Diverse Peptides Research Articles

Improved Large-Scale Synthesis of Acridonylalanine for Diverse Peptide and Protein Applications.

Fluorescent unnatural amino acids give biochemists, biophysicists, and bioengineers new ways to probe the properties of proteins and peptides. Here, the synthesis of acridon-2-ylalanine (Acd) is optimized for large-scale production to enable ribosomal incorporation through genetic code expansion (GCE), and fluorenylmethoxycarbonyl (Fmoc)-protected Acd is prepared for solid-phase peptide synthesis (SPPS). We demonstrate the utility of Acd in several applications: first, Acd quenching by Tyr is used in the design of fluorescent protease sensors made by SPPS. Second, we demonstrate Acd incorporation into a lanthanide-binding peptide that is generated either by GCE or by SPPS and demonstrate the utility of Acd for sensitizing the emission of Eu3+. Finally, Acd is inserted into the intrinsically disordered protein, α-synuclein, using GCE and used to study ion binding and aggregation.

An mRNA Display Approach for Covalent Targeting of a Staphylococcus aureus Virulence Factor.

Staphylococcus aureus ( S. aureus ) is an opportunistic human pathogen that causes over one million deaths around the world each year. We recently identified a family of serine hydrolases termed fluorophosphonate binding hydrolases (Fphs) that play important roles in lipid metabolism and colonization of a host. Because many of these enzymes are only expressed in Staphylococcus bacteria, they are valuable targets for diagnostics and therapeutics. Here we developed and screened highly diverse cyclic peptide libraries using mRNA display with a genetically encoded oxadiazolone (Ox) electrophile that was previously shown to potently and covalently inhibit multiple Fph enzymes. By performing multiple rounds of counter selections with WT and catalytic dead FphB, we were able to tune the selectivity of the resulting selected cyclic peptides containing the Ox residue towards the desired target. From our mRNA display hits, we developed potent and selective fluorescent probes that label the active site of FphB at single digit nanomolar concentrations in live S. aureus bacteria. Taken together, this work demonstrates the potential of using direct genetically encoded electrophiles for mRNA display of covalent binding ligands and identifies potent new probes for FphB that have the potential to be used for diagnostic and therapeutic applications.

The Hydrophobic Amino Acid-Rich Fish Collagen Peptide Ameliorates Dextran Sulfate Sodium-Induced Ulcerative Colitis in Mice via Repairing the Intestinal Barrier, Regulating Intestinal Flora and AA Metabolism.

The incidence of ulcerative colitis (UC) is increasing annually, but treatment option is limited. Fish collagen peptide (FCP) is a food source collagen peptide that has shown promise in alleviating UC symptoms. However, its impact on the intestinal barrier and intestinal metabolic homeostasis in UC remains unclear. This study aimed to analyze the peptide sequences and absolute amino acid (AA) content of FCP, assessing its effects on UC in mice induced by dextran sulfate sodium (DSS). FCP was examined by liquid chromatography and tandem mass spectrometry (LC-MS/MS) analysis. The 3% DSS was utilized to induce UC in murine models, followed by the assessment of the therapeutic efficacy of FCP. Clinical manifestations of UC mice were meticulously evaluated and scored. Subsequently, samples were procured for histological examination and intestinal epithelial barrier integrity analysis as well as macrogenomic and metabolomic profiling. Here, it shows that abundant peptide sequences and AAs were in FCP, particularly enriched in hydrophobic AAs (HAAs). Furthermore, it was observed that FCP effectively reversed colon shortening and reduced the extent of histological damage. Additionally, FCP suppressed the abnormal expression of inflammatory factors and intestinal barrier proteins and modulated the dysbiosis of gut microbiota toward a balanced state. These alterations led to the activation of intestinal alkaline AA and various AA metabolisms, ultimately contributing to the mitigation of UC symptoms. In summary, the diverse peptide sequences and high AAs in FCP, particularly rich in HAAs, can alleviate DSS-induced UC via preserving intestinal barrier integrity, regulating gut microbiota, and modulating AA metabolism.

Metabolomic Analysis Reveals the Linkage between Sleep-Enhancing Effects and Metabolite Biomarkers and Pathways of Different Casein Hydrolysates in Chronic Unpredictable Mild Stressed Mice.

Casein hydrolysates have been proven to exert varying sleep-enhancing and anxiolytic effects due to their distinct release of potential peptides. However, their underlying sleep-enhancing mechanisms at the metabolic level remain unclear. This study aims to investigate the potential sleep-enhancing mechanism of casein hydrolysates through an integrated approach of untargeted and targeted metabolomics in CUMS-induced anxiety mice for the first time. The results showed seven potential biomarkers were identified and screened using orthogonal partial least-squares discriminant analysis, random forest model, and pathway analysis, including ornithine, l-proline, l-prolinamide, inhibitory neurotransmitters gamma-aminobutyric acid, 5-HIAA, fumaric acid, and 4-oxoglutaramate. Moreover, casein hydrolysates exerted sleep-enhancing effects through multiple metabolic pathways, mainly including the GABAergic system, tryptophan metabolism, and cAMP response signaling pathway, which was validated by targeted metabolomics and vital protein expressions. It was interesting that casein hydrolysates with diverse representative peptide compositions exhibited varying activity, which could be attributed to distinct alterations in metabolites via different pathways.

Structure, Function and Engineering of the Nonribosomal Peptide Synthetase Condensation Domain.

The nonribosomal peptide synthetase (NRPS) is a highly precise molecular assembly machinery for synthesizing structurally diverse peptides, which have broad medicinal applications. Withinthe NRPS, the condensation (C) domain is a core catalytic domain responsible for the formation of amide bonds between individual monomer residues during peptide elongation. This review summarizes various aspects of the C domain, including its structural characteristics, catalytic mechanisms, substrate specificity, substrate gating function, and auxiliary functions. Moreover, through case analyses of the NRPS engineering targeting the C domains, the vast potential of the C domain in the combinatorial biosynthesis of peptide natural product derivatives is demonstrated.

Discovery and Heterologous Expression of Trilenodin, an Antimicrobial Lasso Peptide with a Unique Tri-Isoleucine Motif.

Lasso peptides are an increasingly relevant class of peptide natural products with diverse biological activities, intriguing physical properties, and unique chemical structures. Most characterized lasso peptides have been from Actinobacteria and Proteobacteria, despite bioinformatic analyses suggesting that other bacterial taxa, particularly those from Firmicutes, are rich in biosynthetic gene clusters (BGCs) encoding lasso peptides. Herein, we report the bioinformatic identification of a lasso peptide BGC from Paenibacillus taiwanensis DSM18679 which we termed pats. We used a bioinformatics-guided isolation approach and high-resolution tandem mass spectrometry (HRMS/MS) to isolate and subsequently characterize a new lasso peptide produced from the pats BGC, which we named trilenodin, after the tri-isoleucine motif present in its primary sequence. This tri-isoleucine motif is unique among currently characterized lasso peptides. We confirmed the connection between the pats BGC and trilenodin production by establishing the first Bacillus subtilis 168-based heterologous expression system for expressing Firmicutes lasso peptides. We finally determined that trilenodin exhibits potent antimicrobial activity against B. subtilis and Klebsiella pneumoniae, making trilenodin the first characterized biologically active lasso peptide from Firmicutes. Collectively, we demonstrate that bacteria from Firmicutes can serve as high-potential sources of chemically and biologically diverse lasso peptides.

Chemically diverse antimicrobial peptides induce hyperpolarization of the E. coli membrane

The negative membrane potential within bacterial cells is crucial in various essential cellular processes. Sustaining a hyperpolarised membrane could offer a novel strategy to combat antimicrobial resistance. However, it remains uncertain which molecules are responsible for inducing hyperpolarization and what the underlying molecular mechanisms are. Here, we demonstrate that chemically diverse antimicrobial peptides (AMPs) trigger hyperpolarization of the bacterial cytosolic membrane when applied at subinhibitory concentrations. Specifically, these AMPs adopt a membrane-induced amphipathic structure and, thereby, generate hyperpolarization in Escherichia coli without damaging the cell membrane. These AMPs act as selective ionophores for K+ (over Na+) or Cl− (over H2PO4− and NO3−) ions, generating diffusion potential across the membrane. At lower dosages of AMPs, a quasi-steady-state membrane polarisation value is achieved. Our findings highlight the potential of AMPs as a valuable tool for chemically hyperpolarising bacteria, with implications for antimicrobial research and bacterial electrophysiology.

Functional characterization of CCHamides and deorphanization of their receptors in the yellow fever mosquito, Aedes aegypti

As a widely distributed anthropophilic mosquito species and vector of various arboviruses, Aedes aegypti poses a significant threat to human health on a global scale. Investigating mosquito neuropeptides allows us to better understand their physiology. The neuropeptides CCHamide1 (CCHa1) and CCHamide2 (CCHa2) along with their associated G protein-coupled receptors (CCHa1R and CCHa2R) were recently identified and studied across insects. However, expression profiles and physiological roles of CCHamides and their receptors in many other insects, including A. aegypti, remain unclear. This research aimed to quantify and localize the expression of CCHamides along with their receptors and gain insight on their physiological function in the yellow fever mosquito. RT-qPCR analysis revealed transcript abundance of CCHamides and receptors changes over development. Differential expression was also observed in tissues/organs of adult mosquitoes indicating CCHa1 and CCHa2 transcripts are enriched in the midgut, while receptors are expressed across various tissues. CCHamide immunoreactivity was observed in neurons in the brain and ventral nerve cord along with enteroendocrine cells in the posterior midgut adjacent to the midgut-hindgut junction, corroborating their transcript expression profiles. Using different mass spectrometrical approaches, presence of CCHamides were confirmed in the brain of both sexes, including the pars intercerebralis of female mosquitoes, as well as in the gut of adult mosquitoes. For chemical identification of predicted CCHamides, we analyzed brain and gut extracts by ESI-Q Exactive Orbitrap MS and resulting fragmentations confirmed CCHa1 and CCHa2 in brain and midgut samples of both male and female mosquitoes. A heterologous functional assay was used to confirm the specificity and sensitivity of the two CCHamide receptors by assessing their activation in response to diverse mosquito peptides, which confirmed CCHa1 and CCHa2 as natural ligands. Finally, using a capillary feeder (CAFE) bioassay, our results suggest that CCHa2 modulates feeding behaviour in female mosquitoes.

Direct mapping of tyrosine sulfation states in native peptides by nanopore.

Sulfation is considered the most prevalent post-translational modification (PTM) on tyrosine; however, its importance is frequently undervalued due to difficulties in direct and unambiguous determination from phosphorylation. Here we present a sequence-independent strategy to directly map and quantify the tyrosine sulfation states in universal native peptides using an engineered protein nanopore. Molecular dynamics simulations and nanopore mutations reveal specific interactions between tyrosine sulfation and the engineered nanopore, dominating identification across diverse peptide sequences. We show a nanopore framework to discover tyrosine sulfation in unknown peptide fragments digested from a native protein and determine the sequence of the sulfated fragment based on current blockade enhancement induced by sulfation. Moreover, our method allows direct observation of peptide sulfation in ultra-low abundance, down to 1%, and distinguishes it from isobaric phosphorylation. This sequence-independent strategy suggests the potential of nanopore to explore specific PTMs in real-life samples and at the omics level.

The Archetypal Gamma-Core Motif of Antimicrobial Cys-Rich Peptides Inhibits H+-ATPases in Target Pathogens.

Human lactoferrin (hLf) is an innate host defense protein that inhibits microbial H+-ATPases. This protein includes an ancestral structural motif (i.e., γ-core motif) intimately associated with the antimicrobial activity of many natural Cys-rich peptides. Peptides containing a complete γ-core motif from hLf or other phylogenetically diverse antimicrobial peptides (i.e., afnA, SolyC, PA1b, PvD1, thanatin) showed microbicidal activity with similar features to those previously reported for hLf and defensins. Common mechanistic characteristics included (1) cell death independent of plasma membrane (PM) lysis, (2) loss of intracellular K+ (mediated by Tok1p K+ channels in yeast), (3) inhibition of microbicidal activity by high extracellular K+, (4) influence of cellular respiration on microbicidal activity, (5) involvement of mitochondrial ATP synthase in yeast cell death processes, and (6) increment of intracellular ATP. Similar features were also observed with the BM2 peptide, a fungal PM H+-ATPase inhibitor. Collectively, these findings suggest host defense peptides containing a homologous γ-core motif inhibit PM H+-ATPases. Based on this discovery, we propose that the γ-core motif is an archetypal effector involved in the inhibition of PM H+-ATPases across kingdoms of life and contributes to the in vitro microbicidal activity of Cys-rich antimicrobial peptides.

Diastereoselective Synthesis of Phosphinyl Peptides via Rh-Catalyzed 1,4-Addition in Coparticipation of a P-Chiral Moiety and Difluorphos.

The asymmetric Rh-catalyzed 1,4-addition of aryl/heteroaryl moieties to α,β-unsaturated esters was achieved in high diastereoselectivity via the coparticipation of a P-chiral phosphinyl moiety at Cβ to the prochiral center and (R)- or (S)-Difluorphos. This methodology expands the synthetic toolbox available for the preparation of structurally diverse chiral phosphinyl peptides.

Impact of N-Terminal PEGylation on Synthesis and Purification of Peptide-Based Cancer Epitopes for Pancreatic Ductal Adenocarcinoma (PDAC).

Peptide-based cancer vaccines have shown promising results in preclinical trials focusing on tumor immunotherapy. However, the presence of hydrophobic amino acid segments within these peptide sequences poses challenges in their synthesis, purification, and solubility, thereby hindering their potential use as cancer vaccines. In this study, we successfully synthesized peptide sequences derived from mesothelin (MSLN), a tumor-associated antigen overexpressed in pancreatic ductal adenocarcinoma (PDAC) by conjugating them with monodisperse polyethylene glycol (PEG). By PEGylating mesothelin epitopes of varying lengths (ranging from 9 to 38 amino acids) and hydrophobicity (60-90%), we achieved an effective method to improve the peptide yield and facilitate the processes of synthesis and purification. PEGylation significantly enhanced the solubility, facilitating the single-step purification of lengthy hydrophobic peptides. Most importantly, PEGylation did not compromise cell viability and had little to no effect on the immunogenicity of the peptides. In contrast, the addition of a palmitoyl group to increase immunogenicity led to reduced yield and solubility. Overall, PEGylation proves to be an effective technique for enhancing the solubility and broadening the range of utility of diverse long hydrophobic peptides.

Structural basis of divergent substrate recognition and inhibition of human neurolysin

A zinc metallopeptidase neurolysin (Nln) processes diverse bioactive peptides to regulate signaling in the mammalian nervous system. To understand how Nln interacts with various peptides with dissimilar sequences, we determined crystal structures of Nln in complex with diverse peptides including dynorphins, angiotensin, neurotensin, and bradykinin. The structures show that Nln binds these peptides in a large dumbbell-shaped interior cavity constricted at the active site, making minimal structural changes to accommodate different peptide sequences. The structures also show that Nln readily binds similar peptides with distinct registers, which can determine whether the peptide serves as a substrate or a competitive inhibitor. We analyzed the activities and binding of Nln toward various forms of dynorphin A peptides, which highlights the promiscuous nature of peptide binding and shows how dynorphin A (1–13) potently inhibits the Nln activity while dynorphin A (1–8) is efficiently cleaved. Our work provides insights into the broad substrate specificity of Nln and may aid in the future design of small molecule modulators for Nln.

Predicting Peptide Permeability Across Diverse Barriers: A Systematic Investigation.

Peptide-based therapeutics hold immense promise for the treatment of various diseases. However, their effectiveness is often hampered by poor cell membrane permeability, hindering targeted intracellular delivery and oral drug development. This study addressed this challenge by introducing a novel graph neural network (GNN) framework and advanced machine learning algorithms to build predictive models for peptide permeability. Our models offer systematic evaluation across diverse peptides (natural, modified, linear and cyclic) and cell lines [Caco-2, Ralph Russ canine kidney (RRCK) and parallel artificial membrane permeability assay (PAMPA)]. The predictive models for linear and cyclic peptides in Caco-2 and RRCK cell lines were constructed for the first time, with an impressive coefficient of determination (R2) of 0.708, 0.484, 0.553, and 0.528 in the test set, respectively. Notably, the GNN framework behaved better in permeability prediction with larger data sets and improved the accuracy of cyclic peptide prediction in the PAMPA cell line. The R2 increased by about 0.32 compared with the reported models. Furthermore, the important molecular structural features that contribute to good permeability were interpreted; the influence of cell lines, peptide modification, and cyclization on permeability were successfully revealed. To facilitate broader use, we deployed these models on the user-friendly KNIME platform (https://github.com/ifyoungnet/PharmPapp). This work provides a rapid and reliable strategy for systematically assessing peptide permeability, aiding researchers in drug delivery optimization, peptide preselection during drug discovery, and potentially the design of targeted peptide-based materials.

Pyocins and Beyond: Exploring the World of Bacteriocins in Pseudomonas aeruginosa.

Pseudomonas aeruginosa significantly induces health-associated infections in a variety of species other than humans. Over the years, the opportunistic pathogen has developed resistance against commonly used antibiotics. Since most P. aeruginosa strains are multi-drug resistant, regular antibiotic treatment of its infections is becoming a dire concern, shifting the global focus towards the development of alternate antimicrobial approaches. Pyocins are one of the most diverse antimicrobial peptide combinations produced by bacteria. They have potent antimicrobial properties, mainly against bacteria from the same phylogenetic group. P. aeruginosa, whether from clinical or environmental origins, produce several different pyocins that show inhibitory activity against other multi-drug-resistant strains of P. aeruginosa. They are, therefore, good candidates for alternate therapeutic antimicrobials because they have a unique mode of action that kills antibiotic-resistant bacteria by attacking their biofilms. Here, we review pseudomonas-derived antimicrobial pyocins with great therapeutic potential against multi-drug-resistant P. aeruginosa.

Bioactive Peptides Derived from Whey Proteins for Health and Functional Beverages

Milk serves as a crucial source of natural bioactive compounds essential for human nutrition and health. The increased production of high-protein dairy products is a source of whey—a valuable secondary product that, along with other biologically valuable substances, contains significant amounts of whey proteins and is often irrationally used or not utilized at all. Acid whey, containing almost all whey proteins and approximately one-quarter of casein, presents a valuable raw material for generating peptides with potential health benefits. These peptides exhibit properties such as antioxidant, antimicrobial, anti-inflammatory, anticarcinogenic, antihypertensive, antithrombotic, opioid, mineral-binding, and growth-stimulating activities, contributing to improved human immunity and the treatment of chronic diseases. Bioactive peptides can be produced by enzymatic hydrolysis using a variety of proteolytic enzymes, plant extracts, and microbial fermentation. With the participation of plant enzymes, peptides that inhibit angiotensin-converting enzyme are most often obtained. The use of enzymatic hydrolysis and microbial fermentation by lactic acid bacteria (LAB) produces more diverse peptides from different whey proteins with α-lactalbumin and β-lactoglobulin as the main targets. The resulting peptides of varying lengths often have antimicrobial, antioxidant, antihypertensive, and antidiabetic characteristics. Peptides produced by LAB are promising for use in medicine and the food industry as antioxidants and biopreservatives. Other beneficial properties of LAB-produced, whey-derived peptides have not yet been fully explored and remain to be studied. The development of whey drinks rich in bioactive peptides and based on the LAB proteolytic activity is underway. The strain specificity of LAB proteases opens up broad prospects for combining microorganisms to obtain products with the widest range of beneficial properties.

Electrochemical synthesis of peptide aldehydes via C‒N bond cleavage of cyclic amines

Peptide aldehydes are crucial biomolecules essential to various biological systems, driving a continuous demand for efficient synthesis methods. Herein, we develop a metal-free, facile, and biocompatible strategy for direct electrochemical synthesis of unnatural peptide aldehydes. This electro-oxidative approach enabled a step- and atom-economical ring-opening via C‒N bond cleavage, allowing for homoproline-specific peptide diversification and expansion of substrate scope to include amides, esters, and cyclic amines of various sizes. The remarkable efficacy of the electro-synthetic protocol set the stage for the efficient modification and assembly of linear and macrocyclic peptides using a concise synthetic sequence with racemization-free conditions. Moreover, the combination of experiments and density functional theory (DFT) calculations indicates that different N-acyl groups play a decisive role in the reaction activity.

Molecular and structural basis of oligopeptide recognition by the Ami transporter system in pneumococci.

ATP-binding cassette (ABC) transport systems are crucial for bacteria to ensure sufficient uptake of nutrients that are not produced de novo or improve the energy balance. The cell surface of the pathobiont Streptococcus pneumoniae (pneumococcus) is decorated with a substantial array of ABC transporters, critically influencing nasopharyngeal colonization and invasive infections. Given the auxotrophic nature of pneumococci for certain amino acids, the Ami ABC transporter system, orchestrating oligopeptide uptake, becomes indispensable in host compartments lacking amino acids. The system comprises five exposed Oligopeptide Binding Proteins (OBPs) and four proteins building the ABC transporter channel. Here, we present a structural analysis of all the OBPs in this system. Multiple crystallographic structures, capturing both open and closed conformations along with complexes involving chemically synthesized peptides, have been solved at high resolution providing insights into the molecular basis of their diverse peptide specificities. Mass spectrometry analysis of oligopeptides demonstrates the unexpected remarkable promiscuity of some of these proteins when expressed in Escherichia coli, displaying affinity for a wide range of peptides. Finally, a model is proposed for the complete Ami transport system in complex with its various OBPs. We further disclosed, through in silico modelling, some essential structural changes facilitating oligopeptide transport into the cellular cytoplasm. Thus, the structural analysis of the Ami system provides valuable insights into the mechanism and specificity of oligopeptide binding by the different OBPs, shedding light on the intricacies of the uptake mechanism and the in vivo implications for this human pathogen.

Bacteriocin diversity, function, discovery and application as antimicrobials.

Bacteriocins are potent antimicrobial peptides that are produced by bacteria. Since their discovery almost a century ago, diverse peptides have been discovered and described, and some are currently used as commercial food preservatives. Many bacteriocins exhibit extensively post-translationally modified structures encoded on complex gene clusters, whereas others have simple linear structures. The molecular structures, mechanisms of action and resistance have been determined for a number of bacteriocins, but most remain incompletely characterized. These gene-encoded peptides are amenable to bioengineering strategies and heterologous expression, enabling metagenomic miningand modification of novel antimicrobials. The ongoing global antimicrobial resistance crisis demands that novel therapeutics be developed to combat infectious pathogens. New compounds that are target-specific and compatible with the resident microbiota would be valuable alternatives to current antimicrobials. As bacteriocins can be broad or narrow spectrum in nature, they are promising tools for this purpose. However, few bacteriocins have gone beyond preclinical trials and none is currently used therapeutically in humans. In this Review, we explore the broad diversity in bacteriocin structure and function, describe identification and optimization methods and discuss the reasons behind the lack of translation beyond the laboratory of these potentially valuable antimicrobials.

Development of Peptide Displacement Assays to Screen for Antagonists of DDB1 Interactions.

The DNA damage binding protein 1 (DDB1) is an essential component of protein complexes involved in DNA damage repair and the ubiquitin-proteasome system (UPS) for protein degradation. As an adaptor protein specific to Cullin-RING E3 ligases, DDB1 binds different receptors that poise protein substrates for ubiquitination and subsequent degradation by the 26S proteasome. Examples of DDB1-binding protein receptors are Cereblon (CRBN) and the WD-repeat containing DDB1- and CUL4-associated factors (DCAFs). Cognate substrates of CRBN and DCAFs are involved in cancer-related cellular processes or are mimicked by viruses to reprogram E3 ligases for the ubiquitination of antiviral host factors. Thus, disrupting interactions of DDB1 with receptor proteins might be an effective strategy for anticancer and antiviral drug discovery. Here, we developed fluorescence polarization (FP)-based peptide displacement assays that utilize full-length DDB1 and fluorescein isothiocyanate (FITC)-labeled peptide probes derived from the specific binding motifs of DDB1 interactors. A general FP-based assay condition applicable to diverse peptide probes was determined and optimized. Mutagenesis and biophysical analyses were then employed to identify the most suitable peptide probe. The FITC-DCAF15 L49A peptide binds DDB1 with a dissociation constant of 68 nM and can be displaced competitively by unlabeled peptides at sub-μM to low nM concentrations. These peptide displacement assays can be used to screen small molecule libraries to identify novel modulators that could specifically antagonize DDB1 interactions toward development of antiviral and cancer therapeutics.