Search Results (295)

Accurate annotation of secreted and membrane proteins requires detecting N-terminal secretion signals, locating their cleavage sites, and distinguishing secretion-signal classes, while also predicting full transmembrane topology for both alpha-helical and beta-barrel proteins. Current tools typically address either whole-protein topology with a generic signal-peptide category or signal-peptide type classification without integrated topology annotation, leaving end-to-end labels incomplete when both features must be resolved together. Here, we present PRED-TMSdeep, a deep learning method that jointly predicts transmembrane topology and three signal peptide classes: secretory pathway/signal peptidase I (Sec/SPI), secretory pathway/signal peptidase II (Sec/SPII), and twin-arginine translocation/signal peptidase I (Tat/SPI). We introduce a two-step constrained decoding procedure that first detects transmembrane segments and signal peptides and then resolves global orientation and refines boundaries under stricter biological constraints. On redundancy-reduced datasets curated from the Orientation of Proteins in Membranes and the Protein Data Bank of Transmembrane Proteins, PRED-TMSdeep matches leading predictors for segment-level topology while improving signal peptide classification and yielding the highest overall top-1 cleavage-site accuracy. Top-1 cleavage-site accuracy reached 89.2%, compared with 84.7% for TMbed and 86.2% for SignalP 6.0, mainly reflecting strong performance on the predominant Sec/SPI class. The software is available as a web server and a batch command-line tool with pretrained models and reproducible workflows. Full article

Background: Eggshell membrane peptides (ESMPs) are natural bioactive compounds with reported chondroprotective properties. However, their regulatory effects on canine chondrocytes remain unclear. This study investigated ESMP in an interleukin-1β (IL-1β)-induced inflammatory model of canine chondrocytes. Methods: Chondrocytes were assigned to control (Cont), IL-1β, and ESMP + IL-1β groups. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay. NF-κB p65 nuclear translocation was evaluated by immunofluorescence staining. Real-time quantitative PCR (RT-qPCR) and Western blotting (WB) were used to measure mRNA and protein expression levels, respectively. Results: ESMP inhibited IL-1β-induced NF-κB p65 nuclear translocation and reduced the IL-1β-induced increases in interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and matrix metalloproteinase-13 (MMP-13) at both mRNA and protein levels. ESMP also decreased IL-6, nitric oxide (NO), and prostaglandin E₂ (PGE₂) levels in culture supernatants. ESMP reversed the IL-1β-induced reduction in type II collagen α1 chain (COL2A1) and aggrecan (ACAN) expression at both transcriptional and protein levels. Conclusions: ESMP attenuates IL-1β-induced inflammatory responses and extracellular matrix degradation in canine chondrocytes, potentially associated with suppression of NF-κB p65 nuclear translocation. This supports its potential application in promoting joint health in dogs. Full article

The plastid stroma-localized chaperone HSP90C is essential for maintaining chloroplast proteostasis and facilitating protein translocation. Prior research has established HSP90C’s imperative role in the SEC translocase-dependent transport of the photosystem II subunit PsbO1 and its interaction with the SEC1 translocase motor protein SecA1. However, the exact mechanism of this interaction remains to be explored. In this study, we delineated the interactional mode of HSP90C and SecA1 with the model client protein. Yeast two-hybrid and in vitro ATPase activity analyses with purified proteins revealed PsbO1 may bind to HSP90C at multiple sites, including the DPW motif within the C-terminal extension (CTE) region, suggesting a possible client-loading mechanism unique to plastid orthologs. We also confirmed that glycine-646 is important in mediating substrate interaction, though it conferred a much weaker binding than the CTE region, thereby elucidating a critical role for the amino acid whose mutation resulted in visible plant phenotypes. Our in vitro biochemical assays also demonstrated that the stromal intermediate form of PsbO1 with the thylakoid signal peptide (tSP) significantly enhanced SecA1 ATPase activity, suggesting a preferential binding to the motor protein. On the other hand, the mature domain of the PsbO1, excluding the tSP sequence, inhibited HSP90C ATPase activity. We also observed the HSP90C-PsbO1-SecA1 ternary complex was stabilized by the presence of the client tSP. This work therefore provides new insights into the functional mechanisms of HSP90C and its contribution to chloroplast stromal protein stabilization and thylakoid protein transport. Full article

Lactylation is an emerging lactate-derived post-translational modification that may link tumour metabolic reprogramming, epigenetic regulation and DNA damage repair. Enhanced glycolysis and lactate accumulation are common in many tumours, and lactate has been reported to induce histone and non-histone lactylation in specific experimental contexts. Recent studies suggest that lactylation is associated with several DNA repair pathways, including base excision repair/single-strand break repair, nucleotide excision repair, homologous recombination and non-homologous end joining, and may contribute to therapy resistance in selected cancer models. Specifically, XRCC1 lactylation has been reported to promote nuclear translocation and repair activity in glioblastoma models; H4K12 lactylation has been linked to PARP inhibitor resistance through RAD23A activation in ovarian cancer models; and BLM lactylation has been associated with enhanced homologous recombination repair in bladder cancer models. Lactylation of NBS1, RAD51 and XLF has also been implicated in DNA repair regulation in specific experimental systems, although some mechanistic links are inferred from pathway activation or functional rescue experiments rather than directly demonstrated across multiple tumour types. These findings suggest that lactylation may modulate DNA repair and therapeutic response in a context-dependent manner. Targeting lactate metabolism, transport and lactylation regulators, including LDHA, MCT1/4, ACAT1, AARS1 and GCN5, or using site-specific lactylation-inhibiting peptides may improve chemotherapy and PARP inhibitor efficacy, but clinical translation remains limited by heterogeneity, metabolic plasticity, toxicity and insufficient validation. Full article

Background: The translocation of diet-derived antigens from the maternal intestine to breast milk represents a primary gateway for neonatal immune priming, yet the structural basis for why certain proteins survive this transit while others do not remains poorly understood. This review introduces the “Survivor Peptide” hypothesis, proposing that specific food allergens possess intrinsic “stability architectures” that enable them to resist maternal digestion and navigate the gut–mammary axis to reach the infant in an immunologically active form. Methods: We analyzed the current literature regarding the detection and structural characteristics of food allergens in human milk. Integrating evidence from 26 major sources, we performed an in silico structural analysis of five representative “survivor” proteins: Gal d 1 (egg white), Bos d 5 (cow’s milk), Gal d 6 (egg yolk), Tri a 19 (wheat), and tropomyosin (Der p 10-mite/shellfish). High-resolution 3D models were retrieved from the Protein Data Bank and AlphaFold2, and then visualized in UCSF ChimeraX to map stability anchors, including disulfide bonds and hydrophobic clusters, against solvent-accessible IgE-binding epitopes. Results: We identified and categorized allergens into distinct Molecular Resilience Architectures: the “Covalent Cage” (Gal d 1), defined by dense disulfide stapling, the “Glycoprotein Shield” (Gal d 6), utilizing yolk-matrix structural anchors, the “Topological Shield” (Bos d 5), characterized by a stable β-barrel, and “Coiled-Coil Rigidity” (Der p 10). These frameworks protect large, immunogenic fragments that maintain the spatial arrangement required for IgE cross-linking. Conclusions: Allergen persistence in the gut–mammary axis is dictated by a protein’s intrinsic structural architecture. Identifying these stability fingerprints provides a unified theory for allergen persistence and offers a path for refining component-resolved diagnostics and neonatal oral tolerance strategies. Full article

Skin wound healing is a complex and highly coordinated biological process involving inflammation, cell migration and proliferation, angiogenesis, extracellular matrix remodeling and tissue regeneration. While the zebrafish-derived antimicrobial peptide AP10W exhibits broad-spectrum antimicrobial properties, its potential in tissue repair remains unexplored. Herein, we demonstrate that AP10W possesses intrinsic wound-healing capabilities, providing a preliminary investigation into its underlying mechanisms. In this study, using a full-thickness murine wound model and in vitro cell-based assays to evaluate the effects of AP10W on fibroblasts, keratinocytes, endothelial cells, and macrophages, we found that AP10W significantly promoted fibroblast and keratinocyte migration and proliferation. Furthermore, it enhanced endothelial cell motility, survival, and tube formation, while upregulating key pro-angiogenic factors, including Vascular endothelial growth factor A (VEGFA), Platelet-derived growth factor (PDGF), and Fibroblast growth factor 2 (FGF2). Concurrently, AP10W drove macrophage reprogramming from a pro-inflammatory M1 phenotype toward a pro-healing M2 state, as evidenced by upregulated Arginase-1 (Arg-1) and Interleukin-10 (Il-10) expression, alongside attenuated Tumor necrosis factor-alpha (Tnf-α), Interleukin-1 beta (Il-1β), Interleukin-6 (Il-6), and Inducible nitric oxide synthase (iNOS) levels. In vivo, the topical application of AP10W accelerated wound closure, markedly improving re-epithelialization, collagen deposition, vascularization, tissue perfusion, and skin appendage regeneration. Preliminary mechanistic studies revealed that AP10W increased YAP expression and nuclear translocation; conversely, the pharmacological inhibition of YAP significantly abrogated these pro-healing effects. Collectively, our findings identify AP10W as a multifunctional peptide with potent wound-healing properties, positioning it as a promising candidate for wound therapy. Full article

CARP-1, a perinuclear phospho-protein, is a biphasic regulator of cell survival and apoptosis signaling. We previously found that UV cross-linking of proteins from HeLa cervical cancer cells resulted in STAT3 interacting with the CARP-1 (614–638) peptide. Mutagenesis and co-IP-WB experiments revealed that CARP-1 interacts with a 40-amino-acid epitope from positions 441–480 (CE Epitope) located in the STAT3 DNA-binding domain. Overexpression of mutant STAT3 with in-frame deletion of the CE epitope (Gst-STAT3 (ΔCE) mutant), but not Gst-STAT3 (WT), failed to translocate to the nucleus in IL-6-treated cells. The small GTPase p21Rac1 interacts with and regulates STAT3 activation and nuclear translocation. Here we report the interaction of p21Rac1 with the CE epitope of STAT3 and the CARP-1 (600–650) region, suggesting that CARP-1 is part of a dynamic STAT3-p21Rac1 complex that functions in STAT3 activation and nuclear translocation. Expression of a STAT3 (ΔCE) mutant abolished STAT3 Y705 phosphorylation in cells that were treated with EGF or IL-6. Fine mapping revealed that scrambling the CE epitope peptide or a small peptide from positions 456–465 within the CE epitope resulted in abrogation of STAT3 Y705 phosphorylation by IL-6. Moreover, STAT3 phosphorylation by EGF or IL-6 was diminished in multiple CARP-1 null cancer cells. Importantly, incubation of a TAT-tagged STAT3 (454–467) peptide but not its scrambled version resulted in a reduction in STAT3 Y705 phosphorylation by IL-6/EGF. Taken together, our data demonstrates that the STAT3 CE epitope interacts with CARP-1 and p21Rac1, harbors novel sequences that activate STAT3 and promotes its nuclear translocation by IL-6/EGF. Full article

Background/Objectives: Mitochondria are dynamic organelles that continuously undergo balanced cycles of fusion and division to maintain optimal function. Mitochondrial division is mediated by Dynamin-Related Protein 1 (DRP1), a cytosolic large GTPase whose phosphorylation at serine 616 (DRP1-S616Ⓟ) promotes its translocation to the outer mitochondrial membrane and organelle division. Dysregulated mitochondrial division disrupts cellular homeostasis and contributes to disease pathogenesis, including cancer. Our prior work demonstrated that the oncogene-induced mitogen-activated protein kinase (MAPK) pathway constitutively phosphorylates DRP1 at serine 616, which is essential to cellular transformation and correlates with oncogene status in patient tissues. Similarly, DRP1-S616Ⓟ is subject to pharmacologic control by targeted therapies against oncogenic MAPK signaling. Methods: Building upon this foundation, we developed and characterized a recombinant murine monoclonal antibody (referred to as 3G11) with high specificity for human DRP1-S616Ⓟ, raised against a peptide derived from the human DRP1 sequence. Results: Using diverse experimental platforms, we demonstrate the robust utility of 3G11 to detect DRP1-S616Ⓟ in melanoma cell extracts and isolated organelles. Immunofluorescence revealed that pharmacologic inhibition of oncogenic MAPK signaling reduces DRP1-S616Ⓟ levels, which correlates with mitochondrial hyperfusion, while immunohistochemistry showed that elevated DRP1-S616Ⓟ expression in human tissues correlates with BRAF^V600E disease. Conclusions: 3G11 is a new recombinant antibody for detecting DRP1-S616Ⓟ and supports studies of mitochondrial division in cancer. Together, these findings establish 3G11 as a specific, versatile, renewable, and cost-effective tool for studying mitochondrial division, with strong potential for clinical applications. Full article

Inflammation occurs throughout the entire process of atherosclerosis. So, reducing the inflammatory response remains one of the main therapeutic strategies for atherosclerosis. Chensinin-1b, a derivative of the natural antimicrobial peptide extracted from the skin secretions of the Rana chensinensis, has been shown to effectively mitigate the occurrence of inflammatory responses. Here, the anti-inflammatory activity of the peptide by suppressing the canonical Wnt/β-catenin signaling pathway was investigated in ox-LDL-induced AS. The results showed that the classical Wnt/β-catenin signaling pathway was activated in ox-LDL-induced THP-1-derived foam cells. The inhibitor of Wnt reduced the release of pro-inflammatory cytokines by downregulating the NF-κB signaling pathway. Cyclooxygenase-2, a target protein of the canonical Wnt/β-catenin signaling pathway, inhibited the phosphorylation of NF-κB. Chensinin-1b and its analogs effectively downregulated the expression of Wnt signaling proteins by inhibiting the nuclear translocation of the key pathway protein β-catenin, resulting in a decrease in COX-2 expression and simultaneously reducing the release of pro-inflammatory cytokines. In summary, our study suggests the potential of chensinin-1b and its analogs as therapeutic agents for AS. Full article

Botulinum neurotoxin injections are used off-label to treat chronic pain, but their efficacy is limited and paralytic effects restrict clinical utility in these applications. Here, we investigated whether combining the light chain and translocation domains of botulinum neurotoxin A (BoNT/A) with the GM1-binding B subunit of cholera toxin would be beneficial in silencing pain-associated sensory neurons. Chimeric ChoBot was assembled via a coiled-coil linking technology and was shown to retain the enzymatic activity of BoNT/A in vitro and in vivo. In cultured dorsal root ganglion neurons, ChoBot cleaved SNAP25 in a calcitonin gene-related peptide (CGRP)-rich subpopulation of sensory neurons, resulting in marked inhibition of CGRP release. ChoBot had a lesser effect on the compound muscle action potentials of the rat gastrocnemius muscle than BoNT/A following subcutaneous injections. In rat models of pain, including chemotherapy-induced peripheral neuropathy, intraplantar administration of ChoBot significantly attenuated mechanical allodynia. Immunohistochemical analysis confirmed SNAP25 cleavage in NF200- and CGRP-expressing sensory fibres in the epidermis following a single injection. ChoBot also mediated SNAP25 cleavage in human neuroblastoma cells in culture. Together, these findings indicate that ChoBot enables a silencing of pain-associated sensory pathways, providing a new strategy for the development of new long-lasting analgesics for chronic pain. Full article

Alcohol exposure affects immune regulation and tissue homeostasis. Antimicrobial peptides (AMPs) are essential components of innate immunity, not only defending against pathogens but also modulating processes such as inflammation. However, their tissue-specific regulation in response to alcohol remains poorly characterized, particularly in humans after acute intoxication. We evaluated the expression of AMPs in the peripheral blood of patients with alcohol use disorder (AUD, n = 9), individuals with acute alcohol consumption (AAC, n = 9), and controls using quantitative polymerase chain reaction (qPCR). Additionally, we analyzed AMP expression in selected tissues of mice exposed to chronic ethanol feeding (National Institute on Alcohol Abuse and Alcoholism model for 5 days) and performed a systematic review of AMP regulation in alcohol-related disorders (2005–2025; n = 36 studies, reflecting a limited and heterogeneous body of available evidence). Human cathelicidin antimicrobial peptide (LL-37), lipopolysaccharide-binding protein (LBP), and bactericidal/permeability-increasing protein (BPI) were significantly upregulated in patients with AUD, whereas LL-37 and LBP were significantly upregulated in AAC. In the livers of ethanol-fed mice, LEP2, LCN2, and LBP levels were markedly increased, whereas LL-37 and LEP1 were downregulated. Duodenal tissue exhibited upregulation of DEFB1. In adipose tissue, DEFA2 was significantly increased in peripheral depots, whereas only LCN2 was upregulated in brain tissue. The systematic review demonstrated complex, heterogeneous, and organ-dependent AMP regulation and also highlighted the paucity of human data on AAC, a gap that our study partially addresses. Our results are consistent with the hypothesis that selected AMPs may serve as candidate markers of organ damage or microbial translocation and as possible therapeutic targets, a hypothesis that requires confirmation in larger, adequately powered studies. Full article

Background/Objectives: This study aimed to compare the biological effects of two amelogenin-derived peptides—the leucine-rich amelogenin peptide (LRAP) and a synthetic peptide (SP)—on human dental pulp stem cells (hDPSCs) and human bone marrow–derived mesenchymal stem cells (hBMSCs). The investigation focused on cell viability, osteogenic differentiation, mineralization, gene expression, and β-catenin expression. Methods: hDPSCs and hBMSCs were cultured in osteogenic medium and treated with LRAP and SP at 1, 5, 10, 50, and 100 ng/mL. Cytotoxicity was assessed by MTT assay, while osteogenic differentiation was evaluated by alkaline phosphatase (ALP) activity and Alizarin Red S staining. Gene expression of RUNX2, COL1A1, OCN, MEPE, and DMP1 was quantified by qPCR. β-catenin localization was analyzed by immunofluorescence. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test (p < 0.05). Results: Both peptides exhibited good biocompatibility with hBMSCs, while high concentrations (≥50 ng/mL) reduced hDPSC viability. In both cell types, LRAP and SP increased ALP activity and mineral deposition in a concentration-dependent manner, with the greatest effects at 10 ng/mL. LRAP significantly upregulated osteogenic (RUNX2, COL1A1, OCN) and odontogenic (MEPE, DMP1) gene expression in hDPSCs. Immunofluorescence revealed nuclear β-catenin translocation in hDPSCs and membrane-associated accumulation in hBMSCs, indicating activation of canonical and non-canonical pathways, respectively. Conclusions: LRAP and SP promote osteogenic differentiation through distinct cell-type-specific signaling mechanisms, highlighting their potential as biomimetic agents for mineralized tissue regeneration. Full article

Background: Chronic kidney disease (CKD) represents a state of persistent, sterile low-grade inflammation in which sustained innate immune activation accelerates renal decline and cardiovascular complications. Diet-induced gut dysbiosis and intestinal barrier dysfunction lower mucosal immune tolerance, promote metabolic endotoxemia, and position the gut as an upstream modulator of systemic inflammatory signaling along the gut–kidney axis. Scope: Most studies address microbiota-derived metabolites, food-derived bioactive peptides, or omega-3 fatty acids separately. This review integrates evidence across these domains and examines their convergent actions on epithelial barrier integrity, immune polarization, oxidative-inflammatory stress, and inflammasome-dependent pathways relevant to CKD progression. Key mechanisms: CKD-associated dysbiosis is characterized by reduced short-chain fatty acid (SCFA) production and increased generation and accumulation of uremic toxins and co-metabolites, including indoxyl sulfate, p-cresyl sulfate, trimethylamine N-oxide, and altered bile acids. Reduced SCFA availability weakens tight junction-dependent barrier function and regulatory immune programs, favoring Th17-skewed inflammation and endotoxin translocation. Bioactive peptides modulate inflammatory mediator networks and barrier-related pathways through effects on NF-κB/MAPK signaling and redox balance, while omega-3 fatty acids and specialized pro-resolving mediators support resolution-phase immune responses. Across these modalities, shared control points include barrier integrity, metabolic endotoxemia, oxidative stress, and NLRP3 inflammasome activation. Conclusions: Although evidence remains heterogeneous and largely preclinical, combined nutritional modulation targeting these convergent pathways may offer greater immunomodulatory benefit than isolated interventions. Future multi-omics-guided, factorial trials are required to define responder phenotypes and translate precision immunonutrition strategies into clinical CKD care. Full article

This review synthesizes research on nuclear factor erythroid 2-related factor 2 (Nrf2) in intestinal health across human, livestock, and mouse models. The Nrf2 signaling pathway serves as a master regulator of cellular antioxidant defenses and a key therapeutic target for intestinal inflammatory disorders, including ulcerative colitis and Crohn’s disease. The interplay between oxidative stress, Nrf2 signaling, and NF-κB inflammatory cascades represents a critical axis in the pathogenesis and resolution of intestinal inflammation. Under normal physiological conditions, Nrf2 remains sequestered in the cytoplasm by Kelch-like ECH-associated protein 1 (Keap1), which facilitates its ubiquitination and proteasomal degradation. However, during oxidative stress, reactive oxygen species (ROS) and electrophilic compounds modify critical cysteine residues on Keap1, disrupting the Keap1-Nrf2 interaction and enabling Nrf2 nuclear translocation. Once in the nucleus, Nrf2 binds to antioxidant response elements (ARE) in the promoter regions of genes encoding phase II detoxifying enzymes and antioxidant proteins, including heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), and glutamate-cysteine ligase. This comprehensive review synthesizes current evidence demonstrating that activation of Nrf2 signaling confers protection against intestinal inflammation through multiple interconnected mechanisms: suppression of NF-κB-mediated pro-inflammatory cascades, enhancement of cellular antioxidant capacity, restoration of intestinal barrier integrity, modulation of immune cell function, and favorable alteration of gut microbiota composition. We systematically examine a diverse array of therapeutic agents targeting Nrf2 signaling, including bioactive peptides, natural polyphenols, flavonoids, terpenoids, alkaloids, polysaccharides, probiotics, and synthetic compounds. The mechanistic insights and therapeutic evidence presented underscore the translational potential of Nrf2 pathway modulation as a multi-targeted strategy for managing intestinal inflammatory conditions and restoring mucosal homeostasis. Full article

Background: Congenital disorders of glycosylation (CDG) are rare inborn errors of metabolism with multisystemic manifestations. SSR4-CDG is an ultra-rare X-linked subtype caused by pathogenic variants in SSR4, a component of the translocon-associated protein (TRAP) complex essential for protein translocation and N-glycosylation. Case presentation: We report a two-year-old Malaysian male presenting with global developmental delay, central hypotonia, microcephaly with complete agenesis of the corpus callosum, recurrent infections, bilateral vesicoureteral reflux, and failure to thrive. Growth parameters (weight, length, and head circumference) were persistently below the expected percentiles, indicating postnatal growth restriction. Initial metabolic and biochemical investigations for global developmental delay were unremarkable, apart from mild hyperammonemia. Transferrin isoform analysis demonstrated a type I CDG pattern, raising suspicion of a glycosylation defect. Results: Transferrin glycopeptide LC–MS/MS showed impaired N-glycan occupancy at both glycosylation sites (Asn432 and Asn630), with reduced fully sialylated glycoforms and increased non-glycosylated peptides. Targeted metabolomics using triple quadrupole LC–MS/MS revealed systemic abnormalities, including elevated arginine and phenylalanine, reduced glutamate, increased lysophosphatidylcholine (C24:0-LPC), and generalized depletion of free and acylcarnitines. Whole-exome sequencing identified a novel hemizygous SSR4 variant (c.98del; p.Pro33LeufsTer23) on the X chromosome, predicted to produce a truncated, nonfunctional protein. Conclusions: This is the first Malaysian patient with SSR4-CDG, comprehensively characterized using a multi-omics diagnostic workflow. The integration of glycoproteomics, metabolomics, and exome sequencing provided a detailed biochemical fingerprint that expands the clinical, genetic, and metabolic spectrum of SSR4-CDG and demonstrates the diagnostic and translational value of multi-omics approaches in inborn errors of metabolism. Full article