Peptide Research Articles

Amyloid β fragments that suppress oligomers but not fibrils are cytoprotective.

Amyloid β fragments that suppress oligomers but not fibrils are cytoprotective.

Protease-hydrolysis-driven approach towards the quantification of cellular mRNA after drug treatment in protein nanopores.

Protease-hydrolysis-driven approach towards the quantification of cellular mRNA after drug treatment in protein nanopores.

The aggregation tendencies of the signal peptide regions of prone and not prone to aggregate proteins.

The aggregation tendencies of the signal peptide regions of prone and not prone to aggregate proteins.

Serum protein biomarker signature of Duchenne muscular dystrophy.

In contrast to invasive skeletal muscle biopsies and the associated complexity of tissue sampling techniques and potential detrimental side effects, the alternative application of liquid biopsy procedures has considerable advantages concerning minimal invasiveness, repeated sampling options, assay robustness and cost effectiveness. This article outlines the current status of serum biomarkers used for diagnosing and characterizing Duchenne muscular dystrophy (DMD), a primary muscle wasting disease of early childhood due to primary abnormalities in the extremely large DMD gene. Reviewed are important aspects of the discovery, characterization and diagnostic value of biofluid-based protein markers of dystrophinopathy. This includes an overview of traditional general skeletal muscle damage markers, such as creatine kinase, myoglobin and lactate dehydrogenase, which have been used for many decades in clinical applications to evaluate patients with muscular weakness. In addition, this article outlines the biochemical identification of novel biomarker candidates focusing on the usage of mass spectrometry-based proteomic surveys to establish comprehensive profiles of protein alterations in dystrophinopathy. Pathoproteomic serum markers of myonecrosis with great potential for improved patient screening, differential diagnosis, stage-specific prognosis and therapeutic monitoring include specific isoforms of muscle-derived cytosolic proteins, such as carbonic anhydrase isoform CA3 and fatty acid binding protein FABP3, as well as sarcomeric proteins, including specific isoforms of myosin light chain, myosin binding protein, troponin, and myomesin, in addition to peptide fragments derived from the giant protein titin. Biofluid-associated marker proteins of reactive myofibrosis include the extracellular matrix proteins fibronectin, osteopontin, collagen and matrix-metalloproteinases.

Structural Identification and Molecular Interaction Modeling Analysis of Antioxidant Activity Selenium-Enriched Peptides from Selenium-Enriched Pleurotus eryngii.

This study investigated the structure-activity relationships between SePEPs (selenium-enriched peptides) and PEPs (selenium-free peptides) and compared the antioxidant activities of SePEPs and PEPs. The results showed that SePEPs exhibited higher antioxidant activity than PEPs at the same molecular weight, with the molecular weights of 0-3500 Da exhibiting the highest in vitro antioxidant activity. Chelation between selenium and peptides led to a more compact structure and increased particle density in SePEPs. A spectroscopic analysis revealed new peaks and redshifts in SePEPs, along with a higher content of hydrophobic amino acids than PEPs. A molecular interaction modeling analysis indicated that hydrogen bonding and hydrophobic interactions primarily drove the binding between selenium-containing peptides and 1,1-diphenyl-2-picrylhydrazyl (DPPH). Moreover, the solid-phase synthesized MSePGP exhibited significantly greater antioxidant activity than glutathione at high concentrations. At 10 mg/mL, the DPPH radical scavenging rate of MSePGP was 68.5 ± 2.2%. These findings provide a theoretical basis for the design and synthesis of selenium-enriched peptides with enhanced antioxidant properties.

The intratumoral microbiota heterogenicity is related to the prognosis and tumorigenesis of cervical cancer.

The intratumoral microbe-host interaction plays crucial role in the development of cancer. The microbiome can influence cancer development by modulating inflammation, immune responses and metabolic pathways. Therefore, we aim to delineate the landscape and role of intratumoral microbiota in cervical cancer (CC). The presence of bacterial community in CC tissues was confirmed by fluorescence in situ hybridization (FISH). Then 16s rRNA and RNA-Seq were used to characterize the composition of intratumoral microbiota. Combined with cervical squamous cell carcinoma (CESC) data from the Tumor Cancer Genome Atlas (TCGA), the clinical signatures of intratumoral microbiota and DEGs were further analyzed. Finally, the effect of the up-regulated Fibrinogen beta chain (FGB) expressed fragment peptide on the biological behavior of cancer was verified in vitro. We found the composition heterogeneity of bacteria in CC tumors. Pseudomonas was most highly enriched in CC tissues and grouped according to the relative abundance level. The clinical characteristics of patients with relatively high abundance of Pseudomonas had the higher levels of fibrinogen and lower levels of white blood cell (WBC) and albumin (ALB) expression. Combining transcriptome data from the two our collective CC and TCGA-CESC cohorts, we found that Pseudomonas abundance was significantly associated with fibrinogen beta peptide expression and worse overall survival in CC patients. In vitro experiment revealed that Pseudomonas could promote the proliferation and migration of cervical cancer cells through overexpression of FGB. We characterized the composition of the intratumoral microbiota in CC tissues and identified the most significantly differentially abundant bacteria between cancerous and non-cancerous tissues. Our findings provide novel insights into the relationship between intratumoral Pseudomonas and the tumorigenesis of CC. A deeper understanding of the tumor microenvironment and its associated microbiota may reveal new potential therapeutic targets and improve clinical outcomes.

Strategies for neoantigen screening and immunogenicity validation in cancer immunotherapy (Review).

Cancer immunotherapy stimulates and enhances antitumor immune responses to eliminate cancer cells. Neoantigens, which originate from specific mutations within tumor cells, are key targets in cancer immunotherapy. Neoantigens manifest as abnormal peptide fragments or protein segments that are uniquely expressed in tumor cells, making them highly immunogenic. As a result, they activate the immune system, particularly T cell‑mediated immune responses, effectively identifying and eliminating tumor cells. Certain tumor‑associated antigens that are abnormally expressed in normal host proteins in cancer cells are promising targets for immunotherapy. Neoantigens derived from mutated proteins in cancer cells offer true cancer specificity and are often highly immunogenic. Furthermore, most neoantigens are unique to each patient, highlighting the need for personalized treatment strategies. The precise identification and screening of neoantigens are key for improving treatment efficacy and developing individualized therapeutic plans. The neoantigen prediction process involves somatic mutation identification, human leukocyte antigen (HLA) typing, peptide processing and peptide‑HLA binding prediction. The present review summarizes the major current methods used for neoantigen screening, available computational tools and the advantages and limitations of various techniques. Additionally, the present review aimed to summarize experimental strategies for validating the immunogenicity of the predicted neoantigens, which will determine whether these neoantigens can effectively trigger immune responses, as well as challenges encountered during neoantigen screening, providing relevant recommendations for the optimization of neoantigen‑based immunotherapy.

Studies on Formulations from a Clinical Perspective

This review highlights the author's research conducted at Mukogawa Women's University from April 2002 to March 2024. The work is categorized into following three areas: (1) Evaluation of the bitterness of oral medications using a taste sensor, (2) Development of drug delivery systems utilizing poly(lactic-co-glycolic acid) (PLGA), and (3) Clinical pharmaceutical evaluation of various injectable formulations. In section (1), the bitterness of oral medications, both alone and in combination with food or beverages, was quantitatively assessed. The taste sensor demonstrated high predictive accuracy, with a significant correlation observed between the sensor's bitter-sensitive outputs and the human taste receptor hT2R14, as documented in BitterDB. Recently, an innovative taste sensor featuring lipid/polymer membranes modified with 2,6-dihydroxybenzoic acid (2,6-DHBA), based on an allosteric mechanism, was developed to improve the detection of bitterness in non-charged compounds. In section (2), PLGA microspheres were engineered for the sustained release of prostaglandin derivatives over one month. Furthermore, polymeric micelles under 100 nm in diameter, composed of PLGA and LL-12 (a mutated fragment of human cathelicidin peptide), exhibited potent antibacterial activity and inhibited the proliferation of various cancer cells. Section (3) focuses on injectable formulations, including the development of a quantitative predictive system to evaluate the risk of insoluble particle formation when mixing ceftriaxone with calcium-containing injections. Additionally, the use of minimum inhibitory concentration (MIC) values and nomograms was explored to predict the clinical efficacy of imipenem derivatives. This research significantly contributes to enhancing the safety and efficacy of clinical treatments for patients.

Identification of a target polypeptide of the CD169 receptor of bovine macrophage using a phage display peptide library.

Identification of a target polypeptide of the CD169 receptor of bovine macrophage using a phage display peptide library.

Explore peptides extracted from gliadin hydrolysates suppressing BACE1 activity and restraining Aβ protein deposition.

Explore peptides extracted from gliadin hydrolysates suppressing BACE1 activity and restraining Aβ protein deposition.

Transmembrane clustering of short amyloid peptide fragments: A coarse grained molecular dynamics study.

Transmembrane clustering of short amyloid peptide fragments: A coarse grained molecular dynamics study.

Chickpea protein hydrolysates: Production, bioactivity, functional profile, and technological properties

Chickpea plant protein hydrolysates are an innovative product on the Russian food market. However, they meet many urgent needs and may solve some fundamental food safety problems. This article describes some effective enzymatic biodegradation methods that yield hydrolysates and biopeptides with advanced functional and technological properties that possess antioxidant and other bioactive potentials. The study featured Kabuli chickpea (Cicer arietinum L.) protein isolate, as well as a number of enzyme preparations of animal, plant, and microbial origin. Hydrolysis was followed by a set of FRAP, DPPH, and ORAC analyses to determine the functional, technological, and antioxidant properties. A combined approach made it possible to reveal the proteomic profile, e.g., a combination of two-dimensional electrophoresis and subsequent mass spectrometry was used to identify peptides. The bioactivity of peptide fragments was predicted in silico using bioinformatic databases. The efficiency of protein destruction depended on the degree of hydrolysis. At 10%, it improved the functional and technological properties. The best results regarding the time and enzyme concentration belonged to Alcalase 2.4 L FG (2%). The enzymes of animal origin, e.g., pepsin at a 10% hydrolysis degree, also improved the functional and technological profile. The samples treated with pepsin and Protoferm FP showed the highest antioxidant activity (FRAP, ORAC), increasing it by more than 200% relative to the initial chickpea isolate. Computer densitometry revealed that the hydrolysates treated with trypsin and papain could destroy more than 55% of the initial protein. Biologically active peptides of the hydrolysates obtained were determined using bioinformatic forecasting. In this research, chickpea protein hydrolysates provided new technological processing methods for commercial products. They made it possible to obtain biopeptides with antithrombotic, antitumor, antibacterial, antioxidant, and antiamnetic properties, which indicates excellent prospects in the food industry and pharmacy.

Photolysis of the peptide bond at 193 and 222 nm.

Ultraviolet (UV) light is a well-established tool for fragmenting peptides in vacuum. This study investigates the fragmentation of peptides using 193 and 222nm light in aqueous solution. Changes in the absorption spectra of solutions of the model dipeptide glycylglycine are monitored using a combination of real-time in situ transmission measurements and UV-Vis spectroscopy to report peptide bond scission following UV irradiation. Irradiation by a broadband ultraviolet light source flattens the absorbance peak centered near 193nm, indicating cleavage of peptide bonds. Irradiation with low-intensity, monochromatic 193 and 222nm light enabled measurements of the single-photon quantum yield of peptide bond scission, found to be (1.50 ± 0.12)% at 193nm and (0.16 ± 0.03)% at 222nm. These findings indicate that peptides may be fragmented in solution prior to emission into a mass spectrometer for new types of single-molecule analyses. The susceptibility of peptide bonds to ultraviolet radiation also suggests limited lifetimes for peptides on the early Earth's surface, which are relevant to theories of the origins-of-life, and suggests a role for protein damage in explanations of the germicidal effect of 222nm light exposure.

Programmable 2D Metal‐Organic Framework Nanosheets for Enzyme‐Like Hydrolysis of Large Proteins

AbstractThe development of materials that mimic the catalytic activity of natural enzymes, so called nanozymes, are of crucial importance in biochemical and biotechnological fields. A Zr‐pyridine tribenzoate metal‐organic framework nanosheet (MON), Zr‐PTB with 6‐connected Zr6O8 clusters is synthesized via a formic acid modulated solvothermal synthesis. The catalytic activity of MON, graphene‐like 2D materials which possess large external surface areas and tunable properties, is reported for the first time toward peptide bond hydrolysis of various peptides and proteins. Structure‐reactivity analysis and the comparison with Zr‐BTB (BTB = 1,3,5‐tri(4‐carboxyphenylbenzene)), a structural analogue of Zr‐PTB with a more hydrophobic linker, revealed a delicate interplay between coordination bonds and hydrophobic interactions with the MON's surface as the main driving forces influencing reactivity. Compared to 3D MOFs, Zr‐PTB produced a larger number of peptide fragments, indicating the importance of larger external surface area with easily accessible catalytically active sites for more comprehensive hydrolysis of proteins. The advantage of 2D MONs with respect to 3D MOFs, where pore sizes and diffusion of large substrates is a limiting factor influencing their reactivity, is further reflected by the ability to hydrolyze very large proteins. The exceptional stability of Zr‐PTB allowed for its recyclability for over 5 reaction cycles.

Abstract 2200: Development and optimization of cost-effective non-human primate in vitro assays for immunogenicity screening and functional evaluation of immuno-oncology drug candidates

A surge in demand and a decrease in supply of non-human primate (NHP) test subjects have caused acritical shortage and a major price hike for biomedical research. Therefore, researchers are actively lookinginto refinement and/or alternatives to studies using NHP. The development of cost-effective NHP in vitroassays with predictive value for the assessment of unwanted immunogenicity as well as the functionalityof drug candidates can provide useful tools in this context. For instance, PBMCs isolated from blood,sourced from Cynomolgus Macaques, can be cryopreserved and thawed thereafter on multiple separateoccasions to be used for isolation of immune cell subsets of interest.The development of customized in vitro assays based on cynomolgus monkey PBMCs can be used for thepre-selection of drug candidates and therefore reduce the number of NHP that are required for in vivostudies. For instance, cynomolgus PBMCs can be used to evaluate the unwanted immunogenicity of testcompounds in vitro. Here, it was shown that ATR-107, an anti-IL21R antibody and Bococizumab triggeredsignificant proliferation of cynomolgus PBMCs. Furthermore, using immunopeptidomics (in collaborationwith ImmuneSpec) Infliximab-derived peptide fragments that are known to be immunogenic in humanwere found to be presented at the surface of cynomolgus dendritic cells after their incubation withInfliximab during their maturation.We previously reported the development of cynomolgus monkey PBMC-derived in vitro assays that canbe used for the functional evaluation of new drug candidates (e.g., macrophage reprogramming, cytokinerelease, M2-suppression assay, ADCP etc.), target binding studies as well as Cynomolgus Macaque PBMCx human dendritic cell (DC) mixed lymphocyte reactions (MLR). Continuous efforts to optimize these assayscan additionally minimize the requirements of cynomolgus monkey material. For instance, it was shownthat by adapting the cell culture conditions for the generation of cynomolgus macrophages, cell yields canbe substantially improved, reducing the number of required PBMC samples, and allowing to perform largerassays for a given amount of NHP material.With our expanding portfolio and continuous optimization of Cynomolgus Macaque assays we aim to aidbiomedical drug development and to refine/reduce NHP usage. Citation Format: Christoph Schifflers, Vlaming Martijn, Emmanuelle Sidot, Jezabel Lefevre, Elise Pepermans, Ellen Boelen, Sofie Pattyn. Development and optimization of cost-effective non-human primate in vitro assays for immunogenicity screening and functional evaluation of immuno-oncology drug candidates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2200.

Gliadin-dependent UPR induction directly triggers the expression of TG2 and pro-inflammatory cytokines, dysregulates intestinal permeability, and reduces CFTR expression in intestinal epithelial cells of celiac disease patients

BackgroundCeliac disease (CD) is an autoimmune disorder that primarily affects the gut of genetically predisposed individuals and is triggered by gliadin peptides (PT) produced by the digestion of gluten. Although inappropriate activation of the immune system is thought to be the main trigger of CD, the interaction between PT and intestinal epithelial cells (IECs) remains a key step. Recently, the possible involvement of ER stress in the pathogenesis of CD has been pointed out, although its role is still largely unclear. Therefore, discovering the molecular mechanism(s) activated in IECs exposed to PT represents a unique opportunity to better understand the disease and define new potential therapeutic targets.MethodsIn this study we used three different experimental set-ups: intestinal biopsies from CD patients and non-CD control subjects, an in vitro model, based on human CaCo-2 cells, and an ex vivo model, based on our recently described mouse gut-ex-vivo system (GEVS), with the latter two systems were studied after stimulation with gliadin peptides (PT). To understand the signaling pathways involved we monitor the expression of a number of proteins by qPCR, Western blotting, IF, ELISA or a combination of tests. Specifically, we have analyzed the level of CD, ER stress, tissue permeability, and inflammation markers.ResultsIndeed, our study demonstrated a prompt induction of the transcription factors ATF4, ATF6 and XBP1 in IECs upon PT exposure. Thus, the upregulation of TG2 and downregulation of CFTR were prevented by ER stress inhibition/buffering by a pharmacological chaperone, also leading to restored physiological expression of OCL, CLD-2 and CLD-15, while preventing the expression of IFNγ, IL-15 and IL-17 A.ConclusionOverall, our analysis has highlighted the key role of ER stress in the pathogenesis of CD and identified the chemical chaperones as a new potential valuable therapeutic treatment for CD patients.

De Novo Design of Cyclic Peptide Binders Based on Fragment Docking and Assembling.

Cyclic peptides offer distinct advantages in modulating protein-protein interactions (PPIs), including enhanced target specificity, structural stability, reduced toxicity, and minimal immunogenicity. However, most cyclic peptide therapeutics currently in clinical development are derived from natural products or the cyclization of protein loops, with few methodologies available for de novo cyclic peptide design based on target protein structures. To fill this gap, we introduce CycDockAssem, an integrative computational platform that facilitates the systematic generation of head-to-tail cyclic peptides made entirely of natural - or -amino acid residues. The cyclic peptide binders are constructed from oligopeptide fragments containing 3-5 amino acids. A fragment library comprising 15 million fragments was created from the Protein Data Bank. The assembly workflow involves dividing the targeted protein surface into two docking boxes; the updated protein-protein docking program SDOCK2.0 is then utilized to identify the best binding fragments for these boxes. The fragments binding in different boxes are concatenated into a ring using two additional peptide fragments as linkers. A ROSETTA script is employed for sequence redesign, while molecular dynamics simulations and MM-PBSA calculations assess the conformational stability and binding free energy. To enhance docking performance, cation-π interactions, backbone hydrogen bonding potential, and explicit water exclusion energy were incorporated into the docking score function of SDOCK2.0, resulting in a significantly improved performance on the updated test set. A mirror design strategy was developed for cyclic peptides composed of -amino acids, where natural amino acid cyclic peptide binders are first designed for the mirror image of the target protein and the resulting complexes are then mirrored back. CycDockAssem was experimentally validated using tumor necrosis factor α (TNFα) as the target. Surface plasmon resonance experiments demonstrated that six of the seven designed cyclic peptides bind TNFα with micromolar affinity, two of which significantly inhibit TNFα downstream gene expression. Overall, CycDockAssem provides a robust strategy for targeted de novo cyclic peptide drug discovery.

Programming a multiplex lanthanide nanoparticle for customized cancer treatment with real-time efficiency feedback.

Customized cancer therapy relies on timely therapeutic effect evaluation to provide prescription adjustment for individual cases. However, currently reported therapeutic reagents are rarely integrated with imaging probes for self-evaluation of effects. Contrast imaging agents to measure tumor size changes must be administrated separately after therapy, complicating the therapeutic process and delaying reporting time. Herein, we design a customized therapy platform (LNPs-RB/Pep/cRGD) by conjugating lanthanide nanoparticles (LNPs) with the photosensitizer rose bengal, a caspase-3 substrate peptide (with Cy7.5 labelled at the terminal), and the tumor-targeting molecule cRGD. LNPs exhibit NIR-IIb downconversion luminescence under 980 nm/808 nm excitations for in vivo imaging, and visible upconversion luminescence under high-power 980 nm excitation for photodynamic therapy (PDT). By sequentially programming NIR excitation wavelength and power, NIR-IIb-imaging guided PDT and real-time cancer cell apoptosis imaging are achieved as therapeutic efficiency feedback. PDT induces cell apoptosis, generating caspase-3, which cleaves Cy7.5-containing peptide fragments from LNPs. This process corresponds to a recovery in vivo of NIR-IIb ratiometric imaging at 808 nm versus 980 nm excitation. The cleaved Cy7.5-containing peptide fragment is cleared into urine for NIR imaging. Both cell apoptosis imaging processes are completed 12 h after PDT, which is 7 days earlier than tumor size measurement. Therefore, customized therapy is achieved by timely adjusting PDT dosage, enhancing therapeutic efficacy.

Applying Absolute Free Energy Perturbation Molecular Dynamics to Diffusively Binding Ligands.

We have developed and tested an absolute free energy perturbation (FEP) protocol, which combines all-atom molecular dynamics, replica exchange with solute tempering (REST) enhanced sampling, and a spherical harmonic restraint applied to a ligand. Our objective was to compute the binding free energy together with the underlying binding mechanism for a ligand, which binds diffusively to a protein. Such ligands represent nearly impossible targets for traditional FEP simulations. To test our FEP/REST protocol, we selected a conserved motif peptide KKPK termed minNLS from the nuclear localization signal sequence of the Venezuelan equine encephalitis virus capsid protein. This peptide fragment binds diffusively to importin-α transport protein without forming well-defined poses. Our FEP/REST simulations with a spherical restraint provided a converged estimate of minNLS binding free energy. We found that minNLS binds with moderate affinity to importin-α utilizing an unusual, purely entropic mechanism in which binding free energy is determined by favorable entropic gain. For this cationic minNLS peptide, a favorable binding entropic gain is primarily associated with the release of water from the solvation shells of charged amino acids. We demonstrated that FEP/REST simulations sample the KKPK bound ensemble well, allowing us to characterize the distribution of bound structures, binding interactions, and locations on the importin-α surface. Analysis of experimental studies offered support to our rationale behind the KKPK entropic binding mechanism.

Interactions of Laurylated and Myristoylated KR12 Fragment of the LL37 Peptide with Polyoxidovanadates.

Isothermal titration calorimetry (ITC), circular dichroism (CD) spectroscopy, and molecular dynamics simulations were applied to describe interactions between lipopeptides and decavanadate ions ([V10O28]6-). The selected lipopeptides are conjugates of the amide of the KR12 peptide, the smallest antimicrobial peptide derived from human cathelicidin LL-37, with lauric acid (C12-KR12) and myristic acid (C14-KR12). The smaller sizes of C12-KR12 and C14-KR12 compared to proteins allow for the rigorous characterization of their non-covalent interactions with highly negatively charged [V10O28]6- ions. The stoichiometry of the resulting decavanadate-peptide complexes and the thermodynamic parameters (ΔG, ΔH, and TΔS) of the interactions were determined. The ITC results, supported by the MD simulation, showed that the binding of cationic lipopeptides for decavanadate is rather non-specific and is driven by enthalpic contributions resulting from electrostatic interactions between the positively charged residues of the peptides and the anionic decavanadate. Furthermore, the influence of temperature and the interactions with decavanadate ions on the stability of the α-helical structure of the lipopeptides were assessed based on CD spectra. Under the experimental conditions (50 mM sodium cacodylate buffer, pH 5), the peptides adopt an α-helical conformation, with C14-KR12 showing greater thermal stability. The interactions with vanadium species disrupt the α-helical structure and reduce its thermal stability.