on graphene, enabling precise and specific odorant detection. The developed sensors exhibit notable enantioselectivity, achieving a remarkable 35-fold signal contrast between d- and l-limonene. Furthermore, these sensors display distinct responses to various other biogenic volatile organic compounds, demonstrating their versatility as robust tools for odor detection. By acting as both a bioprobe and an electrical signal amplifier, the peptide layer represents a novel and effective strategy to achieve selective odorant detection under normal atmospheric conditions using graphene sensors. This study offers valuable insights into the development of practical odor-sensing technologies with potential applications in diverse fields.

Osmotic stress induces the formation of migrasome-like vesicles.

Migrasomes are extracellular vesicles that form on the retraction fibers of migrating cells. In this study, we report the formation of migrasome-like vesicles enriched in tetraspanin 4 and containing cytoplasmic components in response to hypoosmotic stress. When migrating cells were subjected to hypoosmotic stress, vesicles with a size distribution of 0.5 to 2 µm formed on the retraction fibers, and vanished in a few minutes. The vesicles are rich in cholesterol, and their number was reduced when cells were pretreated with lipoprotein-deficient serum. The formation of migrasome-like vesicles upon hypoosmotic stress may provide biophysical cues regarding the cellular response to this external stimulus in cells and tissues.

Screening of novel DPP-IV inhibitory peptides derived from bovine milk proteins using a peptide array platform.

Dipeptidyl peptidase IV (DPP-IV) has become an important target in the prevention and treatment of diabetes. Although many DPP-IV inhibitory peptides have been identified by a general approach involving the repeated fractionation of food protein hydrolysates, the obtained results have been dependent on the content of each peptide and fractionation conditions. In the present study, a peptide array that provides comprehensive assays of peptide sequences was used to identify novel DPP-IV inhibitory peptides derived from bovine milk proteins; these peptides were then compared with those identified using the general approach. While the general approach identified only known peptides that were abundant in the hydrolysate, the peptide array-based approach identified 10 novel DPP-IV inhibitory peptides, all of which had proline at the second residue from the N-terminus. The proper or combined use of these two approaches, which have different advantages, will enable the efficient development of novel bioactive foods and drugs.

Screening of EWI-2-Derived Peptides for Targeting Tetraspanin CD81 and Their Effect on Cancer Cell Migration.

CD81, a transmembrane protein belonging to the tetraspanin family, has recently been suggested as a therapeutic target for cancers. Here, we screened peptides that bind to the tetraspanin CD81 protein, and evaluated their inhibitory activity in cancer cell migration. To screen for CD81-binding peptides (CD81-BP), a peptide array membrane was prepared from the amino acid sequence of the EWI-2 protein, a major partner of CD81, before binding to fluorescently labeled CD81. As a result, four candidate CD81-BPs were identified and characterized. In particular, the CFMKRLRK peptide (called P152 in this study) was found to be the best candidate that preferentially binds to the extracellular loop of CD81, with an estimated dissociation constant of 0.91 µM. Since CD81 was reported to promote cancer cell migration, an initial step in metastasis, the Boyden chamber assay, was next performed to assess the effect of CD81-BP candidates on the migration of MDA-MB-231 human breast cancer cells. Interestingly, our result indicated that P152 could suppress MDA-MB-231 cell migration at the level comparable to that of an anti-human CD81 antibody (5A6). Thus, we propose these CD81-BPs with the anti-migration property against cancer cells for the development of novel therapeutic strategies.

Volatile Organic Compound Detection by Graphene Field-Effect Transistors Functionalized with Fly Olfactory Receptor Mimetic Peptides.

An olfactory receptor mimetic peptide-modified graphene field-effect transistor (gFET) is a promising solution to overcome the principal challenge of low specificity graphene-based sensors for volatile organic compound (VOC) sensing. Herein, peptides mimicking a fruit fly olfactory receptor, OR19a, were designed by a high-throughput analysis method that combines a peptide array and gas chromatography for the sensitive and selective gFET detection of the signature citrus VOC, limonene. The peptide probe was bifunctionalized via linkage of a graphene-binding peptide to facilitate one-step self-assembly on the sensor surface. The limonene-specific peptide probe successfully achieved highly sensitive and selective detection of limonene by gFET, with a detection range of 8-1000 pM, while achieving facile sensor functionalization. Taken together, our target-specific peptide selection and functionalization strategy of a gFET sensor demonstrates advancement of a precise VOC detection system.

Designable peptides on graphene field-effect transistors for selective detection of odor molecules.

Gas sensing based on graphene field-effect transistors (GFETs) has gained broad interest due to their high sensitivity. Further progress in gas sensing with GFETs requires to detection of various odor molecules for applications in the environmental monitoring, healthcare, food, and cosmetic industries. To develop the ubiquitous odor-sensing system, establishing an artificial sense of smell with electronic devices by mimicking olfactory receptors will be key. Although the application of olfactory receptors to GFETs is straightforward for odor sensing, synthetic molecules with a similar function to olfactory receptors would be desirable to realize the robust performance of sensing. In this work, we designed three new peptides consisting of two domains: a bio-probe to the target molecules and a molecular scaffold. These peptides were rationally designed based on a motif sequence in olfactory receptors and self-assembled into a molecular thin film on GFETs. Limonene, methyl salicylate, and menthol were employed as representative odor molecules of plant flavors to demonstrate the biosensing of odor molecules. The conductivity change of GFETs against the binding to odor molecules with various concentrations and the dynamic response revealed a distinct signature of three different peptides against individual species of the target molecules. The kinetic response of each peptide exhibited characteristic time constants in the adsorption and desorption process, also supported by the principal component analysis. Our demonstration of the graphene odor sensors with the designed peptides opens a way to establish future peptide-array sensors with multi-sequence of peptide, realizing an odor sensing system with higher selectivity.

A peptide binding to the tetraspanin CD9 reduces cancer metastasis.

As an organizer of multi-molecular membrane complexes, the tetraspanin CD9 has been implicated in a number of biological processes, including cancer metastasis, and is a candidate therapeutic target. Here, we evaluated the suppressive effects of an eight-mer CD9-binding peptide (CD9-BP) on cancer cell metastasis and its mechanisms of action. CD9-BP impaired CD9-related functions by adversely affecting the formation of tetraspanin webs-networks composed of CD9 and its partner proteins. The anti-cancer metastasis effect of CD9-BP was evidenced by the in vitro inhibition of cancer cell migration and invasion as well as exosome secretion and uptake, which are essential processes during metastasis. Finally, using a mouse model, we showed that CD9-BP reduced lung metastasis in vivo. These findings provide insight into the mechanism by which CD9-BP inhibits CD9-dependent functions and highlight its potential application as an alternative therapeutic nano-biomaterial for metastatic cancers.

Non-competitive fluorescence polarization immunosensing for CD9 detection using a peptide as a tracer.

This paper is the first report of a non-competitive fluorescence polarization immunoassay (NC-FPIA) using a peptide as a tracer. The NC-FPIA can easily and quickly quantify the target after simply mixing them together. This feature is desirable for point-of-need applications such as clinical diagnostics, infectious disease screening, on-site analysis for food safety, etc. In this study, the NC-FPIA was applied to detect CD9, which is one of the exosome markers. We succeeded in detecting not only CD9 but also CD9 expressing exosomes derived from HeLa cells. This method can be applied to various targets if a tracer for the target can be prepared, and expectations are high for its future uses.

Sensitive and specific capture of polystyrene and polypropylene microplastics using engineered peptide biosensors.

Owing to increased environmental pollution, active research regarding microplastics circulating in the ocean has attracted significant interest in recent times. Microplastics accumulate in the bodies of living organisms and adversely affect them. In this study, a new method for the rapid detection of microplastics using peptides was proposed. Among the various types of plastics distributed in the ocean, polystyrene and polypropylene were selected. The binding affinity of the hydrophobic peptides suitable for each type of plastic was evaluated. The binding affinities of peptides were confirmed in unoxidized plastics and plasma-oxidized plastics in deionised or 3.5% saline water. Also, the detection of microplastics in small animals' intestine extracts were possible with the reported peptide biosensors. We expect plastic-binding peptides to be used in sensors to increase the detection efficiency of microplastics and potentially help separate microplastics from seawater.

Surface glycan targeting for cancer nano-immunotherapy.

Cancer immunotherapy is an emerging therapeutic strategy for cancer treatment. Most of the immunotherapeutics approved by the FDA regulate the innate immune system and associated immune cell activity, with immune check inhibitors in particular having transformed the field of cancer immunotherapy due to their significant clinical potential. However, previously reported immunotherapeutics have exhibited undesirable side effects, including autoimmune toxicity and inflammation. Controlling these deleterious responses and designing therapeutics that can precisely target specific regions are thus crucial to improving the efficacy of cancer immunotherapies. Recent studies have reported that cancer cells employ glycan-immune checkpoint interactions to modulate immune cell activity. Thus, the recognition of cancer glycan moieties such as sialoglycans may improve the anticancer activity of immune cells. In this review, we discuss recent advances in cancer immunotherapies involving glycans and glycan-targeting technologies based on nanomaterial-assisted local delivery systems.

Enrichment of membrane curvature-sensing proteins from Escherichia coli using spherical supported lipid bilayers.

Bacteria display dynamically organized curved membrane structures, especially during cell division. The importance of membrane curvature-sensing (MCS) proteins for the recognition and regulation of biological membrane morphologies has predominately been investigated in eukaryotic cells. Recently, a technique for screening MCS proteins from solutions that contain peripheral membrane proteins was developed, and MCS protein candidates were identified from mammalian cells. The technique uses differently sized spherical supported lipid bilayers (SSLBs), which consist of spherical SiO2 particles covered with a lipid bilayer. To discriminate between proteins possessing the MCS property, SSLBs with the same surface area were used in a comparative sedimentation assay with shotgun proteome analysis. In this study, to prove that the technique could be applied to other samples, MCS proteins in Escherichia coli were investigated. Through a comparative proteomic study, 35 and 47 proteins were enriched as candidate MCS proteins preferentially bound to SSLBs of 100 nm and 1000 nm, respectively. Among the identified MCS candidate proteins, FtsZ and SecA were further examined for their MCS properties using the two SSLB sizes, which revealed a high binding affinity for the low membrane curvature (large SSLB). This is the first study to explore MCS proteins in prokaryotic cells and the MCS property of the SecA protein. The results demonstrate a method to enrich MCS proteins that could be utilized to better elucidate membrane dynamics and protein function expression on curved membrane structures in prokaryotic cells.

Peptide-modified substrate enhances cell migration and migrasome formation.

Extracellular vesicles (EVs) are cell-to-cell communication tools. Migrasomes are recently discovered microscale EVs formed at the rear ends of migrating cells, and thus are suggested to be involved in communicating with neighboring cells. In cell culture, peptide scaffolds on substrates have been used to demonstrate cellular function for regenerative medicine. In this study, we evaluated peptide scaffolds, including cell penetrating, virus fusion, and integrin-binding peptides, for their potential to enable the formation of migrasome-like vesicles. Through structural and functional analyses, we confirmed that the EVs formed on these peptide-modified substrates were migrasomes. We further noted that the peptide interface comprising cell-penetrating peptides (pVEC and R9) and virus fusion peptide (SIV) have superior properties for enabling cell migration and migrasome formation than fibronectin protein, integrin-binding peptide (RGD), or bare substrate. This is the first report of migrasome formation on peptide-modified substrates. Additionally, the combination of 95% RGD and 5% pVEC peptides provided a functional interface for effective migrasome formation and desorption of cells from the substrate via a simple ethylenediaminetetraacetic acid treatment. These results provide a functional substrate for the enhancement of migrasome formation and functional analysis.

Synthesis of near-infrared absorbing triangular Au nanoplates using biomineralisation peptides.

Triangular Au nanoplates (TrAuNPls) possessing strong plasmonic properties can be used as photothermal agents in cancer therapy. However, the controlled preparation of such morphologies typically requires harsh synthetic conditions. Biomolecules offer an alternative route to developing biocompatible synthetic protocols. In particular, peptides offer a novel route for inorganic synthesis under ambient conditions. Herein, using the previously isolated peptide, ASHQWAWKWE, for Au nanoparticle (AuNP) synthesis, the conditions for preparing TrAuNPls via a one-pot synthetic process of mixing HAuCl4 and peptides at room temperature were investigated to effectively obtain particles possessing near-infrared absorbance for non-invasive optical diagnosis and phototherapy. By adjusting the peptide concentration, the size and property of TrAuNPls were controlled under neutral pH conditions. The synthesised particles showed potential as photothermal therapeutic agents in vitro. In addition, peptide characterisation using B3 derivatives revealed the importance of the third amino acid histidine in morphological regulation and potential circular Au nanoplates (AuNPl) synthesis with ASEQWAWKWE and ASAQWAWKWE peptides. These findings provide not only an easy and green synthetic method for TrAuNPls and circular AuNPls, but also some insight to help elucidate the regulation of peptide-based nanoparticle synthesis for use in cancer therapy. STATEMENT OF SIGNIFICANCE: Biological molecules have received increasing attention as a vehicle to synthesise inorganic materials with specific properties under ambient conditions; particularly, short peptides have the potential to control the synthesis of nanoscale materials with tailored functions. Here, the application of a previously isolated peptide was assessed in synthesising Au nanoparticles containing decahedral and triangular nanoplates with near-infrared absorbance. The size and absorbance peaks of the triangular nanoplates observed were peptide concentration-dependent. In addition, these fine-tuned triangular nanoplates exhibited potential as a phototherapeutic agent. Moreover, the peptide derivatives indicated the possibility of synthesising circular nanoplates. These findings may offer insight into development of new techniques for synthesising functional nanoparticles having biological applications using non-toxic molecules under mild conditions stituted in the original B3 peptide is underlined.

Inhibition of cancer-cell migration by tetraspanin CD9-binding peptide.

A CD9-binding peptide (RSHRLRLH), screened from EWI-2, was characterized, and its effect on cellular migration and invasion was evaluated. As CD9 protein is overexpressed in cancer cells and plays an important role in cellular migration, the CD9-binding peptide preferentially inhibited the migration of cancer cells. Unlike conventional antiproliferative drugs, this CD9-binding peptide is promising as a novel precision antimigratory agent for cancer therapeutics.

Microfluidic-based capture and release of cancer-derived exosomes via peptide-nanowire hybrid interface.

Cancer-derived circulating exosomes or nanoscale extracellular vesicles are emerging biomarkers for disease detection and treatment because of their cell-specific constituents and unique intercellular pathways. For efficient exosome isolation from bio-fluids, the design of high-affinity nanointerfaces is of great importance in the development of miniaturized systems for the collection of exosomes. Herein, we report peptide-functionalized nanowires as a biorecognition interface for the capture and release of cancer-derived exosomes within a microfluidic channel. Based on the amino-acid sequence of EWI-2 protein, a partial peptide that bound to the CD9 exosome marker and thus targeted cancer exosomes was screened. Linkage of the exosome-targeting peptide with a ZnO-binding sequence allowed one-step and reagent-free peptide modification of the ZnO nanowire array. As a result of peptide functionalization, the exosome-capturing ability of ZnO nanowires was significantly improved. Furthermore, the captured exosomes could be subsequently released from the nanowires under a neutral salt condition for downstream applications. This engineered surface that enhances the nanowires' efficiency in selective and controllable collection of cancer-derived exosomes provides an alternative foundation for developing microfluidic platforms for exosome-based diagnostics and therapeutics.

Proteomic Exploration of Membrane Curvature Sensors Using a Series of Spherical Supported Lipid Bilayers.

Membrane curvature-sensing (MCS) proteins recognize and regulate the morphologies of biological membranes. As these proteins lack characteristic sequence motifs in their primary structure, they are not instantly recognizable by genomic databases. Overcoming this technological challenge toward the agile identification of new proteins can promote the elucidation of membrane morphological regulation. Here, for the selective identification of MCS proteins, comparative proteomic analysis was performed using different sizes of the spherical supported lipid bilayer (SSLB), which consists of spherical SiO2 particles covered with a lipid bilayer. Because of the presence of SiO2 core, the curvature of the surrounding membrane is well-controlled and stable even on a micron scale. To prove this concept, known membrane curvature-sensing protein domains, Bin/Amphiphysin/Rvs (BAR) and Epsin N-terminal homology (ENTH), were evaluated by performing a binding assay using SSLBs, and the preferential binding to the highly curved membrane was confirmed. Peripheral membrane proteins obtained from normal human dermal fibroblast (NHDF) and human breast cancer (MDA-MB-231) cells were used in shotgun proteomic analysis, and 786 and 949 proteins were identified from SSLBs as lipid membrane binders, respectively. Statistical quantitative analyses of proteins detected from each SSLB with a different size revealed 118 candidate proteins, including 23 proteins unique to MDA-MB-231 cells, as membrane curvature sensors, including some previously reported curvature sensors. Functional clustering analysis based on the KEGG orthology database revealed that the protein-binding property to specific high or low membrane curvature correlated with their functions. Further investigation of candidate proteins will lead to the identification of new MCS proteins as well as cancer biomarkers.

Machine learning-driven electronic identifications of single pathogenic bacteria.

A rapid method for screening pathogens can revolutionize health care by enabling infection control through medication before symptom. Here we report on label-free single-cell identifications of clinically-important pathogenic bacteria by using a polymer-integrated low thickness-to-diameter aspect ratio pore and machine learning-driven resistive pulse analyses. A high-spatiotemporal resolution of this electrical sensor enabled to observe galvanotactic response intrinsic to the microbes during their translocation. We demonstrated discrimination of the cellular motility via signal pattern classifications in a high-dimensional feature space. As the detection-to-decision can be completed within milliseconds, the present technique may be used for real-time screening of pathogenic bacteria for environmental and medical applications.

Peptide array-based inhibition ELISA for evaluating antigenicity in infant formulas.

With increased awareness among consumers regarding food safety and security, food allergen control has become an indispensable requirement in the food industry. Although several methods for detecting allergens in food products are available, highly sensitive techniques are required. In this study, we developed a technique named as peptide array-based inhibition enzyme-linked immunosorbent assay (ELISA), Pep-iEIA, for evaluating antigenicity and detecting cow's milk antigen in infant formula products, using a peptide array consisting of a series of overlapping peptides found in allergenic milk proteins. Pep-iEIA was used to examine five cow's milk-based infant formulas with different degrees of hydrolyzation, and the assay offered both more sensitive detection and detailed analysis of remaining antigenic peptides in allergen compared to conventional ELISA. The antigenicity level of the allergenic peptides identified using Pep-iEIA was confirmed by surface plasmon resonance assay. We believe that Pep-iEIA will be highly useful for antigenicity evaluation of dairy products consumed by infants and patients with cow's milk allergy.