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PubMed2026

Integrated Multi-omics Profiling of 2,4-dinitrochlorobenzene (DNCB)-induced Atopic Dermatitis in Mice Reveals a Coordinated Network of Barrier Dysfunction, Immune Activation, and Metabolic Reprogramming.

Atopic dermatitis (AD) is caused by a combination of epidermal barrier defect and immune imbalance. However, the molecular networks between these structural abnormalities and metabolic variations are unclear. This study aim of this research was to examine the concurrent molecular alterations in skin barrier damage and metabolic disorders in an AD-like mouse model by a multi-omics strategy. A 2,4-dinitrochlorobenzene (DNCB)-induced AD-like mouse model was established and the skin tissues were examined through the combination of transcriptomic, quantitative proteomic, and metabolomic analyses. Cross-omics correlation and network analyses were performed to identify consistently abnormal molecular pathways and crucial regulatory molecules. DNCB treatment caused severe epidermal hyperplasia, and prominent infiltration of CD3⁺ T cells, F4/80⁺ macrophages, and mast cells. Transcriptomic and proteomic analysis indicated significant disruption in keratinocyte differentiation, extracellular matrix organization, and cornified envelope formation pathways. Combined analysis detected 171 molecules which were simultaneously altered at both mRNA and protein levels, and network analysis identified FLG2 and KRT6B as central barrier-related molecules. Pathway enrichment analysis consistently showed the participation of AMPK and PPAR signaling pathways. Metabolomic analysis also revealed coordinated changes in lipid and amino acid metabolism which were closely associated with cornified envelope-associated genes and collagen-modifying enzymes. These findings indicate a close relationship between barrier, immune and metabolic regulation in DNCB-induced dermatitis and provide a multi-omics resource for future mechanistic studies of atopic skin inflammation.

Animals↗
PubMed2026

Multi-omics analysis reveals Protein Kinase A-associated regulatory remodeling during adaptation of Trichoderma reesei to lignocellulosic substrate.

The filamentous fungus Trichoderma reesei is a major industrial source of holocellulolytic enzymes, and its response to complex carbon sources is regulated by nutrient-sensing mechanisms, including the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway. Here, we integrated transcriptomics, quantitative proteomics, and phosphoproteomics to analyze PKAc1-associated responses in the parental strain QM9414 and a Δpkac1 strain cultivated under glucose or sugarcane bagasse conditions. Deletion of pkac1 was associated with altered growth-related phenotypes and reduced extracellular activities of selected biomass-depolymerizing enzymes. Multi-omics profiling revealed condition-dependent changes affecting subsets of carbohydrate-active enzymes (CAZymes) genes and proteins, nutrient transporters, stress-associated proteins, and regulatory factors. Phosphoproteomics identified phosphorylation-state changes associated with pkac1 deletion, including reduced phosphorylation at sites enriched for the PKA consensus motif. In silico peptide docking was used to prioritize candidate PKAc1-associated substrates for future validation, including a Sec 7-derived peptide with favorable docking behavior relative to the control peptide. Together, these data support a working model in which PKAc1 contributes to regulatory and phosphorylation-state remodeling during adaptation to sugarcane bagasse, with effects on the magnitude and/or timing of selected CAZyme-related outputs in T. reesei.

Trichoderma↗
PubMed2005

Data analysis methods for detection of differential protein expression in two-dimensional gel electrophoresis.

The recent development of microarray technology has led statisticians and bioinformaticians to develop new statistical methodologies for comparing different biological samples. The objective is to identify a small number of differentially expressed genes from among thousands. In quantitative proteomics, analysis of protein expression using two-dimensional gel electrophoresis shows some similarities with transcriptomic studies. Thus, the goal of this study was to evaluate different data analysis methodologies widely used in array analysis using different proteomic data sets of hundreds of proteins. Even with few replications, the significance analysis of microarrays method appeared to be more powerful than the Student's t test in truly declaring differentially expressed proteins. This procedure will avoid wasting time due to false positives and losing information with false negatives.

Animals↗
PubMed2026

Functional mapping of the Trypanosoma cruzi serinome by fluorophosphonate activity-based protein profiling.

Serine hydrolases (SHs) constitute one of the largest enzyme superfamilies in eukaryotes, yet their roles in Trypanosoma cruzi, the causative agent of Chagas disease, remain largely uncharacterized. Here, we report an activity-based chemoproteomic map of the T. cruzi epimastigote serinome by combining genome-informed in silico curation with whole-cell activity-based protein profiling (ABPP) using a panel of cell-permeable fluorophosphonate (FP)-alkyne probes. Whole-cell labelling followed by label-free quantitative proteomics (LFQ-MS) identified 37 enriched SH-like proteins, including 35 with conserved or partially conserved catalytic triad/dyad features, spanning lipases, peptidases, esterases, and previously uncharacterized hydrolases. The 35 SHs represent approximately 63% of the 56 predicted SHs retained after catalytic-site curation. Domain architecture analysis revealed broad structural diversity, while orthologue-based localization data suggested association with multiple subcellular compartments, including glycosomal, mitochondrial, and endosomal localizations. Gene Ontology enrichment highlighted lipid metabolic and catabolic processes as dominant functional themes, and protein-protein interaction network analysis supported functional connectivity among the captured enzymes. Several identified SHs, including oligopeptidase B, prolyl oligopeptidase Tc80, serine carboxypeptidase CPB1, and phospholipase A1 (PLA1) have previously been characterized in trypanosomatids, with roles linked to parasite virulence or host-pathogen interactions. Together, these findings establish a fluorophosphonate-based chemoproteomic resource for the kinetoplastid community and prioritize probe-accessible active T. cruzi SHs for future functional validation and antiparasitic inhibitor discovery.

Activity-based protein profiling↗
PubMed2026

Glycogen-dependent demixing of frog egg cytoplasm at increased crowding.

Crowding increases the tendency of macromolecules to aggregate and phase separate, and regulated crowding contributes to subcellular organization and stress response. To explore the effect of crowding in a well-characterized model cytoplasm, we developed methods to concentrate the macromolecule components of Xenopus egg extracts without changing small molecules. Egg cytoplasm contains a high concentration of glycogen that serves as an energy store for early development. When crowding was increased 1.4×, the egg cytoplasm demixed into two liquid phases of approximately equal volume, one of which was highly enriched in glycogen. Glycogen hydrolysis prevented and reversed demixing. Quantitative proteomics showed that the glycogen-rich phase was enriched in proteins that bind glycogen, participate in carbohydrate metabolism, or are part of very high-molecular-weight complexes. The glycogen-depleted phase was enriched in ribosomes, endoplasmic reticulum (ER), and mitochondria. Smaller soluble proteins were approximately equipartitioned. Glycogen is usually observed in aggregates in intact cells, and recent work suggested a role for phase separation in its localization. Our results show that glycogen particles can spontaneously demix and suggest that demixing may be regulated by crowding.

Animals↗
PubMed2024

Specificity profiling of deubiquitylases against endogenously generated ubiquitin-protein conjugates.

Deubiquitylating enzymes (DUBs) remove ubiquitin from proteins thereby regulating their stability or activity. Our understanding of DUB-substrate specificity is limited because DUBs are typically not compared to each other against many physiological substrates. By broadly inhibiting DUBs in Xenopus egg extract, we generated hundreds of ubiquitylated proteins and compared the ability of 30 DUBs to deubiquitylate them using quantitative proteomics. We identified five high-impact DUBs (USP7, USP9X, USP36, USP15, and USP24) that each reduced ubiquitylation of over 10% of the isolated proteins. Candidate substrates of high-impact DUBs showed substantial overlap and were enriched for disordered regions, suggesting this feature may promote substrate recognition. Other DUBs showed lower impact and non-overlapping specificity, targeting distinct non-disordered proteins including complexes such as the ribosome or the proteasome. Altogether our study identifies candidate DUB substrates and defines patterns of functional redundancy and specificity, revealing substrate characteristics that may influence DUB-substrate recognition.

Substrate Specificity↗
PubMed2026

A FERONIA-MPK3/6-WRKY3/4 module links auxin signaling to lateral root development in Arabidopsis.

The phytohormone auxin orchestrates root development through intricate signaling networks. In the non-canonical auxin pathway, both the transmembrane kinase (TMK)-mediated signaling and the mitogen-activated protein kinase (MAPK) cascade are shown to be involved in the auxin-regulated lateral root (LR) formation. However, the role and mechanism of the receptor-like kinase FERONIA (FER) in this process remain unclear. Here, quantitative proteomic and phosphoproteomic analyses of Arabidopsis roots identify FER, MPK3/6, and WRKY3/4 as auxin-responsive components. Further analyses reveal that FER functions as a negative regulator of LR development by modulating cell division patterns within LR primordia. FER interacts with and phosphorylates MPK3/6, which then phosphorylate the transcription factors WRKY3 and WRKY4 to form a repressive module that ultimately suppresses LR organogenesis. Collectively, our findings define a FER-MPK3/6-WRKY3/4 signaling module that negatively regulates LR formation, demonstrating a previously unknown integration of FER-mediated signaling into the MAPK cascade in auxin-triggered organogenesis.

Arabidopsis↗
PubMed2026

Tanshinone IIA impairs platelet function and thrombus formation.

BACKGROUND: Tanshinone IIA (T-IIA) is a fat-soluble active ingredient derived from the traditional Chinese medicine Danshen and possesses cardioprotective property. However, its exact role in platelet function is unknown. OBJECTIVES: This study investigated T-IIA's role in platelet aggregation, granules release, spreading, clot retraction, as well as in vivo hemostasis and thrombus formation. METHODS: Human platelets were treated with different doses of T-IIA (10, 50, and 100 μM) to measure platelet function and activation. In addition, T-IIA was administrated into wild-type mice to evaluate hemostasis and thrombus formation. RESULTS: T-IIA significantly impaired platelet aggregation, adenosine triphosphate secretion, P-selectin expression, and spreading and clot retraction dose dependently without affecting the expression profiles of αIIbβ3 and glycoprotein VI or Ibα. Administration of T-IIA significantly prolonged mice tail bleeding time and inhibited arterial and venous thrombosis. Further analysis showed that T-IIA dose dependently reduced platelet reactive oxygen species generation. Quantitative proteomic and phosphoproteimic assays analyzing T-IIA-treated vs vehicle-treated platelets after stimulation identified dysregulated phosphorylation of several proteins, which were enriched in platelet activation. Among the downregulated phosphoproteins, Rho-associated protein kinase (ROCK)1, integrin β3, and talin1 exhibited the lower fold change of phosphorylation in T-IIA-treated platelets compared with those in vehicle-treated platelets. Consistently, T-IIA treatment inhibited the phosphorylation of ROCK1, p47phox, integrin β3, and talin1 in activated platelets. CONCLUSION: T-IIA impairs platelet function and thrombosis via inhibition of several signaling pathways including ROCK1/p47phox, β3, and talin1, implying that T-IIA may represent a promising therapeutic candidate for treating thrombotic diseases.

Animals↗
PubMed2026

H-NOX and NosP Regulate Flagellar Protein and Virulence Factor Production in Vibrio cholerae.

The ability of Vibrio cholerae to transition between motile and sessile forms in the environment and in the host is critical to its survival and virulence. The molecular cues, sensor proteins, and signaling pathways mediating these transitions are highly complex and often overlapping. Nevertheless, a detailed understanding of them is critical for understanding the persistence and pathogenesis of this deadly pathogen. Nitric oxide (NO) functions as an important signaling molecule in many bacteria, affecting biofilm formation, motility, and virulence, often through interaction with heme protein sensors. The genome of V. cholerae encodes two such sensors called H-NOX and NosP. Here we constructed a Δhnox/nosP mutant and employed a multi-omics methodology that combines tandem-mass-tag (TMT)-based quantitative proteomics, phosphoproteomics, and targeted metabolomics to investigate the function of these sensors. A set of 258 proteins was differentially expressed in the mutant that included many proteins involved in flagellar biosynthesis and motility as well as critical virulence factors, iron acquisition systems, and metabolic enzymes. Many of the identified genes are also part of the ferric uptake regulator (Fur) regulon and iron-dependent transcriptional repression of several Fur targets was disrupted. Phosphoproteomics analysis also revealed proteins involved in motility and virulence as differentially phosphorylated in the mutant strain. In most cases, these phosphoproteins have not been previously observed and provide a wealth of new targets for investigating mechanisms of V. cholerae signaling. Taken together, this work illustrates a role for H-NOX and NosP in promoting factors important for infection while suppressing those important for environmental survival, suggesting a function in priming the organism for infection and/or maintaining the infectious phenotype.

Journal Article↗
PubMed2005

Regulation of hepatocyte activator inhibitor-1 expression by androgen and oncogenic transformation in the prostate.

Hepatocyte activator inhibitor-1 (HAI-1) is a transmembrane serine protease inhibitor that regulates the conversion of latent to active hepatocyte growth factor (HGF). Studies supporting a role for the HGF pathway in prostate carcinogenesis prompted an analysis of HAI-1 expression in the prostate. Here we analyze the regulation of HAI-1 expression by androgen, oncogenic transformation, and cancer progression. Immunohistochemical analysis revealed that HAI-1 expression was restricted to prostate epithelium, where staining occurred primarily in basal and atrophic luminal epithelial cells. Compared to normal glands, HAI-1 expression was significantly increased in localized prostate cancer and was present in most prostate cancer metastases. HAI-1 protein expression levels were sensitive to androgen in normal epithelium but not in cancer. Although androgen did not increase HAI-1 protein expression levels in LNCaP cells, it decreased HAI-1 surface expression, consistent with previous data from our group (Martin DB, Gifford DR, Wright ME, Keller A, Yi E, Goodlett DR, Aebersold R, Nelson PS: Quantitative proteomic analysis of proteins released by neoplastic prostate epithelium. Cancer Res 2004, 64:347-355). HAI-1 overexpression in cancer was predictive of prostate-specific antigen recurrence (relative risk, 1.24). These results suggest that HAI-1 regulates the HGF Met axis on prostate epithelial cells and influences HGF mediated tumor invasion and metastasis.

Androgens↗
PubMed2000

Four years of post-genomic life with 6,000 yeast genes.

Four years after disclosure of the full yeast genome sequence, a series of resources including tens of thousands of mutant strains, plasmids bearing isolated genes and disruption cassettes are becoming publicly available. Deletions of each of the 6,000 putative yeast genes are being screened systematically for dozens of phenotypic traits. In addition, new global approaches such as DNA hybridization arrays, quantitative proteomics and two-hybrid interactions are being steadily improved. They progressively build up an immense computation network of billions of data points which will, within the next decade, characterize all molecular interactions occurring in a simple eukaryotic cell. In this process of acquisition of new basic knowledge, an international community of over 1,000 laboratories cooperates with a remarkable willingness to share projects and results.

DNA Transposable Elements↗
PubMed2005

De novo sequencing of neuropeptides using reductive isotopic methylation and investigation of ESI QTOF MS/MS fragmentation pattern of neuropeptides with N-terminal dimethylation.

A stable-isotope dimethyl labeling strategy was previously shown to be a useful tool for quantitative proteomics. More recently, N-terminal dimethyl labeling was also reported for peptide sequencing in combination with database searching. Here, we extend these previous studies by incorporating N-terminal isotopic dimethylation for de novo sequencing of neuropeptides directly from tissue extract without any genomic information. We demonstrated several new sequencing applications of this method in addition to the identification of the N-terminal residue using the enhanced a(1) ion. The isotopic labeling also provides easier and more confident de novo sequencing of peptides by comparing similar MS/MS fragmentation patterns of the isotopically labeled peptide pairs. The current study on neuropeptides shows several distinct fragmentation patterns after N-terminal dimethylation which have not been reported previously. The y((n-1)) ion is enhanced in multiply charged peptides and is weak or missing in singly charged peptides. The MS/MS spectra of singly charged peptides are simplified due to the enhanced N-terminal fragments and suppressed internal fragments. The neutral loss of dimethylamine is also observed. The mechanisms for the above fragmentations are proposed. Finally, the structures of the immonium ion and related ions of N(alpha), N(epsilon)-tetramethylated lysine and N(epsilon)-dimethylated lysine are explored.

Amino Acid Sequence↗
PubMed2026

Hyperlactate-Associated Lysine Lactylome Remodeling in Laryngeal Squamous Cell Carcinoma.

Laryngeal squamous cell carcinoma (LSCC) lacks reliable biomarkers, and the roles of lactate metabolism and lysine lactylation (Kla) remain largely unknown. We profiled the lysine lactylome of LSCC, paired it with adjacent normal tissues, and integrated the data with quantitative proteomic and transcriptomic analyses. LSCC exhibited a hyperlactate-associated phenotype characterized by dysregulated lactate-related genes (LRGs), altered protein abundance, increased tissue lactate, and globally increased Kla levels. Data-independent acquisition mass spectrometry (DIA-MS) identified 1616 Kla sites on 1468 peptides from 688 proteins, with most differential sites being upregulated in tumors. Differentially lactylated proteins were enriched in cell-matrix adhesion, cell migration, chromatin remodeling, and gene-regulatory processes and were clustered into cytoskeletal and nuclear regulatory modules. Multiple Kla sites were also detected on the core histones. Immunoblotting and tissue microarray analyses confirmed increased pan-Kla expression in the LSCC. Pan-Kla levels were independent of sex and age but positively correlated with the tumor stage and lymph-node metastasis. These findings provide a systematic resource for hyperlactate-associated lactylome remodeling in LSCCs and identify candidate Kla-related molecular features associated with clinicopathological progression for future functional and clinical evaluation.

Humans↗
PubMed2025

Marine-Inspired Antimicrobial Peptides Disrupt Gene Expression at the DNA Level.

Genome mining of Streptomyces sp. H-KF8 combined with sequence engineering yielded two serum-stable, noncytotoxic, nonlytic antimicrobial peptides, L3 and L3-K. Initial studies in uropathogenic Escherichia coli suggested membrane effects and nucleoid relaxation, prompting a comprehensive investigation of their mode of action. In this study tandem mass tag (TMT)-based quantitative proteomics revealed extensive proteome remodeling, with 175 and 120 differentially expressed proteins (DEPs) after treatment with L3 and L3-K, respectively. L3 induced predominantly upregulated responses linked to metabolism, RNA processing, transport, and homeostasis, whereas L3-K mainly caused the downregulation of proteins involved in metabolism, transport, and cell structure. Both peptides disrupted ABC transporter-mediated nutrient uptake and elicited stress responses, while L3 specifically perturbed the mal regulon, indicative of broader transcriptional dysregulation. Complementary fluorescent dye displacement and in vitro transcription/translation assays demonstrated nonspecific DNA binding, stronger for L3 than L3-K, and potent inhibition of transcriptional and translational processes. Strikingly, inhibitory concentrations paralleled their minimum inhibitory concentrations, directly linking DNA binding and interference with central information processing to antimicrobial activity. These findings reveal that L3 and L3-K primarily act by targeting DNA and interfering with the transcription-translation machinery. Beyond offering mechanistic insights, this study underscores peptides' potential to act as scaffolds for next-generation antimicrobial peptides with DNA-binding and nonmembrane-lytic activity.

Antimicrobial Peptides↗
PubMedDate not supplied

Integrated approach for manual evaluation of peptides identified by searching protein sequence databases with tandem mass spectra.

Quantitative proteomics relies on accurate protein identification, which often is carried out by automated searching of a sequence database with tandem mass spectra of peptides. When these spectra contain limited information, automated searches may lead to incorrect peptide identifications. It is therefore necessary to validate the identifications by careful manual inspection of the mass spectra. Not only is this task time-consuming, but the reliability of the validation varies with the experience of the analyst. Here, we report a systematic approach to evaluating peptide identifications made by automated search algorithms. The method is based on the principle that the candidate peptide sequence should adequately explain the observed fragment ions. Also, the mass errors of neighboring fragments should be similar. To evaluate our method, we studied tandem mass spectra obtained from tryptic digests of E. coli and HeLa cells. Candidate peptides were identified with the automated search engine Mascot and subjected to the manual validation method. The method found correct peptide identifications that were given low Mascot scores (e.g., 20-25) and incorrect peptide identifications that were given high Mascot scores (e.g., 40-50). The method comprehensively detected false results from searches designed to produce incorrect identifications. Comparison of the tandem mass spectra of synthetic candidate peptides to the spectra obtained from the complex peptide mixtures confirmed the accuracy of the evaluation method. Thus, the evaluation approach described here could help boost the accuracy of protein identification, increase number of peptides identified, and provide a step toward developing a more accurate next-generation algorithm for protein identification.

Algorithms↗
PubMed2006

pH dependency of the carboxyl oxygen exchange reaction catalyzed by lysyl endopeptidase and trypsin.

The pH dependency of the carboxyl oxygen exchange reaction catalyzed by lysyl endopeptidase (Lys-C) and trypsin has been studied. The reaction was quantitatively monitored by measuring the incorporation of 18O atom into the alpha-carboxyl group of N(alpha)-acetyl-L-lysine from H2(18)O solvent. The optimum pHs of the carboxyl oxygen exchange reaction catalyzed by Lys-C and trypsin were found to be pH 5.0 and 6.0, respectively, which were significantly shifted toward acidic pHs compared to the most favorable pHs of their amidase activities for N(alpha)-acetyl-L-lysine amide in the pHs examined. Steady-state kinetics parameters were also determined for both enzymes at two different pHs, one at the pH optimum for their carboxyl oxygen exchange activity (pH 5-6) and the other at the favorable pH for their amidase activity (pH 8-9). Significantly lower Km (2-fold lower for Lys-C, 3-fold lower for trypsin), and higher kcat values (1.5-fold higher for Lys-C, 5-fold higher for trypsin) were obtained at the acidic pHs compared to the alkaline pHs, suggesting that Lys-C and trypsin have higher substrate binding affinities and higher catalytic rates at the acidic pHs than at the alkaline pHs. The higher carboxyl oxygen exchange activities at the acidic pHs were also confirmed with peptide substrates derived from apomyoglobin. These findings are significant toward the goal of improving the efficiency of the Lys-C and trypsin catalyzed 18O labeling reactions and are thus pertinent to improving the accuracy and reliability of quantitative proteomic experiments utilizing 18O labeling.

Carbon Dioxide↗
PubMed2005

Phosphotyrosine interactome of the ErbB-receptor kinase family.

Interactions between short modified peptide motifs and modular protein domains are central events in cell signal-transduction. We determined interaction partners to all cytosolic tyrosine residues of the four members of the ErbB-receptor family in an unbiased fashion by quantitative proteomics using pull-down experiments with pairs of phosphorylated and nonphosphorylated synthetic peptides. Each receptor had characteristic preferences for interacting proteins and most interaction partners had multiple binding sites on each receptor. EGFR and ErbB4 had several docking sites for Grb2, while ErbB3 was characterized by six binding sites for PI3K. We identified STAT5 as a direct binding partner to EGFR and ErbB4 and discovered new recognition motifs for Shc and STAT5. The overall pattern of interaction partners of EGFR and ErbB4 suggests similar roles during signaling through their respective ligands. Phosphorylation kinetics of several tyrosine resides was measured by mass spectrometry and correlated with interaction partner preference. Our results demonstrate that system-wide mapping of peptide-protein interactions sites is possible, and suggest shared and unique roles of ErbB-receptor family members in downstream signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry.

Quantitative proteome profiling using stable isotope protein tagging and automated tandem mass spectrometry (MS/MS) is an emerging technology with great potential for the functional analysis of biological systems and for the detection of clinical diagnostic or prognostic marker proteins. Owing to the enormous complexity of proteomes, their comprehensive analysis is an as-yet-unresolved technical challenge. However, biologically or clinically important information can be obtained if specific, information-rich protein classes, or sub-proteomes, are isolated and analyzed. Glycosylation is the most common post-translational modification. Here we describe a method for the selective isolation, identification and quantification of peptides that contain N-linked carbohydrates. It is based on the conjugation of glycoproteins to a solid support using hydrazide chemistry, stable isotope labeling of glycopeptides and the specific release of formerly N-linked glycosylated peptides via peptide- N-glycosidase F (PNGase F). The recovered peptides are then identified and quantified by MS/MS. We applied the approach to the analysis of plasma membrane proteins and proteins contained in human blood serum.

Amino Acid Sequence↗