Search Results (80)

Seed germination is a critical phase in rice production and is highly sensitive to environmental and chemical stresses. Chloramphenicol (CAM), a known phytotoxic antibiotic, has been reported to suppress rice seedling establishment, yet its underlying molecular mechanisms remain poorly understood. In this study, we investigated the effects of varying CAM concentrations on rice germination and early seedling establishment. While CAM significantly retarded germination speed and seedling growth, the final germination rates remained largely unaffected, even at high concentrations. To uncover the molecular basis of CAM phytotoxicity, we conducted time-resolved phosphoproteomic profiling during both the germination and early seedling stages. Our analyses revealed dynamic, stage-specific phosphorylation changes: moderate alterations affecting metabolic and cytokinesis-related processes during germination, and extensive disruptions in metabolic pathways, stress response mechanisms, DNA replication, and hormone signaling during early seedling establishment. Collectively, these findings demonstrate that CAM disrupts rice development by remodeling phosphorylation networks and modulating key physiological and signaling pathways. This study provides novel insights into the molecular mechanisms underlying antibiotic-induced growth inhibition and advances our understanding of plant stress responses during early development. Full article

Soil salinity severely impairs plant growth, and polyamines such as spermidine (Spd) are known to bolster stress tolerance by acting as osmoprotectants and signaling molecules. Using TiO₂ enrichment, iTRAQ quantification, and bioinformatics analysis, we identified 870 proteins and 157 differentially phosphorylated proteins. Functional annotation showed that salt stress activated key components of the Salt Overly Sensitive pathway, particularly serine threonine kinases (SOS2) and Ca²⁺ binding sensors (SOS3). Among thirty-six SOS-associated kinases detected, eight SOS2 isoforms, four MAPKs, and two SOS3 homologs were significantly upregulated by NaCl, and Spd further increased the phosphorylation of six SOS2 proteins and one SOS3 protein under salt stress, with no detectable effect on SOS1. qRT PCR revealed enhanced expression of MAPKs and calcium-dependent protein kinases, suggesting a phosphorylation-centered model in which Spd amplifies Ca²⁺-mediated SOS signaling and reinforces ion homeostasis through coordinated transcriptional priming and post-translational control. Additional, proteins involved in protein synthesis and turnover (ribosomal subunits, translation initiation factors, ubiquitin–proteasome components), DNA replication and transcription, and RNA processing showed differential expression under salt or Spd treatment. Central metabolic pathways were reprogrammed, involving glycolysis, the TCA cycle, the pentose phosphate pathway, as well as ammonium transporters and amino acid biosynthetic enzymes. These findings indicate that exogenous Spd regulated phosphorylation-mediated networks involving the SOS signaling pathway, protein homeostasis, and metabolism, thereby enhancing cucumber salt tolerance. Full article

Heat stress severely inhibited the flower bud growth and development of passion fruit (Passiflora edulis Sims) in summer, resulting in severe production damage. Protein phosphorylation plays a key role in plant protein regulatory networks in response to abiotic stress, while the mechanism of phosphorylation regulation response to heat stress in passion fruit is still unknown. In this study, 97.62% of passion fruit floral buds withered and fell off after 2 h of heat stress, compared to 3.33% after 0.5 h. A total of 10,614 phosphorylation sites across 2906 proteins were identified by phosphoproteomic analysis. Among them, 1343 differentially regulated phosphoproteins (DRPPs) were mainly located in the nucleus, cytoplasm, and chloroplast. The DRPPs whose phosphorylation sites were induced by heat stress were mainly involved in the ‘ABC transporters’, ‘Plant hormone signal transduction’, and ‘MAPK signaling’ pathways. In addition, the accumulations of ABA and H₂O₂ were induced under heat stress for 0.5 h. Through protein interaction prediction and qRT-PCR analyses, we identified a key protein PePP2C1, in which the levels of gene expression, protein expression, and phosphorylation were induced by heat stress. The transient assays showed that the overexpression of PePP2C1 inhibited the accumulation of H₂O₂. Our results suggested the potential role of phosphoproteins under heat stress in the floral buds of passion fruit. The findings in this study contribute to a better understanding of the molecular mechanism of phosphoproteins in response to heat stress. Full article

The cell wall integrity (CWI) pathway is responsible for transcriptional regulation of cell wall remodeling in response to cell wall stress. How cell wall remodeling mediated by the CWI pathway is effected by inputs from other signaling pathways is not well understood. Here, we demonstrate that the Mck1 kinase cooperates with Slt2, the MAP kinase of the CWI pathway, to promote cell wall thickening in glucose-starved cells. Integrative analyses of the transcriptome, proteome and metabolic profiling indicate that Mck1 is required for the accumulation of UDP-glucose (UDPG), the substrate for β-glucan synthesis, through the activation of two regulons: the Msn2/4-dependent stress response and the Cat8-/Adr1-mediated metabolic reprogram dependent on the SNF1 complex. Analysis of the phosphoproteome suggests that similar to mammalian Gsk-3 kinases, Mck1 is involved in the regulation of cytoskeleton-dependent cellular processes, metabolism, signaling and transcription. Specifically, Mck1 may be implicated in the Snf1-dependent metabolic reprogram through PKA inhibition and SAGA (Spt-Ada-Gcn5 acetyltransferase)-mediated transcription activation, a hypothesis further underscored by the significant overlap between the Mck1- and Gcn5-activated transcriptomes. Phenotypic analysis also supports the roles of Mck1 in actin cytoskeleton-mediated exocytosis to ensure plasma membrane homeostasis and cell wall remodeling in cell wall-stressed cells. Together, these findings not only reveal the novel functions of Mck1 in metabolic reprogramming and polarized growth but also provide valuable omics resources for future studies to uncover the underlying mechanisms of Mck1 and other Gsk-3 kinases in cell growth and stress response. Full article

Rapid ascent to high altitudes by unacclimatized individuals significantly increases the risk of brain damage, given the brain’s heightened sensitivity to hypoxic conditions. Investigating hypoxia-tolerant animals can provide insights into adaptive mechanisms and guide prevention and treatment of hypoxic-ischemic brain injury. In this study, we exposed Brandt’s voles to simulated altitudes (100 m, 3000 m, 5000 m, and 7000 m) for 24 h and performed quantitative proteomic and phosphoproteomic analyses of brain tissue. A total of 3990 proteins and 9125 phosphorylation sites (phospho-sites) were quantified. Differentially expressed (DE) analysis revealed that while protein abundance changes were relatively modest, phosphorylation levels exhibited substantial alterations, suggesting that Brandt’s voles rapidly regulate protein structure and function through phosphorylation to maintain cellular homeostasis under acute hypoxia. Clustering analysis showed that most co-expressed proteins exhibited non-monotonic responses with increasing altitude, which were enriched in pathways related to cytokine secretion regulation and glutathione metabolism, contributing to reduced inflammation and oxidative stress. In contrast, most co-expressed phospho-sites showed monotonic changes, with phospho-proteins enriched in glycolysis and vascular smooth muscle contraction regulation. Kinase activity prediction identified nine hypoxia-responsive kinases, four of which belonging to the CAMK family. Immunoblot validated that the changes in CAMK2A activity were consistent with predictions, suggesting that CAMK may play a crucial role in hypoxic response. In conclusion, this work discovered that Brandt’s voles may cope with hypoxia through three key strategies: (1) vascular regulation to enhance cerebral blood flow, (2) glycolytic activation to increase energy production, and (3) activation of neuroprotective mechanisms. Full article

Influenza A virus (IAV) is a widespread human respiratory pathogen that contributes significantly to morbidity and mortality worldwide. The adsorption of the virus into the cell surface is the earliest stage of its replication cycle. The key role of N-linked sialic acids (SIAs) as receptors for binding to IAV’s hemagglutinin (HA) has long been acknowledged. The molecular specificity of this interaction is a key factor in host range, pathogenicity, and transmissibility of various IAV subtypes. Along with this, a number of recent studies have introduced significant complexity into the picture of IAV adsorption and revealed a multitude of new molecules on host cell surfaces to serve as receptors and/or co-receptors for IAV attachment. For successful internalization of the adsorbed virus, downstream signal transduction is necessary to activate effector endocytosis mechanisms. In recent years, our understanding of the sophistication and variability of signal transduction pathways in the virus attachment site has significantly expanded, with the help of research techniques like fluorescence imaging of individual viruses in real-time, dominant-negative mutants, siRNA knockdowns, protein kinase selective inhibitors, phosphoproteome profiling, and others. These approaches deepen our knowledge of the molecules involved in the early stages of the IAV life cycle and also serve as the basis for the development of new effective antiviral drugs. In our review, we analyze recent publications on the mechanisms of IAV adsorption, newly discovered receptors for virus attachment, and signal transmission in the site of the adsorbed virion. Besides this, we consider new data on the development of selective inhibitors as antiviral drugs aimed at both viral and cellular factors of IAV adsorption. Full article

Peanut (Arachis hypogaea L.) is one of the most important crops for oil and protein production. The unique characteristic of peanut is geocarpy, which means that it blooms aerially and the peanut gynophores (pegs) penetrate into the soil, driving the fruit underground. In order to fully understand this phenomenon, we investigated the dynamic proteomic and phosphoproteomic profiling of the pegs aerially and underground in this study. A total of 6859 proteins and 4142 unique phosphoproteins with 10,070 phosphosites were identified. The data were validated and quantified using samples randomly selected from arial pegs (APs) and underground pegs (UPs) by parallel reaction monitoring (PRM). Function analyses of differentially abundant proteins (DAPs) and differentially regulated phosphoproteins (DRPPs) exhibited that they were mainly related to stress response, photosynthesis, and substance metabolism. Once the pegs successfully entered the soil, disease-resistant and stress response proteins, such as glutathione S-transferase, peroxidase, and cytochrome P450, significantly increased in the UP samples in order to adapt to the new soil environment. The increased abundance of photosynthesis-associated proteins in the UP samples provided more abundant photosynthetic products, which provided the preparation for subsequent pod development. Phosphoproteomics reveals the regulatory network of the synthesis of nutrients such as starch, protein, and fatty acid (FA). These results provide new insights into the mechanism, indicating that after the pegs are inserted into the soil, phosphorylation is involved in the rapid elongation of the pegs, accompanied by supplying energy for pod development and preparing for the synthesis of metabolites during pod development following mechanical stimulation and darkness. Full article

Background: Macrophage-mediated cancer cell phagocytosis has demonstrated considerable therapeutic potential. While the initiation of phagocytosis, facilitated by interactions between cancer cell surface signals and macrophage receptors, has been characterized, the mechanisms underlying its sustentation and attenuation post-initiation remain poorly understood. Methods: Through comprehensive phosphoproteomic profiling, we interrogated the temporal evolution of the phosphorylation profiles within macrophages during cancer cell phagocytosis. Results: Our findings reveal that activation of the mTOR pathway occurs following the initiation of phagocytosis and is crucial in sustaining phagocytosis of cancer cells. mTOR inhibition impaired the phagocytic capacity, but not affinity, of the macrophages toward the cancer cells by delaying phagosome maturation and impeding the transition between non-phagocytic and phagocytic states of macrophages. Conclusions: Our findings delineate the intricate landscape of macrophage phagocytosis and highlight the pivotal role of the mTOR pathway in mediating this process, offering valuable mechanistic insights for therapeutic interventions. Full article

Recent advancements in mass spectrometry-based proteomics have made it possible to conduct comprehensive protein analysis. In particular, the emergence of the data-independent acquisition (DIA) method powered by machine learning has significantly improved protein identification efficiency. However, compared with the conventional data-dependent acquisition (DDA) method, the degree to which peptides are uniquely identified by DIA and DDA has not been thoroughly examined. In this study, we identified over 10,000 proteins using the DDA and DIA methods and analyzed the characteristics of unique peptides identified by each method. Results showed that the number of peptides uniquely identified by DDA and DIA using the same column type was 19% and 32%, respectively, with shorter peptides preferentially detected by the DIA method. In addition, more DIA-specific peptides were identified, especially during the first 10% of elution time, and the overall 1/K₀ and m/z shifted toward smaller values than in the DDA method. Furthermore, comparing the phosphorylation and ubiquitination proteome profiles with those of whole-cell lysates by DDA showed that the enrichment of post-translationally modified peptides resulted in wider m/z and 1/K₀ ranges. Notably, the ubiquitin peptide-enriched samples displayed lower m/z values than the phospho-proteome. These findings suggest a bias in the types of peptides identified by the acquisition method and the importance of setting appropriate ranges for DIA based on the post-translational modification of peptide characteristics. Full article

Seminal plasma is rich in proteins originating from various male reproductive organs. The phosphorylation of these proteins can significantly impact sperm motility, capacitation, and acrosome reaction. Phosphoproteomics identifies, catalogues, and characterizes phosphorylated proteins. The phosphoproteomic profiling of seminal plasma offers valuable insights into the molecular mechanisms that influence semen quality and male fertility. Thus, the aim of this study was a phosphoproteomic analysis of white and yellow turkey seminal plasma. The experimental material consisted of 100 ejaculates from BIG-6 turkeys between 39 and 42 weeks of age. The collected white and yellow turkey seminal plasmas were analyzed for total protein content; the activity of selected enzymes, i.e., alkaline phosphatase (ALP), acid phosphatase (ACP), superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT); and the content of reduced glutathione (GSH) and malondialdehyde (MDA). Phosphoproteins were isolated from white and yellow seminal fluids, and the resulting protein fractions were separated by SDS-PAGE and Western blotting. Phosphorylated residues were immunodetected, and the isolated phosphoproteins were identified (nano LC-MS/MS). Yellow seminal plasmas were characterized by higher levels of total protein, GSH, and MDA, as well as higher levels of ALP, ACP, and GPx activity. There were no significant differences in the activity of SOD and CAT. A total of 113 phosphoproteins were identified in turkey seminal fluids. The functional analysis demonstrated that these phosphoproteins were mainly involved in oocyte fertilization, organization and metabolism of the actin cytoskeleton, amplification of the intracellular signal transduction pathway, general regulation of transport, vesicular transport, proteome composition of individual cellular compartments, and the organization and localization of selected cellular components and macromolecules. Increased phosphorylation of the fractions containing proteins encoded by SPARC, PPIB, TRFE, QSOX1, PRDX1, PRDX6, and FASN genes in white plasmas and the proteins encoded by CKB, ORM2, APOA1, SSC5D, RAP1B, CDC42, FTH, and TTH genes in yellow plasmas was observed based on differences in the optical density of selected bands. The obtained results indicate that the phosphorylation profiles of turkey seminal plasma proteins vary depending on the type of ejaculate. Full article

Epsilon toxin (ETX), a potential agent of biological and toxic warfare, causes the death of many ruminants and threatens human health. It is crucial to understand the toxic mechanism of such a highly lethal and rapid course toxin. In this study, we detected the effects of ETX on the proteome and phosphoproteome of MDCK cells after 10 min and 30 min. A total of 44 differentially expressed proteins (DEPs) and 588 differentially phosphorylated proteins (DPPs) were screened in the 10 min group, while 73 DEPs and 489 DPPs were screened in the 30 min group. ETX-induced proteins and phosphorylated proteins were mainly located in the nucleus, cytoplasm, and mitochondria, and their enrichment pathways were related to transcription and translation, virus infection, and intercellular junction. Meanwhile, the protein–protein interaction network screened out several hub proteins, including SRSF1/2/6/7/11, SF3B1/2, NOP14/56, ANLN, GTPBP4, THOC2, and RRP1B. Almost all of these proteins were present in the spliceosome pathway, indicating that the spliceosome pathway is involved in ETX-induced cell death. Next, we used RNAi lentiviruses and inhibitors of several key proteins to verify whether these proteins play a critical role. The results confirmed that SRSF1, SF3B2, and THOC2 were the key proteins involved in the cytotoxic effect of ETX. In addition, we found that the common upstream kinase of these key proteins was SRPK1, and a reduction in the level of SRPK1 could also reduce ETX-induced cell death. This result was consistent with the phosphorylated proteomics analysis. In summary, our study demonstrated that ETX induces phosphorylation of SRSF1, SF3B2, THOC2, and SRPK1 proteins on the spliceosome pathway, which inhibits normal splicing of mRNA and leads to cell death. Full article

Phosphoglycerylation is a non-enzymatic protein modification in which a phosphoglyceryl moiety is covalently bound to the ε-amino group of lysine. It is enriched in glycolytic enzymes from humans and mice and is thought to provide a feedback mechanism for regulating glycolytic flux. We report the first proteomic analysis of this post-translational modification in bacteria by profiling phosphoglyceryl-lysine during the growth of Streptococcus pyogenes in different culture media. The identity of phosphoglyceryl-lysine was confirmed by a previously unknown diagnostic cyclic immonium ion generated during MS/MS. We identified 370 lysine phosphoglycerylation sites in 123 proteins of S. pyogenes. Growth in a defined medium on 1% fructose caused a significant accumulation of phosphoglycerylation compared to growth in a rich medium containing 0.2% glucose. Re-analysis of phosphoproteomes from 14 bacterial species revealed that phosphoglycerylation is generally widespread in bacteria. Many phosphoglycerylation sites were conserved in several bacteria, including S. pyogenes. There was considerable overlap between phosphoglycerylation, acetylation, succinylation, and other acylations on the same lysine residues. Despite some exceptions, most lysine phosphoglycerylations in S. pyogenes occurred with low stoichiometry. Such modifications may be meaningless, but it is also conceivable that phosphoglycerylation, acetylation, and other acylations jointly contribute to the overall regulation of metabolism. Full article

Understanding signaling patterns of transformation and controlling cell phenotypes is a challenge of current biology. Here we applied a cell State Transition Assessment and Regulation (cSTAR) approach to a perturbation dataset of single cell phosphoproteomic patterns of multiple breast cancer (BC) and normal breast tissue-derived cell lines. Following a separation of luminal, basal, and normal cell states, we identified signaling nodes within core control networks, delineated causal connections, and determined the primary drivers underlying oncogenic transformation and transitions across distinct BC subtypes. Whereas cell lines within the same BC subtype have different mutational and expression profiles, the architecture of the core network was similar for all luminal BC cells, and mTOR was a main oncogenic driver. In contrast, core networks of basal BC were heterogeneous and segregated into roughly four major subclasses with distinct oncogenic and BC subtype drivers. Likewise, normal breast tissue cells were separated into two different subclasses. Based on the data and quantified network topologies, we derived mechanistic cSTAR models that serve as digital cell twins and allow the deliberate control of cell movements within a Waddington landscape across different cell states. These cSTAR models suggested strategies of normalizing phosphorylation networks of BC cell lines using small molecule inhibitors. Full article

Proteomics offers a robust method for quantifying proteins and elucidating their roles in cellular functions, surpassing the insights provided by transcriptomics. The Clinical Proteomic Tumor Analysis Consortium database, enriched with comprehensive cancer proteomics data including phosphorylation and ubiquitination profiles, alongside transcriptomics data from the Genomic Data Commons, allow for integrative molecular studies of cancer. The ProteoCancer Analysis Suite (PCAS), our newly developed R package and Shinyapp, leverages these resources to facilitate in-depth analyses of proteomics, phosphoproteomics, and transcriptomics, enhancing our understanding of the tumor microenvironment through features like immune infiltration and drug sensitivity analysis. This tool aids in identifying critical signaling pathways and therapeutic targets, particularly through its detailed phosphoproteomic analysis. To demonstrate the functionality of the PCAS, we conducted an analysis of GAPDH across multiple cancer types, revealing a significant upregulation of protein levels, which is consistent with its important biological and clinical significance in tumors, as indicated in our prior research. Further experiments were used to validate the findings performed using the tool. In conclusion, the PCAS is a powerful and valuable tool for conducting comprehensive proteomic analyses, significantly enhancing our ability to uncover oncogenic mechanisms and identify potential therapeutic targets in cancer research. Full article

Lysophosphatidic acid (LPA) species, prevalent in the tumor microenvironment (TME), adversely impact various cancers. In ovarian cancer, the 18:0 and 20:4 LPA species are selectively associated with shorter relapse-free survival, indicating distinct effects on cellular signaling networks. Macrophages represent a cell type of high relevance in the TME, but the impact of LPA on these cells remains obscure. Here, we uncovered distinct LPA-species-specific responses in human monocyte-derived macrophages through unbiased phosphoproteomics, with 87 and 161 phosphosites upregulated by 20:4 and 18:0 LPA, respectively, and only 24 shared sites. Specificity was even more pronounced for downregulated phosphosites (163 versus 5 sites). Considering the high levels 20:4 LPA in the TME and its selective association with poor survival, this finding may hold significant implications. Pathway analysis pinpointed RHO/RAC1 GTPase signaling as the predominantly impacted target, including AHRGEF and DOCK guanine exchange factors, ARHGAP GTPase activating proteins, and regulatory protein kinases. Consistent with these findings, exposure to 20:4 resulted in strong alterations to the actin filament network and a consequent enhancement of macrophage migration. Moreover, 20:4 LPA induced p38 phosphorylation, a response not mirrored by 18:0 LPA, whereas the pattern for AKT was reversed. Furthermore, RNA profiling identified genes involved in cholesterol/lipid metabolism as selective targets of 20:4 LPA. These findings imply that the two LPA species cooperatively regulate different pathways to support functions essential for pro-tumorigenic macrophages within the TME. These include cellular survival via AKT activation and migration through RHO/RAC1 and p38 signaling. Full article