ABSTRACT
Lung cancer in East Asia is characterized by a high percentage of never-smokers, early onset and predominant EGFR mutations. To illuminate the molecular phenotype of this demographically distinct disease, we performed a deep comprehensive proteogenomic study on a prospectively collected cohort in Taiwan, representing early stage, predominantly female, non-smoking lung adenocarcinoma. Integrated genomic, proteomic, and phosphoproteomic analysis delineated the demographically distinct molecular attributes and hallmarks of tumor progression. Mutational signature analysis revealed age- and gender-related mutagenesis mechanisms, characterized by high prevalence of APOBEC mutational signature in younger females and over-representation of environmental carcinogen-like mutational signatures in older females. A proteomics-informed classification distinguished the clinical characteristics of early stage patients with EGFR mutations. Furthermore, integrated protein network analysis revealed the cellular remodeling underpinning clinical trajectories and nominated candidate biomarkers for patient stratification and therapeutic intervention. This multi-omic molecular architecture may help develop strategies for management of early stage never-smoker lung adenocarcinoma.
Subject(s)
Disease Progression , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Proteogenomics , Smoking/genetics , Adenocarcinoma of Lung/genetics , Adenocarcinoma of Lung/pathology , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Carcinogens/toxicity , Cohort Studies , Cytosine Deaminase/metabolism , Asia, Eastern , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Genome, Human , Humans , Matrix Metalloproteinases/metabolism , Mutation/genetics , Principal Component AnalysisABSTRACT
Epitranscriptomic RNA modifications have emerged as important regulators of the fate and function of viral RNAs. One prominent modification, the cytidine methylation 5-methylcytidine (m5C), is found on the RNA of HIV-1, where m5C enhances the translation of HIV-1 RNA. However, whether m5C functionally enhances the RNA of other pathogenic viruses remains elusive. Here, we surveyed a panel of commonly found RNA modifications on the RNA of hepatitis B virus (HBV) and found that HBV RNA is enriched with m5C as well as ten other modifications, at stoichiometries much higher than host messenger RNA (mRNA). Intriguingly, m5C is mostly found on the epsilon hairpin, an RNA element required for viral RNA encapsidation and reverse transcription, with these m5C mainly deposited by the cellular methyltransferase NSUN2. Loss of m5C from HBV RNA due to NSUN2 depletion resulted in a partial decrease in viral core protein (HBc) production, accompanied by a near-complete loss of the reverse transcribed viral DNA. Similarly, mutations introduced to remove the methylated cytidines resulted in a loss of HBc production and reverse transcription. Furthermore, pharmacological disruption of m5C deposition led to a significant decrease in HBV replication. Thus, our data indicate m5C methylations as a critical mediator of the epsilon elements' function in HBV virion production and reverse transcription, suggesting the therapeutic potential of targeting the m5C methyltransfer process on HBV epsilon as an antiviral strategy.
Subject(s)
Cytidine , Hepatitis B virus , RNA, Viral , Reverse Transcription , Hepatitis B virus/genetics , Hepatitis B virus/metabolism , Hepatitis B virus/physiology , RNA, Viral/genetics , RNA, Viral/metabolism , Cytidine/analogs & derivatives , Cytidine/metabolism , Cytidine/genetics , Humans , Reverse Transcription/genetics , Methylation , Virus Replication/genetics , Epigenesis, Genetic , Virion/metabolism , Virion/genetics , TranscriptomeABSTRACT
Protein post-translational modifications (PTMs) are crucial in plant cellular processes, particularly in protein folding and signal transduction. N-glycosylation and phosphorylation are notably significant PTMs, playing essential roles in regulating plant responses to environmental stimuli. However, current sequential enrichment methods for simultaneous analysis of phosphoproteome and N-glycoproteome are labor-intensive and time-consuming, limiting their throughput. Addressing this challenge, this study introduces a novel tandem S-Trap-IMAC-HILIC (S-Trap: suspension trapping; IMAC: immobilized metal ion affinity chromatography; HILIC: hydrophilic interaction chromatography) strategy, termed TIMAHAC, for simultaneous analysis of plant phosphoproteomics and N-glycoproteomics. This approach integrates IMAC and HILIC into a tandem tip format, streamlining the enrichment process of phosphopeptides and N-glycopeptides. The key innovation lies in the use of a unified buffer system and an optimized enrichment sequence to enhance efficiency and reproducibility. The applicability of TIMAHAC was demonstrated by analyzing the Arabidopsis phosphoproteome and N-glycoproteome in response to abscisic acid (ABA) treatment. Up to 1954 N-glycopeptides and 11,255 phosphopeptides were identified from Arabidopsis, indicating its scalability for plant tissues. Notably, distinct perturbation patterns were observed in the phosphoproteome and N-glycoproteome, suggesting their unique contributions to ABA response. Our results reveal that TIMAHAC offers a comprehensive approach to studying complex regulatory mechanisms and PTM interplay in plant biology, paving the way for in-depth investigations into plant signaling networks.
Subject(s)
Arabidopsis , Chromatography, Affinity , Phosphoproteins , Proteomics , Workflow , Proteomics/methods , Arabidopsis/metabolism , Phosphoproteins/metabolism , Phosphoproteins/analysis , Chromatography, Affinity/methods , Arabidopsis Proteins/metabolism , Glycopeptides/metabolism , Glycopeptides/analysis , Hydrophobic and Hydrophilic Interactions , Protein Processing, Post-Translational , Proteome/metabolism , Phosphorylation , Phosphopeptides/metabolism , Phosphopeptides/analysis , Tandem Mass Spectrometry , Plant Proteins/metabolismABSTRACT
Mass spectrometry (MS)-based single-cell proteomics (SCP) provides us the opportunity to unbiasedly explore biological variability within cells without the limitation of antibody availability. This field is rapidly developed with the main focuses on instrument advancement, sample preparation refinement, and signal boosting methods; however, the optimal data processing and analysis are rarely investigated which holds an arduous challenge because of the high proportion of missing values and batch effect. Here, we introduced a quantification quality control to intensify the identification of differentially expressed proteins (DEPs) by considering both within and across SCP data. Combining quantification quality control with isobaric matching between runs (IMBR) and PSM-level normalization, an additional 12% and 19% of proteins and peptides, with more than 90% of proteins/peptides containing valid values, were quantified. Clearly, quantification quality control was able to reduce quantification variations and q-values with the more apparent cell type separations. In addition, we found that PSM-level normalization performed similar to other protein-level normalizations but kept the original data profiles without the additional requirement of data manipulation. In proof of concept of our refined pipeline, six uniquely identified DEPs exhibiting varied fold-changes and playing critical roles for melanoma and monocyte functionalities were selected for validation using immunoblotting. Five out of six validated DEPs showed an identical trend with the SCP dataset, emphasizing the feasibility of combining the IMBR, cell quality control, and PSM-level normalization in SCP analysis, which is beneficial for future SCP studies.
Subject(s)
Proteomics , Quality Control , Single-Cell Analysis , Single-Cell Analysis/methods , Proteomics/methods , Humans , Mass Spectrometry/methods , Data Analysis , Proteome/metabolismABSTRACT
Secondary mutation, T790M, conferring tyrosine kinase inhibitors (TKIs) resistance beyond oncogenic epidermal growth factor receptor (EGFR) mutations presents a challenging unmet need. Although TKI-resistant mechanisms are intensively investigated, the underlying responses of cancer cells adapting drug perturbation are largely unknown. To illuminate the molecular basis linking acquired mutation to TKI resistance, affinity purification coupled mass spectrometry was adopted to dissect EGFR interactome in TKI-sensitive and TKI-resistant non-small cell lung cancer cells. The analysis revealed TKI-resistant EGFR-mutant interactome allocated in diverse subcellular distribution and enriched in endocytic trafficking, in which gefitinib intervention activated autophagy-mediated EGFR degradation and thus autophagy inhibition elevated gefitinib susceptibility. Alternatively, gefitinib prompted TKI-sensitive EGFR translocating toward cell periphery through Rab7 ubiquitination which may favor efficacy to TKIs suppression. This study revealed that T790M mutation rewired EGFR interactome that guided EGFR to autophagy-mediated degradation to escape treatment, suggesting that combination therapy with TKI and autophagy inhibitor may overcome acquired resistance in non-small cell lung cancer.
Subject(s)
Antineoplastic Agents , Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Humans , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Gefitinib/pharmacology , ErbB Receptors/genetics , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Mutation/genetics , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Drug Resistance, Neoplasm/genetics , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Line, TumorABSTRACT
Data analysis is a critical part of quantitative proteomics studies in interpreting biological questions. Numerous computational tools for protein quantification, imputation and differential expression (DE) analysis were generated in the past decade and the search for optimal tools is still going on. Moreover, due to the rapid development of RNA sequencing (RNA-seq) technology, a vast number of DE analysis methods were created for that purpose. The applicability of these newly developed RNA-seq-oriented tools to proteomics data remains in doubt. In order to benchmark these analysis methods, a proteomics dataset consisting of proteins derived from humans, yeast and drosophila, in defined ratios, was generated in this study. Based on this dataset, DE analysis tools, including microarray- and RNA-seq-based ones, imputation algorithms and protein quantification methods were compared and benchmarked. Furthermore, applying these approaches to two public datasets showed that RNA-seq-based DE tools achieved higher accuracy (ACC) in identifying DEPs. This study provides useful guidelines for analyzing quantitative proteomics datasets. All the methods used in this study were integrated into the Perseus software, version 2.0.3.0, which is available at https://www.maxquant.org/perseus.
Subject(s)
Benchmarking , Proteomics , Algorithms , Proteins , Proteomics/methods , Sequence Analysis, RNA , SoftwareABSTRACT
A series of novel benzimidazole derivatives were designed and synthesised based on the structures of reported oral available ALK inhibitor and HDAC inhibitor, pracinostat. In enzymatic assays, compound 3b, containing a 2-acyliminobenzimidazole moiety and hydroxamic acid side chain, could inhibit both ALK and HDAC6 (IC50 = 16 nM and 1.03 µM, respectively). Compound 3b also inhibited various ALK mutants known to be involved in crizotinib resistance, including mutant L1196M (IC50, 4.9 nM). Moreover, 3b inhibited the proliferation of several cancer cell lines, including ALK-addicted H2228 cells. To evaluate its potential for treating cancers in vivo, 3b was used in a human A549 xenograft model with BALB/c nude mice. At 20 mg/kg, 3b inhibited tumour growth by 85% yet had a negligible effect on mean body weight. These results suggest a attracting route for the further research and optimisation of dual ALK/HDAC inhibitors.
Subject(s)
Antineoplastic Agents , Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Mice , Animals , Humans , Anaplastic Lymphoma Kinase , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/metabolism , Histone Deacetylase Inhibitors/pharmacology , Mice, Nude , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Cell Proliferation , Protein Kinase Inhibitors/chemistry , Antineoplastic Agents/chemistry , Cell Line, TumorABSTRACT
Cornea transplantation is one of the most commonly performed allotransplantations worldwide. Prolonged storage of donor corneas leads to decreased endothelial cell viability, severe stromal edema, and opacification, significantly compromising the success rate of corneal transplantation. Corneal stroma, which constitutes the majority of the cornea, plays a crucial role in maintaining its shape and transparency. In this study, we conducted proteomic analysis of corneal stroma preserved in Optisol-GS medium at 4 °C for 7 or 14 days to investigate molecular changes during storage. Among 1923 identified proteins, 1634 were quantifiable and 387 were significantly regulated with longer preservation. Compared to stroma preserved for 7 days, proteins involved in ocular surface immunomodulation were largely downregulated while proteins associated with extracellular matrix reorganization and fibrosis were upregulated in those preserved for 14 days. The increase in extracellular matrix structural proteins together with upregulation of growth factor signaling implies the occurrence of stromal fibrosis, which may compromise tissue clarity and cause vision impairments. This study is the first to provide insights into how storage duration affects corneal stroma from a proteomic perspective. Our findings may contribute to future research efforts aimed at developing long-term preservation techniques and improving the quality of preserved corneas, thus maximizing their clinical application.
Subject(s)
Cryopreservation , Proteomics , Humans , Cryopreservation/methods , Cornea , Corneal Stroma/metabolism , Extracellular Matrix , Gentamicins/metabolism , Complex Mixtures/metabolismABSTRACT
Plant phosphoproteomics provides a global view of phosphorylation-mediated signaling in plants; however, it demands high-throughput methods with sensitive detection and accurate quantification. Despite the widespread use of protein precipitation for removing contaminants and improving sample purity, it limits the sensitivity and throughput of plant phosphoproteomic analysis. The multiple handling steps involved in protein precipitation lead to sample loss and process variability. Herein, we developed an approach based on suspension trapping (S-Trap), termed tandem S-Trap-IMAC (immobilized metal ion affinity chromatography), by integrating an S-Trap micro-column with a Fe-IMAC tip. Compared with a precipitation-based workflow, the tandem S-Trap-IMAC method deepened the coverage of the Arabidopsis (Arabidopsis thaliana) phosphoproteome by more than 30%, with improved number of multiply phosphorylated peptides, quantification accuracy, and short sample processing time. We applied the tandem S-Trap-IMAC method for studying abscisic acid (ABA) signaling in Arabidopsis seedlings. We thus discovered that a significant proportion of the phosphopeptides induced by ABA are multiply phosphorylated peptides, indicating their importance in early ABA signaling and quantified several key phosphorylation sites on core ABA signaling components across four time points. Our results show that the optimized workflow aids high-throughput phosphoproteome profiling of low-input plant samples.
Subject(s)
Arabidopsis , Arabidopsis/metabolism , Workflow , Chromatography, Affinity/methods , Phosphopeptides/chemistry , PhosphorylationABSTRACT
For decades, major difficulties in analyzing histidine phosphorylation have limited the study of phosphohistidine signaling. Here we report a method revealing widespread and abundant protein histidine phosphorylation in Escherichia coli. We generated an extensive E. coli phosphoproteome data set, in which a remarkably high percentage (â¼10%) of phosphorylation sites are phosphohistidine sites. This resource should help enable a better understanding of the biological function of histidine phosphorylation.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Histidine/metabolism , Mass Spectrometry/methods , Proteomics/methods , PhosphorylationABSTRACT
Helicobacter pylori, a pathogen of various gastric diseases, has many genome sequence variants. Thus, the pathogenesis and infection mechanisms of the H. pylori-driven gastric diseases have not been elucidated. Here, we carried out a large-scale proteome analysis to profile the heterogeneity of the proteome expression of 7 H. pylori strains by using an LC/MS/MS-based proteomics approach combined with a customized database consisting of nonredundant tryptic peptide sequences derived from full genome sequences of 52 H. pylori strains. The nonredundant peptide database enabled us to identify more peptides in the database search of MS/MS data compared with a simply merged protein database. Using this approach, we carried out proteome analysis of genome-unknown strains of H. pylori at as large a scale as genome-known ones. Clustering of the H. pylori strains using proteome profiling slightly differed from the genome profiling and more clearly divided the strains into two groups based on the isolated area. Furthermore, we identified phosphorylated proteins and sites of the H. pylori strains and obtained the phosphorylation motifs located in the N-terminus that are commonly observed in bacteria.
Subject(s)
Bacterial Proteins/metabolism , Genetic Variation , Helicobacter Infections/microbiology , Helicobacter pylori/metabolism , Phosphoproteins/metabolism , Proteome/analysis , Bacterial Proteins/genetics , Gene Expression Profiling , Genome, Bacterial , Geography , Helicobacter Infections/genetics , Helicobacter Infections/metabolism , Helicobacter pylori/classification , Helicobacter pylori/genetics , Humans , Phosphoproteins/genetics , Phylogeny , Proteome/metabolismABSTRACT
Here we demonstrate that biomolecular contaminants, such as nucleic acid molecules, can seriously interfere with immobilized metal ion affinity chromatography (IMAC)-based phosphopeptide enrichments. We address and largely solve this issue, developing a robust protocol implementing methanol/chloroform protein precipitation and enzymatic digestion using benzonase, which degrades all forms of DNA and RNA, before IMAC-column loading. This simple procedure resulted in a drastic increase of enrichment sensitivity, enabling the identification of around 17,000 unique phosphopeptides and 12,500 unambiguously localized phosphosites in human cell-lines from a single LC-MS/MS run, constituting a 50% increase when compared with the standard protocol. The improved protocol was also applied to bacterial samples, increasing the number of identified bacterial phosphopeptides even more strikingly, by a factor 10, when compared with the standard protocol. For E. coli we detected around 1300 unambiguously localized phosphosites per LC-MS/MS run. The preparation of these ultra-pure phosphopeptide samples only requires marginal extra costs and sample preparation time and should thus be adoptable by every laboratory active in the field of phosphoproteomics.
Subject(s)
Chromatography, Affinity/methods , Iron/chemistry , Phosphopeptides/metabolism , HEK293 Cells , HeLa Cells , Humans , Ions , Reference StandardsABSTRACT
The rapid emergence of antimicrobial resistance is a major threat to human health. Antibiotics modulate a wide range of biological processes in bacteria and as such, the study of bacterial cellular signaling could aid the development of urgently needed new antibiotic agents. Due to the advances in bacterial phosphoproteomics, such a systemwide analysis of bacterial signaling in response to antibiotics has recently become feasible. Here we present a dynamic view of differential protein phosphorylation upon antibiotic treatment and antibiotic resistance. Most strikingly, differential phosphorylation was observed on highly conserved residues of resistance regulating transcription factors, implying a previously unanticipated role of phosphorylation mediated regulation. Using the comprehensive phosphoproteomics data presented here as a resource, future research can now focus on deciphering the precise signaling mechanisms contributing to resistance, eventually leading to alternative strategies to combat antimicrobial resistance.
Subject(s)
Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Drug Resistance, Multiple, Bacterial , Escherichia coli/drug effects , Escherichia coli/metabolism , Signal Transduction/drug effects , Bacterial Proteins/metabolism , Chromatography, Liquid , Escherichia coli Proteins , Humans , Phosphorylation , Proteomics/methods , Tandem Mass Spectrometry , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
Protein phosphorylation can induce signal transduction to change sperm motility patterns during sperm capacitation. However, changes in the phosphorylation of sperm proteins in mice are still incompletely understood. Here, capacitation-related phosphorylation in mouse sperms were firstly investigated by label-free quantitative (LFQ) phosphoproteomics coupled with bioinformatics analysis using ingenuity pathway analysis (IPA) methods such as canonical pathway, upstream regulator, and network analysis. Among 1632 phosphopeptides identified at serine, threonine, and tyrosine residues, 1050 novel phosphosites, corresponding to 402 proteins, were reported. Gene heatmaps for IPA canonical pathways showed a novel role for GSK-3 in GP6 signaling pathways associated with capacitation for 60 min. At the same time, the reduction of the abundant isoform-specific GSK-3α expression was shown by western blot (WB) while the LFQ pY of this isoform slightly decreased and then increased. The combined results from WB and LFQ methods explain the less inhibitory phosphorylation of GSK-3α during capacitation and also support the predicted increases in its activity. In addition, pAKAP4 increased at the Y156 site but decreased at the Y811 site in a capacitated state, even though IPA network analysis and WB analysis for overall pAKAP revealed upregulated trends. The potential roles of GSK-3 and AKAP4 in fertility are discussed.
Subject(s)
A Kinase Anchor Proteins/genetics , Glycogen Synthase Kinase 3/genetics , Proteomics , Sperm Capacitation/genetics , Animals , Computational Biology , Gene Expression Regulation, Developmental/genetics , Male , Mice , Phosphoproteins/genetics , Phosphorylation/genetics , Signal Transduction/genetics , Spermatozoa/growth & developmentABSTRACT
Recent technological advances have made it possible to investigate the hitherto rather elusive protein histidine phosphorylation. However, confident site-specific localization of protein histidine phosphorylation remains challenging. Here, we address this problem, presenting a mass-spectrometry-based approach that outperforms classical HCD fragmentation without compromising sensitivity. We use the phosphohistidine immonium ion as a diagnostic tool as well as ETD-based fragmentation techniques to achieve unambiguous identification and localization of histidine-phosphorylation sites. The work presented here will allow more confident investigation of the phosphohistidine proteome to reveal the roles of histidine phosphorylation in cellular signaling events.
Subject(s)
Histidine , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Proteomics/methods , Amino Acid Sequence , Mass SpectrometryABSTRACT
Thermus thermophilus HB27 is an extremely thermophilic eubacteria with a high frequency of natural competence. This organism is therefore often used as a thermophilic model to investigate the molecular basis of type IV pili-mediated functions, such as the uptake of free DNA, adhesion, twitching motility, and biofilm formation, in hot environments. In this study, the phosphoproteome of T. thermophilus HB27 was analyzed via a shotgun approach and high-accuracy mass spectrometry. Ninety-three unique phosphopeptides, including 67 in vivo phosphorylated sites on 53 phosphoproteins, were identified. The distribution of Ser/Thr/Tyr phosphorylation sites was 57%/36%/7%. The phosphoproteins were mostly involved in central metabolic pathways and protein/cell envelope biosynthesis. According to this analysis, the ATPase motor PilF, a type IV pili-related component, was first found to be phosphorylated on Thr-368 and Ser-372. Through the point mutation of PilF, mimic phosphorylated mutants T368D and S372E resulted in nonpiliated and nontwitching phenotypes, whereas nonphosphorylated mutants T368V and S372A displayed piliation and twitching motility. In addition, mimic phosphorylated mutants showed elevated biofilm-forming abilities with a higher initial attachment rate, caused by increasing exopolysaccharide production. In summary, the phosphorylation of PilF might regulate the pili and biofilm formation associated with exopolysaccharide production.
Subject(s)
Biofilms/growth & development , Fimbriae Proteins/physiology , Fimbriae, Bacterial/physiology , Phosphoproteins/physiology , Thermus thermophilus/physiology , Biopolymers/metabolism , Escherichia coli/genetics , Phosphopeptides/physiology , Phosphorylation , Polysaccharides/metabolism , ProteomicsABSTRACT
In adult mammals, wound healing predominantly follows a fibrotic pathway, culminating in scar formation. However, cutaneous microwounds generated through fractional photothermolysis, a modality that produces a constellation of microthermal zones, exhibit a markedly different healing trajectory. Our study delineates the cellular attributes of these microthermal zones, underscoring a temporally limited, subclinical inflammatory milieu concomitant with rapid re-epithelialization within 24 hours. This wound closure is facilitated by the activation of genes associated with keratinocyte migration and differentiation. In contrast to macrothermal wounds, which predominantly heal through a robust myofibroblast-mediated collagen deposition, microthermal zones are characterized by absence of wound contraction and feature delayed collagen remodeling, initiating 5-6 weeks after injury. This distinct wound healing is characterized by a rapid re-epithelialization process and a muted inflammatory response, which collectively serve to mitigate excessive myofibroblast activation. Furthermore, we identify an initial reparative phase characterized by a heterogeneous extracellular matrix protein composition, which precedes the delayed collagen remodeling. These findings extend our understanding of cutaneous wound healing and may have significant implications for the optimization of therapeutic strategies aimed at mitigating scar formation.
ABSTRACT
Rhodopseudomonas palustris (R. palustris) is a purple nonsulfur anoxygenic phototrophic bacterium with metabolic versatility and is able to grow under photoheterotrophic and chemoheterotrophic states. It has uses in carbon management, carbon recycling, hydrogen generation, and lipid production; therefore, it has the potential for bioenergy production and biodegradation. This study is the first to identify the phosphoproteome of R. palustris including 100 phosphopeptides from 54 phosphoproteins and 74 phosphopeptides from 42 phosphoproteins in chemoheterotrophic and photoheterotrophic growth conditions, respectively. In the identified phosphoproteome, phosphorylation at the threonine residue, Thr487, of pyruvate phosphate dikinase (PPDK, RPA1051) was found to participate in the regulation of carbon metabolism. Here, we show that PPDK enzyme activity is higher in photoheterotrophic growth, with Thr487 phosphorylation as a possible mediator. Under the same photoheterotrophic conditions, R. palustris with overexpressed wild-type PPDK showed an enhanced accumulation of total lipids than those with mutant PPDK (T487V) form. This study reveals the role of the PPDK in the production of biodiesel material, lipid content, with threonyl-phosphorylation as one of the possible regulatory events during photoheterotrophic growth in R. palustris.
Subject(s)
Bacterial Proteins/metabolism , Lipids/biosynthesis , Phosphoproteins/metabolism , Proteome , Pyruvate, Orthophosphate Dikinase/metabolism , Rhodopseudomonas/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Base Sequence , Chromatography, Liquid , Cloning, Molecular , DNA Primers , Escherichia coli/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphorylation , Pyruvate, Orthophosphate Dikinase/genetics , Rhodopseudomonas/enzymology , Tandem Mass SpectrometryABSTRACT
Bacterial polysaccharides are known to induce the immune response in macrophages. Here we isolated a novel extracellular polysaccharide from the biofilm of Thermus aquaticus YT-1 and evaluated its structure and immunomodulatory effects. The size of this polysaccharide, TA-1, was deduced by size-exclusion chromatography as 500 kDa. GC-MS, high performance anion-exchange chromatography with pulsed amperometric detection, electrospray ionization-MS/MS, and NMR revealed the novel structure of TA-1. The polysaccharide is composed of tetrasaccharide-repeating units of galactofuranose, galactopyranose, and N-acetylgalactosamine (1:1:2) and lacked acidic sugars. TA-1 stimulated macrophage cells to produce the cytokines TNF-α and IL-6. Screening of Toll-like receptors and antibody-blocking experiments indicated that the natural receptor of TA-1 in its immunoactivity is TLR2. Recognition of TA-1 by TLR2 was confirmed by TA-1 induction of IL-6 production in peritoneal macrophages from wild-type mice but not from TLR2(-/-) mice. TA-1, as a TLR2 agonist, could possibly be used as an adjuvant and could enhance cytokine release, which increases the immune response. Furthermore, TA-1 induced cytokine release is dependent on MyD88/TIRAP.
Subject(s)
Biofilms , Macrophage Activation/immunology , Macrophages/immunology , Polysaccharides, Bacterial/immunology , Thermus/physiology , Toll-Like Receptor 2/immunology , Adjuvants, Immunologic/pharmacology , Animals , Carbohydrate Conformation , HEK293 Cells , Humans , Interleukin-6/genetics , Interleukin-6/immunology , Macrophage Activation/drug effects , Mice , Mice, Knockout , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/immunology , Polysaccharides, Bacterial/pharmacology , Toll-Like Receptor 2/genetics , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunologyABSTRACT
Encapsulated Klebsiella pneumoniae is the predominant causative agent of pyogenic liver abscess, an emerging infectious disease that often complicates metastatic meningitis or endophthalmitis. The capsular polysaccharide on K. pneumoniae surface was determined as the key to virulence. Although the regulation of capsular polysaccharide biosynthesis is largely unclear, it was found that protein-tyrosine kinases and phosphatases are involved. Therefore, the identification and characterization of such kinases, phosphatases, and their substrates would advance our knowledge of the underlying mechanism in capsule formation and could contribute to the development of new therapeutic strategies. Here, we analyzed the phosphoproteome of K. pneumoniae NTUH-K2044 with a shotgun approach and identified 117 unique phosphopeptides along with 93 in vivo phosphorylated sites corresponding to 81 proteins. Interestingly, three of the identified tyrosine phosphorylated proteins, namely protein-tyrosine kinase (Wzc), phosphomannomutase (ManB), and undecaprenyl-phosphate glycosyltransferase (WcaJ), were found to be distributed in the cps locus and thus were speculated to be involved in the converging signal transduction of capsule biosynthesis. Consequently, we decided to focus on the lesser studied ManB and WcaJ for mutation analysis. The capsular polysaccharides of WcaJ mutant (WcaJY5F) were dramatically reduced quantitatively, and the LD(50) increased by 200-fold in a mouse peritonitis model compared with the wild-type strain. However, the capsular polysaccharides of ManB mutant (ManBY26F) showed no difference in quantity, and the LD(50) increased by merely 6-fold in mice test. Our study provided a clear trend that WcaJ tyrosine phosphorylation can regulate the biosynthesis of capsular polysaccharides and result in the pathogenicity of K. pneumoniae NTUH-K2044.