Comparative analysis of proteomics and transcriptomics reveals novel mechanism underlying the antibacterial activity and immune-enhancing properties of horse milkChen, Gulbahar, Ding
et alFront Nutr (2025) 12, 1512669
Abstract: Horse milk is a highly valuable organic food that is a promising alternative to cow milk, exhibiting plenty of healthy and immune benefits to human. However, identification of proteins associated human wellness and underlying molecular mechanism in horse milk remain unclear.Label-free mass spectrometry-based protein quantification technology was employed to investigate protein composition of animal milk, including cow, goat, camel and horse milk. Prokaryotic expression and disk diffusion assay were applied to acquire and evaluate in vitro antimicrobial activity of candidate proteins. RAW264.7 macrophage model cell line was used to validate effect of proteins on cytotoxicity, apoptosis and immune induction. ROS probe detected cell ROS change and RT-qPCR verified expression of immune response genes induced by proteins. Microscopy was used to observe the effects of protein on the morphological characteristics of bacteria, further transcriptome analysis was performed to investigate transcriptional changes of bacteria induced by candidate proteins.A total of 1,335 proteins was identified in cow, goat, camel and horse milk. GO enrichment analysis showed that the proteins related to protein degradation were highly expressed in horse milk compared to other three types of milk, contributing to easier assimilation and palatability. KEGG analysis showed that horse milk contained abundant antimicrobial associated proteins relevant to pathogenic bacterial resistance, leading to the decreased risk of pathogenic diseases. A higher accumulation of proteins associated with caffeine metabolism, amino acid biosynthesis, and glycolysis/gluconeogenesis in horse milk contributes to its distinctive flavor. Notably, highly expressed proteins in horse milk were closely linked to immune signaling pathways, functioning as immune modulators. Importantly, we identified four highly expressed antimicrobial associated proteins in horse milk including LPO, B2M, CD14 and PGL, among them, PGL functioned dually by in vitro antibacterial activity and immune activation. Further transcriptome analysis demonstrated that PGL exerted significant transcriptional changes to bacteria. Enrichment analysis showed PGL could inhibit growth of P. aeruginosa and E. coli by repressing the biosynthesis of secondary metabolites.Comparative proteomics revealed immune enhancement and nutrient composition of horse milk compared to cow, goat and camel milk. Identification of PGL showed antibacterial activity and potential medicinal value.Copyright © 2025 Chen, Gulbahar, Ding, Nie and Gao.
Lower degree of microsatellite instability in colorectal carcinomas from MSH6-associated Lynch syndrome patientsHelderman, Strobel, Bohaumilitzky
et alMod Pathol (2025)
Abstract: Numerous observational and molecular studies focusing on Lynch syndrome (LS) have revealed significant variation in the phenotype and molecular characteristics among carriers of pathogenic variants in mismatch repair genes (path_MMR). Recently, we demonstrated that colorectal carcinomas in path_MSH6 carriers exhibit fewer insertion/deletion mutations compared to CRCs from other MMR groups, raising the question of whether MSH6-mutated CRCs might display a relatively lower degree of microsatellite instability (MSI). Mutations at twenty coding microsatellites (cMS) were analyzed in 39 MSH6-, 18 MLH1-, 16 MSH2- and 22 PMS2-mutated CRCs and 35 sporadic MSI CRCs, and mutation frequencies and mutant allele ratios were compared among the different MMR-deficient groups. Considering factors such as HLA-A*02:01 type, B2M status, and the anticipated immunogenicity of frameshift peptides derived from cMS mutations, the identified cMS mutation profiles of MSH6-mutated CRCs were further investigated to assess their potential impact on immunotherapeutic strategies. MSH6-mutated CRCs exhibited lower mutation frequencies and mutant allele ratios across most cMS. Variation in cMS mutation patterns was observed both between different tumor regions and between tumor tissue and adjacent adenomatous tissue. The cMS mutations in MSH6-mutated CRCs demonstrated inverse correlations with the predicted immunogenicity of the resulting frameshift peptides, which may suggest negative selection of cell clones bearing highly immunogenic frameshift peptides. Overall, MSH6-mutated CRCs display a relatively lower degree of MSI and represent a biologically distinct subgroup of LS-associated CRCs. This lower MSI level may implicate an altered immune response compared to other MSI CRCs, which could have theoretical implications for the success of immunotherapy in MSH6-mutated CRCs. Future studies should carefully evaluate this possibility. If confirmed, these results would reinforce the notion of classifying LS as distinct syndromes associated with specific MMR genes.Copyright © 2025. Published by Elsevier Inc.
Deleterious knock-outs in the HLA class I antigen processing and presentation machinery induce distinct changes in the immunopeptidomeShapiro, Maschke, Michaux
et alMol Cell Proteomics (2025)
Abstract: The human leukocyte antigen (HLA) processing and presentation machinery (APPM) is altered in various diseases and in response to drug treatments. Defects in the machinery may change presentation levels or alter the repertoire of presented peptides, globally or in an HLA allele restricted manner, with direct implications for adaptive immunity. In this study, we investigated the immunopeptidome landscape across a panel of isogenic HAP1 cell line clones each with a knock-out of a single gene encoding a key protein in the APPM, including B2M, TAP1, TAP2, TAPBP, IRF2, PDIA3, ERAP1, GANAB, SPPL3, CANX, and CALR. We applied immunopeptidomic and proteomic to assess the successful gene knock-outs on the protein level, to understand how these proteins participate in antigen presentation, and to contextualize protein expression and antigen presentation. We validated the absence of the knocked-out proteins in the respective samples and found that knocking-out an APPM component leads to the loss of peptide subsets that are normally presented on the control wild type cells. We assessed the immunopeptidomes qualitatively and quantitatively, considering factors like peptide diversity, peptide length distribution, and binding affinity to the endogenously expressed HLA alleles in HAP1 cells. We demonstrated a prominent HLA allele-specific alterations in several knock-out conditions. The absence of CALR, CANX, and TAP1 led to significant changes in HLA allele-specific presentation levels. Overall, this work represents the first systematic analysis of how the absence of individual APPM components, knocked out in a single cell line under controlled conditions, affects the peptidome. This approach could facilitate the creation of predictive tools capable of prioritizing HLA-bound peptides likely to be presented when presentation defects occur, such as in cancer and viral infections.Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.
Peptidomic profiling of mesenchymal stem cell-derived extracellular vesicles and anti-inflammatory activity of degraded peptidesChu, Xiao, Xun
et alInt Immunopharmacol (2025) 152, 114452
Abstract: Mesenchymal stem cell derived extracellular vesicles (MSC-EVs) are key paracrine mediators involved in various autoimmune diseases. While current research on EVs predominantly focuses on their protein and nucleic acid components, small peptides received less attention. In this study, we found IFN-γ-treated MSC-EVs, as engineered EVs, exhibit better anti-inflammatory effects both in vitro and in vivo. Through LC-MS/MS and bioinformatics analysis, we identified four peptides-C3-1, C3-2, B2M-1, and IFIT3-1-that are highly expressed in IFN-γ-treated MSCs-EVs. These peptides significantly mitigate the proliferation inhibition of HUVEC cells induced by H₂O₂ and enhance their migratory capacity. Furthermore, they downregulate the expression of inflammatory cytokines TNF-α and IL-6 in H₂O₂-induced oxidative stress models of HUVEC and LPS-induced inflammatory models of RAW264.7 macrophages. The peptides also upregulate p-AKT and HIF-1α, with C3-1 demonstrating superior anti-inflammatory efficacy in both cell models. Consistent with the in vitro findings, in vivo experiments revealed that C3-1 reduces LPS-induced inflammatory cell infiltration in liver tissue and restores hepatocyte structural integrity, as evidenced by HE-stained liver sections. Western blot analysis further confirmed that C3-1 upregulates p-AKT expression and suppresses inflammatory cytokines. Collectively, these findings suggest that C3-1 exerts its anti-inflammatory effects via activation of the AKT signaling pathway and regulation of TNF-α and IL-6. This study not only highlights the anti-inflammatory potential of IFN-γ-treated MSC-derived EVs but also identifies C3-1 as a promising candidate for anti-inflammatory drug development. Notably, this is the first study to identify degraded peptides within EVs, providing a foundation for future research in this area.Copyright © 2025 Elsevier B.V. All rights reserved.
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