Individual Peptides Research Articles

Precise Identification of Native Peptides with Posttranslational Proline Hydroxylation by Nanopore.

Hydroxylation, an extensive post-translational modification on proline, is critical for the modulation of protein structures, further dominating their functions in life systems. However, current mass spectrometry-based identification, could hardly distinguish hydroxylation from neighboring oxidation due to the same mass shifts, as well as challenges posed by low abundance and exogenous oxidation during sample preparation. To address these, an engineered nanopore was designed, capable of discriminating single hydroxyl group, to achieve the identification of proline hydroxylation on individual native peptides directly in the mixture. By modeling the interactions between hydroxylated proline and its specific recognition protein, we introduced a hydrophobic region in aerolysin lumen with A224Y/T274W mutations to enhance the sensitivity for proline residue. The results showed that proline hydroxylation on native HIF-1α fragments could be unambiguously identified without purification, which could be maintained even in the presence of neighboring oxidation. The voltage-dependent experiments further demonstrated more relaxed peptide structures induced by hydroxylation, supporting the great impact of hydroxylation on chemical properties of proline and the molecular mechanism of the specific recognition for hydroxylated peptides in nature. These findings highlight the potential of nanopore for precise hydroxylation detection, offering a reliable platform for further uncovering the related functions in biological systems.

Precise Identification of Native Peptides with Posttranslational Proline Hydroxylation by Nanopore

Hydroxylation, an extensive post‐translational modification on proline, is critical for the modulation of protein structures, further dominating their functions in life systems. However, current mass spectrometry‐based identification, could hardly distinguish hydroxylation from neighboring oxidation due to the same mass shifts, as well as challenges posed by low abundance and exogenous oxidation during sample preparation. To address these, an engineered nanopore was designed, capable of discriminating single hydroxyl group, to achieve the identification of proline hydroxylation on individual native peptides directly in the mixture. By modeling the interactions between hydroxylated proline and its specific recognition protein, we introduced a hydrophobic region in aerolysin lumen with A224Y/T274W mutations to enhance the sensitivity for proline residue. The results showed that proline hydroxylation on native HIF‐1α fragments could be unambiguously identified without purification, which could be maintained even in the presence of neighboring oxidation. The voltage‐dependent experiments further demonstrated more relaxed peptide structures induced by hydroxylation, supporting the great impact of hydroxylation on chemical properties of proline and the molecular mechanism of the specific recognition for hydroxylated peptides in nature. These findings highlight the potential of nanopore for precise hydroxylation detection, offering a reliable platform for further uncovering the related functions in biological systems.

P0211 Characterization of drug-specific CD4+ T cells in patients with inflammatory bowel diseases

Abstract Background Biologicals are commonly used to treat chronic inflammatory diseases. Treatment failure is a major limitation for the efficacy of biologicals, which can be caused by the development of anti-drug antibodies. The formation of high-affinity anti-drug antibodies suggests the involvement of specific CD4+ T cell responses. However, the CD4+ T cell reaction against biologicals remains poorly characterized. Therefore, in this study we analyzed CD4+ T cells specific to various biologicals and investigated potential differences of drug-specific CD4+ T cells in patients with continued remission versus those with secondary nonresponses or allergic reactions. Methods Peripheral blood samples were collected at baseline and at week 6, week 14, week 22, week 30 after biological treatment. Drug-specific CD4+ T cells were analyzed using Antigen-Reactive T cell Enrichment (ARTE) directly ex vivo from human peripheral blood. To detect immunogenic regions, drug-specific CD4+ T cells were expanded and restimulated with individual peptides covering the whole variable chain of the therapeutic antibodies. Additionally, single-cell RNA sequencing was performed to investigate the gene expression profiles of drug-specific CD4+ T cells. Results Of the analyzed biological agents, the chimeric antibody infliximab showed significantly higher activation of reactive CD4+ T cells compared to other humanized or fully human biological agents. Notably, this response was characterized by a highly proliferative Th1 phenotype with evidence of long-term memory. In patients with secondary nonresponses or allergic reactions infliximab-specific CD4+ T cells showed elevated gene and protein expression of TNF-a, IFN-g and IL-21. Restimulation experiments indicated that drug-specific CD4+ T cell responses are driven by small immunodominant peptide regions. Conclusion Infliximab exhibits a higher activation of specific CD4+ T cell reactions compared to other biologicals, characterized by a proliferative Th1 response with long-term memory capabilities. Infliximab is a chimeric antibody consisting of murine variable regions, which likely contributes to its immunogenicity. The increased production of TNF-a, IFN-g and IL-21 in patients experiencing secondary nonresponses or allergic reactions highlights the potential role of drug-specific CD4+ T cells in treatment outcomes and formation of anti-drug antibodies. Overall, our data identifies differences in anti-drug CD4+ T cell reactions for different biologicals as well as different treatment outcomes which may contribute to enhance drug efficacy and improve long-term success with immunogenic biologicals.

P0002 Identification of Infliximab and Adalimumab -derived peptides using MHC class II antibody mediated immunopeptidomics and mass spectrometry: implications for drug re-design

Abstract Background Monoclonal antibodies against tumour necrosis factor (TNF) such as Infliximab (IFX) and Adalimumab (ADA) are used as first line therapy against inflammatory bowel disease (IBD) and have proven efficacy. However, in up to 40% of IBD patients these drugs become ineffective after the first year, principally due to the development of anti-drug immunogenicity. Distinct allelic variants at the HLA DQ1 locus predispose patients to develop immunogenicity to IFX or ADA (1, 2, 3). The aim of this study is to identify peptides derived from IFX and ADA that are presented on class II MHC of antigen presenting cells, in order to determine the epitopes that may play a role in T cell mediated immunogenicity against anti-TNF therapy. This may allow rational drug re-design. Methods Monocyte-derived dendritic cells (MDDCS) from 8 different donors, as well as EBV-transformed B cell lines from 3 different donors of known HLA-type were cultured and pulsed with IFX and ADA. Unpulsed cells were used as a negative control. Cells were processed for sequential immune precipitation with HLA-DQ, HLA-DP and HLA-DR antibodies. Peptides were then purified and analysed by mass spectrometry. These were run against a database of known proteins, including IFX and ADA, using the Peaks analytical software and then mapped to the original protein. Peptides were subjected to a binding prediction algorithm, to determine the likelihood of individual peptides binding to each HLA allele combinations. Peptides were then ranked from highest to lowest binder. Results Analysis of peptides from all three MHC class II antibody fractions showed that the highest number of peptides immunoprecipitated were between 14 to 16 amino acids in length, in line with the predicted length for class II MHC proteins. Additionally, there was an enrichment of peptides predicted to bind to specific class II MHC molecules, with an average of 77% binders versus 23% non-binders for MDDCs, and 84% binders versus 16 % non-binders for B-cells, after immune precipitation. Several overlapping peptides from the variable regions of both the heavy and light chain of IFX and ADA were identified, some of which are predicted to bind to risk alleles for anti-TNF immunogenicity, including DQA1*05:01, DRB1*11:01 and HLA-DQA1*05:05 (Figures 1 and 2). Conclusion We have characterised a series of IFX and ADA derived peptides from MDDCs and B cell lines, that were pulsed with anti-TNF antibody drugs. These peptides represent candidate T cell epitopes that may play a role in eliciting anti-drug antibody responses, and these data will therefore contribute to the understanding of the molecular mechanism of anti-TNF treatment failure. References 1)Doherty R, Liao H, Satsangi J, Ternette N. Extended Analysis Identifies Drug-Specific Association of 2 Distinct HLA Class II Haplotypes for Development of Immunogenicity to Adalimumab and Infliximab. Gastroenterology. 2020, 159: 784-787. 2)Sazonovs A, Kennedy N, Moutsianas L, et al. HLA-DQA1*05 Carriage Associated With Development of Anti-Drug Antibodies to Infliximab and Adalimumab in Patients With Crohn’s Disease Gastroenterology. 2020, 158:189-199 3)Ternette N, Liao H., Satsangi J. Association of the HLA DQA1*05 allelic gene variants with immunogenicity to anti-TNF therapeutics - important differences between infliximab and adalimumab. Journal of Crohn's and Colitis. 2024

Designing Short Cardin-Motif Peptide and Biopolymer-Based Multicomponent Hydrogels for Developing Advanced Composite Scaffolds for Improving Cellular Behavior.

Multicomponent self-assembly represents a cutting-edge strategy in peptide nanotechnology, enabling the creation of nanomaterials with enhanced physical and biological characteristics. This approach draws inspiration from the highly complex nature of the native extracellular matrix (ECM) constituting multicomponent biomolecular entities. In recent years, the combination of bioactive peptide with polymer has gained significant attention for the fabrication of novel biomaterials due to their inherent specificity, tunable physiochemical properties, biocompatibility, and biodegradability. This advanced strategy can address the limitation of the lower mechanical strength of the individual peptide hydrogel by incorporating the biopolymer, resulting in the formation of a composite scaffold. In this direction, this advanced strategy is explored using noncovalent interactions between cellulose nano-fiber (CNF) and cationic Cardin-motif peptide to develop advanced composite scaffolds. The bioactive cationic peptide otherwise failed to form hydrogel at physiological conditions. Interestingly, the differential mixing ratio of CNF and peptide modulated the surface charge, functionality, and mechanical properties of the composite scaffolds, resulting in diverse cellular responses. 10:1 (w/w) ratio of CNF and peptide-based composite scaffold demonstrates improved cellular survival and proliferation in 2D culture conditions. Notably, in 3D cultures, cell proliferation on the 10:1 matrix is comparable to Matrigel, highlighting its potential for advanced tissue engineering applications.

Anticarcinogenic cationic peptides derived from tryptic hydrolysis of β-lactoglobulin.

This study investigated the tryptic hydrolysis of β-lactoglobulin (BLG) for 30, 60, 90, and 120 min at 1/200 E/S (enzyme/substrate ratio, w/w) to prepare potentially anticarcinogenic peptides. The properties of hydrolysates were characterized, including degree of hydrolysis, free amino acids, SDS-PAGE, FTIR, and antioxidant activity employing DPPH-assay, β-carotene/linoleic acid, and FRAP assay. BLG tryptic hydrolysate produced after 60 min hydrolysis recorded the highest antioxidant activity, and LCMS analysis revealed 162 peptides of molecular masses ranging from 800 to 5671Da, most of them are of hydrophobic nature. Within the low-MW peptide group (24 peptides), there were nine hydrophobic basic (HB) and seven hydrophobic acidic (HA), representing 38% and 29%, respectively. The HB peptides may be responsible for the considerable biological activity of the hydrolysate. With dominant basic character supporting the carcinogenic activity of this hydrolysate. The in vitro anticancer activity against Mcf-7, Caco-2, and A-549 human cancer cell lines proliferation by MTT assay recorded IC50% at 42.8, 76.92, and 45.93 μg/mL, respectively. Treating each cell line with IC50% of the hydrolysate for 24 h increased the apoptosis by enhancing the expression of caspase-9 by 5.66, 7.97, and 3.28 folds over the untreated control and inhibited angiogenesis by reducing VEGFR-2 expression by about 56, 76, and 70%, respectively, indicating strong anticancer and antiangiogenic actions on human cancer cells. BLG tryptic hydrolysate may serve as a natural anticarcinogenic agent. The results of this study demonstrated that BLG hydrolysates have direct anticancer and antiangiogenic effects on human cancer cells. The chemical composition and characteristics of the BLG tryptic hydrolysate influence these biological and anticancer activities. The tryptic hydrolysates were generally effective against the three cancer cell lines studied (Mcf-7, Caco-2, and A-549). This effectiveness was assessed by measuring cell proliferation using the MTT assay and by evaluating their impact on angiogenesis through inhibition of VEGFR-2 activity. Future studies may focus on enhancing the anticarcinogenic effectiveness of the peptides by isolating and evaluating the most prominent individual peptide and varying the treatment conditions.

Towards a Quantitative Description of Proteolysis: Contribution of Demasking and Hydrolysis Steps to Proteolysis Kinetics of Milk Proteins.

The hydrolysis of proteins by proteases (proteolysis) plays a significant role in biology and food science. Despite the importance of proteolysis, a universal quantitative model of this phenomenon has not yet been created. This review considers approaches to modeling proteolysis in a batch reactor that take into account differences in the hydrolysis of the individual peptide bonds, as well as the limited accessibility (masking) for the enzymes of some hydrolysis sites in the protein substrate. Kinetic studies of the proteolysis of β-casein and β-lactoglobulin by various proteolytic enzymes throughout the whole degree of hydrolysis are reviewed. The two-step proteolysis model is regarded, which includes demasking of peptide bonds as a result of opening of the protein structure at the first stage, then hydrolysis of the demasked peptide bonds. To determine the kinetics of demasking, the shift in Trp fluorescence during opening of the protein substrate is analyzed. Two stages of demasking and secondary masking are also considered, explaining the appearance of unhydrolyzed peptide bonds at the end of proteolysis with decreasing enzyme concentrations. Proteolysis of a nanosized substrate is considered for the example of tryptic hydrolysis of β-CN micelles, leading to the formation and degradation of new nanoparticles and non-monotonic changes in the secondary protein structures during proteolysis.

Binding mechanism of oligopeptides on solid surface: assessing the significance of single-molecule approach.

This paper addresses the complementarity and potential disparities between single-molecule and ensemble-average approaches to probe the binding mechanism of oligopeptides on inorganic solids. Specifically, we explore the peptide/gold interface owing to its significance in various topics and its suitability to perform experiments both in model and real conditions. Experimental results show that the studied peptide adopts a lying configuration upon adsorption on the gold surface and interacts through its peptidic links and deprotonated thiolate extremities, in agreement with theoretical predictions. Single-molecule force spectroscopy (SMFS) measurements revealed the existence of a wide panel of adhesion forces, resulting from the interaction between individual peptide moieties and the abundant surface sites. We therefore propose methodological developments for sorting the events of interest to understand the peptide adsorption mechanism. Thermodynamic and kinetic aspects of the peptide adsorption are probed using both static and dynamic force spectroscopy measurements. Specifically, we show the possibility of providing a reasonable estimate of the peptide free energy of adsorption ΔadsG° by exploring the fluctuations of the adhesion work, based on the Jarzynski equality, and by using a parametric Gamma estimator. The proposed approach offers a relevant method for studying the different factors influencing the peptide adsorption and evaluating their impact on ΔadsG° as an alternative to exploring adhesion forces that may lead to misinterpretations. This is illustrated by the comparison of the adsorption of two peptides with specific amino acids substitution. Our method provides insights into the overall mechanism by which peptides interact with the surface and allows an integration of the single-molecule versus ensemble-average points of view.

A Thin Polymer Layer Enables Peptide-Polycation Complexes with Ultrahigh Efficient Encapsulation.

A monolayer encapsulation is a new opportunity for engineering a system with high drug loading, but immobilizing polymer molecules on the surface of individual peptide nanoparticles is still an ongoing challenge. Herein, an individual peptide nanoparticle encapsulation strategy is proposed via surface adsorption, in which peptide molecules undergo granulation and subsequently aggregate with polymer molecules, forming a network via electrostatic interactions. Under the water phase, surplus polymer molecules dissolve, leading to a single nanoparticle encapsulation with a core-shell structure. As expected, the dense interfacial layer on the peptide nanoparticle surface achieves a superior loading degree of up to 95.4%. What's more, once the core-shell structure is established, the peptide mass fraction in individual encapsulation always exceeds 90% even under fierce external force. Following the individual nanoparticle encapsulation, the insulin-polycation complex (InsNp@PEI) reduces the inflammation from polymer and displays an effective glycemic control in type 1 diabetes. Overall, the newly developed single surface decoration encapsulates peptides with ultrahigh efficiency and opens up the possibility for further encapsulation.

A Matter of Charge: Electrostatically Tuned Coassembly of Amphiphilic Peptides.

Coassembly of peptide biomaterials offers a compelling avenue to broaden the spectrum of hierarchically ordered supramolecular nanoscale structures that may be relevant for biomedical and biotechnological applications. In this work coassemblies of amphiphilic and oppositely charged, anionic and cationic, β-sheet peptides are studied, which may give rise to a diverse range of coassembled forms. Mixtures of the peptides show significantly lower critical coassembly concentration (CCC) values compared to those of the individual pure peptides. Intriguingly, the highest formation of coassembled fibrils is found to require excess of the cationic peptide whereas equimolar mixtures of the peptides exhibited the maximum folding into β-sheet structures. Mixtures of the peptides coassembled sequentially from solutions at concentrations surpassing each peptide's intrinsic critical assembly concentration (CAC), are also found to require a higher portion of the cationic peptide to stabilize hydrogels. This study illuminates a systematic investigation of oppositely charged β-sheet peptides over a range of concentrations, in solutions and in hydrogels. The results may be relevant to the fundamental understanding of such intricate charge-driven assembly systems and to the formulation of peptide-based nanostructures with diverse functionalities.

Targeting the hypothalamus for modeling age-related DNA methylation and developing OXT-GnRH combinational therapy against Alzheimer’s disease-like pathologies in male mouse model

The hypothalamus plays an important role in aging, but it remains unclear regarding the underlying epigenetics and whether this hypothalamic basis can help address aging-related diseases. Here, by comparing mouse hypothalamus with two other limbic system components, we show that the hypothalamus is characterized by distinctively high-level DNA methylation during young age and by the distinct dynamics of DNA methylation and demethylation when approaching middle age. On the other hand, age-related DNA methylation in these limbic system components commonly and sensitively applies to genes in hypothalamic regulatory pathways, notably oxytocin (OXT) and gonadotropin-releasing hormone (GnRH) pathways. Middle age is associated with transcriptional declines of genes which encode OXT, GnRH and signaling components, which similarly occur in an Alzheimer’s disease (AD)-like model. Therapeutically, OXT-GnRH combination is substantially more effective than individual peptides in treating AD-like disorders in male 5×FAD model. In conclusion, the hypothalamus is important for modeling age-related DNA methylation and developing hypothalamic strategies to combat AD.

Proteomic analysis of psychedelic mushroom isolate and exploring potential antimicrobial peptides against bacteria

Psychedelic mushrooms belonging to basidiomycota have gained prominence in research due to the range of hallucinogenic compounds. To combat the challenge of antimicrobial resistance, exploring alternative antimicrobial peptides has become crucial. In this study, we present the proteomic analysis of psychedelic mushroom. Psilocybe cubensis was identified by molecular characterisation using the ITS1 and ITS4 regions. Subsequently, LC-MS/MS and gene ontology analyses were used to characterise the proteome of P. cubensis. The proteomic analysis unveiled several antimicrobial peptides. The results revealed favourable binding scores, suggesting the potential efficacy of these peptides against Staphylococcus aureus. Hence the inhibition of bacterial growth, supporting the antimicrobial properties of the identified peptides. In our findings, the individual peptides from P. cubensis against S. aureus suggest a promising avenue for the discovery of novel antimicrobial peptides.

Research on the Synergistic Inhibition of Angiotensin-Converting Enzyme (ACE) by the Gastrointestinal Digestion Products of the ACE Inhibitory Peptide FPPDVA.

To gain a deeper understanding of the ACE inhibition effect, the inhibitory effect of ACE-inhibiting peptide (ACEIP) FPPDVA's digestive products on ACE was further investigated. Two novel peptides, PD (IC50 = 161.1 ± 1.10 μM) and DV (IC50 = 66.51 ± 0.99 μM) were identified in the digestive products of FPPDVA using LC-MS/MS. The Peptide Mix (FPPDVA, PD, and DV) exhibited a remarkable synergistic effect on ACE inhibition by significantly enhancing it by up to 508% compared to the individual peptides alone. Furthermore, theoretical simulations suggest that the Peptide Mix synergistically inhibits ACE activity by forming more stable complexes with the active site of ACE, facilitated by an increased number of hydrogen bonds. Additionally, Lineweaver-Burk plot analysis and spectroscopic studies further verified the presence of these stable complexes. ITC results show that the combination of Peptides Mix and ACE is a spontaneous exothermic process driven by entropy. The study showed that FPPDVA has a stronger inhibitory effect on ACE after digestion, making it suitable as an antihypertensive peptide in functional foods.

Abstract PR-07: Optimally designed mRNA vaccine encoding tumor-specific antigens identified in a colorectal cancer model leads to complete tumor rejection in mice

Abstract mRNA vaccines can be designed to encode multiple epitopes of choice, which is a crucial advantage when aiming to include a broad coverage immune response. Upon translation, the cell machinery must process the vaccine-encoded poly-epitope protein to release individual peptides. This processing step is key in inducing an immune response toward selected epitopes, as efficient processing yields a higher amount of peptide available for MHC-I presentation. The efficiency of peptide processing is greatly influenced by the peptide surrounding context (i.e. the flanking amino acids in N-and C-terminal). Aberrantly expressed tumor-specific antigens (aeTSAs) are MHC-I-associated peptides (MAPs) resulting from cancer-specific epigenetic changes and splicing aberrations. In contrast to TSAs derived from mutated protein-coding exons, aeTSAs are highly shared by different tumors. Moreover, unlike commonly used tumor-associated antigens (TAAs), they are not expressed by normal healthy cells, which is pivotal for inducing strong CD8+ T cell responses. The present study aimed to determine whether RNA vaccines encoding aeTSAs would elicit protective anti-tumor responses against a colorectal cancer cell line model (MC38). We hypothesized that multiepitope mRNA vaccine efficacy would be improved if the mRNA construct was composed of minimal epitopes (here, cancer antigens) bordered by optimal (rather than natural) flanking sequences. Thus, we designed 2 vaccine constructs encoding 5 aeTSAs identified by mass spectrometry in MC38 tumor cells. The 2 constructs differ in the identity of the amino acids flanking the antigens (natural vs. substituted antigen flanking sequences). Mice were injected subcutaneously with MC38 cells on Day 0. Three mRNA vaccine doses were administered intravenously one week apart, starting at Day 4. Vaccination with our improved mRNA vaccine design showed a clear therapeutic effect. As opposed to the natural flanking sequences design resulting in delayed tumor growth, vaccination with the substituted flanking sequences design led to the complete elimination of tumors and the survival of all mice. Elispot and dextramer stainings revealed that at least 3 of the 5 MC38 aeTSAs were immunogenic and that the amplitude of antigen-specific CD8+ T cell responses was significantly higher in mice vaccinated with the substituted flanking sequences design. Our results confirm our hypothesis that highly proficient flanking regions have intrinsic benefits that can be carried over to different antigens. From a translational perspective, our work provides new insights into the therapeutic potential of optimally designed aeTSA-encoding mRNA vaccines for treating cancers. Citation Format: Marie-Pierre Hardy, Krystel Vincent, Gabriel Ouellet-Lavallée, Chantal Durette, Isabelle Caron, Joel Lanoix, Mathieu Courcelles, Jean-Philippe Laverdure, Pierre Thibault, Claude Perreault. Optimally designed mRNA vaccine encoding tumor-specific antigens identified in a colorectal cancer model leads to complete tumor rejection in mice [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr PR-07.

Metal-antimicrobial peptides combo: promising weapons to combat bacteria invaders

Antimicrobial resistance has significantly increased over the last 30 years, prompting scientists to continuously look for novel, effective ways to combat drug-resistant bacteria and fungi. Due to their broad range of effectiveness, ease of synthesis, and ability to avoid resistance, antimicrobial peptides (AMPs) represent a potential approach. The direct investigation of metal effects on peptide activity has not received much attention. Divalent metal ions such as Zn(II), Cu(II), Ni(II), and Fe(II) do, in fact, influence some AMPs, producing an effect on their mode of action or function. Although the precise process by which metals are involved in microbial death is not well understood, this review goes into detail on several potential strategies to enhance AMPs activity through the synergy with metals. Important variables in these interactions include the individual peptide sequence, the type of metal ion, the capacity of metal to form chelates, and the softness of the ligand/metal complex. This article offers a thorough summary of the ways in which metal ions alter the structure of AMPs to boost their activity or sequester metal to impact on bacteria function.

Structural characterization and bioactivity profiling of the fungal peptaibiotic tolypin reveal protective effects against influenza viruses.

In a bioprospection for new antivirals, we tested nonribosomally biosynthesized polypeptide antibiotics in MDCK II cells for their actions on influenza A and B viruses (IAV/IBV). Only tolypin, a mixture of closely related 16-residue peptaibiotics from the fungus Tolypocladium inflatum IE 1897, showed promising activity. It was selected for further investigation and structural characterization byultrahigh performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HR-MS/MS) and ultrahigh performance liquid chromatography coupled to in-source collision-induced dissociation tandem mass spectrometry (UHPLC-isCID-HR-MS/MS), revealing 12 partially co-eluting individual peptides that were fully sequenced. Since tolypin-related efrapeptins are potent inhibitors of F1/Fo-ATPase, we screened tolypin for its toxicity against MDCK II cells and larvae of the greater wax moth Galleria mellonella. We found that a nontoxic concentration of tolypin (1 µg/mL) reduced the titer of two IBV strains by 4-5 log values, and that of an H3N2 strain by 1-2 log values, but the H1N1pdm strain was not affected. The higher concentrations of tolypin were cytostatic to MDCK II cells, shifted their metabolism from oxidative phosphorylation to glycolysis, and induced paralysis in G. mellonella, supporting the inhibition of F1/Fo-ATPase as the mode of action. Our results lay the foundations for future work to investigate the interplay between viral replication and cellular energy metabolism, as well as the development of drugs that target host factors.

Decoding the impact of neighboring amino acids on ESI-MS intensity output through deep learning

Peptide-level quantification using mass spectrometry (MS) is no trivial task as the physicochemical properties affect both response and detectability. The specific amino acid (AA) sequence affects these properties, however the connection between sequence and intensity output remains poorly understood. In this work, we explore combinations of amino acid pairs (i.e., dimer motifs) to determine a potential relationship between the local amino acid environment and MS1 intensity. For this purpose, a deep learning (DL) model, consisting of an encoder-decoder with an attention mechanism, was built. The attention mechanism allowed to identify the most relevant motifs. Specific patterns were consistently observed where a bulky/aromatic and hydrophobic AA followed by a cationic AA as well as consecutive bulky/aromatic and hydrophobic AAs were found important for the prediction of the MS1 intensity. Correlating attention weights to mean MS1 intensities revealed that some important motifs, particularly containing Trp, His, and Cys, were linked with low responding peptides whereas motifs containing Lys and most bulky hydrophobic AAs were often associated with high responding peptides. Moreover, Asn-Gly was associated with low response. The model predicts MS1 response with a mean average percentage error of ∼11 % and a Pearson correlation coefficient of ∼0.64. While dimer representation of peptide sequences did not improve predictive capacity compared to single AA representation in earlier work, this work adds valuable insight for a better understanding of peptide response in MS analysis. SignificanceMass spectrometry is not inherently quantitative, and the response of a compound relies not only on its concentration but also on the molecular composition. For mass spectrometry-based analysis of peptides, such as in bottom-up proteomics, this directly implies that the response cannot be used directly to quantify individual peptides. Moreover, the dependency of the response on the amino acid sequence of individual peptides remains poorly understood. Using a deep learning model based on a recurrent neural network with an attention mechanism, we here investigate how the presence of dimer motifs within a peptide affects the MS1 response through the analysis of intended equimolar peptide pools comprising almost 200,000 unique peptides in total. Not only do we identify certain dimer classes and specific dimers that substantially affect the MS1 response, but the model is also able to predict peptide intensity with low error rates within the independent test subset. The findings not only improve our understanding of the link between sequence and response for peptides but also highlight the potential of utilizing deep learning for developing methods allowing for absolute, label-free peptide quantification.

Peptide-Bound Glycative, AGE and Oxidative Modifications as Biomarkers for the Diagnosis of Alzheimer's Disease-A Feasibility Study.

Background: The diagnosis of Alzheimer's disease (AD) relies on core cerebrospinal fluid (CSF) biomarkers, amyloid beta (Aβ) and tau. As the brain is then already damaged, researchers still strive to discover earlier biomarkers of disease onset and the progression of AD. Glycation, advanced glycation end products (AGEs) and oxidative modifications on proteins in CSF mirror the underlying biological mechanisms that contribute to early AD pathology. However, analyzing free AGEs in the body fluids of AD patients has led to controversial results. Thus, this pilot study aimed to test the feasibility of detecting, identifying and quantifying differentially glycated, AGE or oxidatively modified peptides in CSF proteins of AD patients (n = 5) compared to a control group (n = 5). Methods: To this end, we utilized a data-dependent (DDA) nano liquid chromatography (LC) linear ion trap-Orbitrap tandem mass spectrometry (MS/MS) ) approach and database search that included over 30 glycative and oxidative modifications in four search nodes to analyze endogenous modifications on individual peptides. Furthermore, we quantified candidate peptide abundance using LC Quan. Results: We identified 299 sites of early and advanced glycation and 53 sites of oxidatively modified tryptophan. From those, we identified 17 promising candidates as putative biomarkers (receiver operating curve-area under the curve (ROC-AUC) > 0.8), albeit without statistical significance. Conclusions: The potential candidates with higher discrimination power showed correlations with established diagnostic markers, thus hinting toward the potential of those peptides as biomarkers.

Mechanically rigid metallopeptide nanostructures achieved by highly efficient folding

Natural proteins must fold into complex three-dimensional structures to achieve excellent mechanical properties vital for biological functions, but this has proven to be exceptionally difficult to control in synthetic systems. As such, the long-standing issue of low mechanical rigidity and stability induced by misfolding constrains the physical and chemical properties of self-assembling peptide materials. Here we introduce a mixed-chirality strategy that enhances folding efficiency in topologically interlocked metallopeptide nanostructures. The orderly entanglement of heterochiral peptide-derived linkers can fold into a compact three-dimensional catenane. These folding-mediated secondary structural changes not only generate biomimetic binding pockets derived from individual peptide strands but also result in strong chiral amplification by the tight interlocking manner. Notably, this strategic ‘chirality mutation’ alters their arrangement into tertiary structures and is pivotal in achieving exceptional mechanical rigidity observed in the metallopeptide crystals, which exhibit a Young’s modulus of 157.6 GPa, approximately tenfold higher than the most rigid proteinaceous materials in nature. This unusual nature is reflected in enhanced peptide-binding properties and heightened antimicrobial activities relative to its unfolded counterpart.

40 Preclinical evaluation of a novel dendritic cell vaccine for kidney cancer

Abstract Background Dendritic cell (DC)-based vaccines have been previously shown to promote therapeutic T cell responses in both preclinical tumor models and in cancer patients, where extended patient overall survival has been noted in many cases. The goal of the present study is to develop a novel DC-based vaccine as an effective immuno-oncology (IO) agent for the prevention/treatment of kidney cancer. We previously demonstrated that actin-binding protein profilin1 (Pfn1) is overexpressed in tumor-associated vascular endothelial cells (ECs), where it may serve as a prognostic factor in human clear cell renal cancer (ccRCC). We further established a direct causal relationship between EC Pfn1 dysregulation, immune microenvironment alterations, and tumor progression in mouse models of RCC. The current work specifically explores whether Pfn1-targeted DC-based vaccines are effective in preventing orthotopic RCC in mice. Methods To generate Pfn1-targeted DC vaccines, we harvested and matured DCs from Balb/C mouse bone marrow-derived precursor cells in cultures containing rmIL4 and rmGM-CSF. DCs loaded with individual Pfn1 synthetic peptides (these sequences were identified using three MHC class I/II peptide-binding algorithms) were injected into syngeneic mice on days 0, 7 and 14. Spleens were harvested from immunized mice on day 21 to isolate CD4+ and CD8+ T cells, with T cells then stimulated with unmanipulated DCs or DCs pulsed with individual immunizing peptides to detect specific T cell activation, as measured by IFN-gamma release quantitated by ELISA. Tissue histology was performed to assess immune-related adverse events (iRAE) in vaccinated mice as a safety index. For cancer studies, Balb/c mice were immunized with DCs loaded with pooled Pfn1 peptides a few days prior to establishing either subcutaneous or orthotopic RCC tumors. Tumor-bearing mice were subjected to an identical booster vaccine prior one week later in advance of euthanasia and study end-point analyses. Results Our studies show that vaccination of Balb/c mice with DCs pulsed with Pfn1 peptides elicit specific CD4+ and/or CD8+ T cell responses without promoting irAEs. When applied in the prophylactic setting, DC-Pfn1 peptide vaccines substantially slow the growth of subcutaneous RCC tumors. In support of these findings, we have also developed preliminary data supporting the therapeutic effects of DC/Pfn1-peptide vaccines in a murine orthotopic model of RCC. Conclusions In summary, our studies provide first evidence for Pfn1-targeted IO agent in the cancer setting. Given that the survival benefit for dual immune-check point inhibitors (ICI)/anti-angiogenic therapy remains modest and is limited to a small minority of ccRCC patients, Pfn1-targeted vaccines could represent a novel therapeutic agent for improved patient outcomes when applied alone or in combination with in-clinic ICI agents. DOD CDMRP Funding: yes