ABSTRACT
Single-nucleotide polymorphism (SNP) is one of the core mechanisms that respond to antibiotic resistance of Escherichia coli (E. coli), which is a major issue in environmental pollution. A specific type of SNPs, synonymous SNPs, have been generally considered as the "silent" SNPs since they do not change the encoded amino acid. However, the impact of synonymous SNPs on mRNA splicing, nucleo-cytoplasmic export, stability, and translation was gradually discovered in the last decades. Figuring out the mechanism of synonymous SNPs in regulating antibiotic resistance is critical to improve antimicrobial therapy strategies in clinics and biological treatment strategies of antibiotic-resistant E. coli-polluted materials. With our newly designed antibiotic resistant SNPs prediction algorithm, Multilocus Sequence Type based Identification for Phenotype-single nucleotide polymorphism Analysis (MIPHA), and in vivo validation, we identified 2 important synonymous SNPs 522 G>A and 972 C>T, located at hisD gene, which was previously predicted as a fluoroquinolone resistance-related gene without a detailed mechanism in the E. coli samples with environmental backgrounds. We first discovered that hisD causes gyrA mutation via the upregulation of sbmC and its downstream gene umuD. Moreover, those 2 synonymous SNPs of hisD cause its own translational slowdown and further reduce the expression levels of sbmC and its downstream gene umuD, making the fluoroquinolone resistance determining region of gyrA remains unmutated, ultimately causing the bacteria to lose their ability to resist drugs. This study provided valuable insight into the role of synonymous SNPs in mediating antibiotic resistance of bacteria and a new perspective for the treatment of environmental pollution caused by drug-resistant bacteria.
Subject(s)
Escherichia coli , Fluoroquinolones , Fluoroquinolones/pharmacology , Escherichia coli/genetics , Polymorphism, Single Nucleotide , Microbial Sensitivity Tests , Anti-Bacterial Agents/pharmacologyABSTRACT
Infections caused by drug-resistant bacteria are a serious threat to public health worldwide, and the discovery of novel antibacterial compounds is urgently needed. Here, we screened an FDA-approved small-molecule library and found that crizotinib possesses good antimicrobial efficacy against Gram-positive bacteria. Crizotinib was found to increase the survival rate of mice infected with bacteria and decrease pulmonary inflammation activity in an animal model. Furthermore, it showed synergy with clindamycin and gentamicin. Importantly, the Gram-positive bacteria showed a low tendency to develop resistance to crizotinib. Mechanistically, quantitative proteomics and biochemical validation experiments indicated that crizotinib exerted its antibacterial effects by reducing ATP production and pyrimidine metabolism. A drug affinity responsive target stability study suggested crizotinib targets the CTP synthase PyrG, which subsequently disturbs pyrimidine metabolism and eventually reduces DNA synthesis. Subsequent molecular dynamics analysis showed that crizotinib binding occurs in close proximity to the ATP binding pocket of PyrG and causes loss of function of this CTP synthase. Crizotinib is a promising antimicrobial agent and provides a novel choice for the development of treatment for Gram-positive infections. IMPORTANCE Infections caused by drug-resistant bacteria are a serious problem worldwide. Therefore, there is an urgent need to find novel drugs with good antibacterial activity against multidrug-resistant bacteria. In this study, we found that a repurposed drug, crizotinib, exhibits excellent antibacterial activity against drug-resistant bacteria both in vivo and in vitro via suppressing ATP production and pyrimidine metabolism. Crizotinib was found to disturb pyrimidine metabolism by targeting the CTP synthase PyrG, thus reducing DNA synthesis. This unique mechanism of action may explain the decreased development of resistance by Staphylococcus aureus to crizotinib. This study provides a potential option for the treatment of drug-resistant bacterial infections in the future.
Subject(s)
Anti-Bacterial Agents , Gram-Positive Bacteria , Adenosine Triphosphate , Animals , Anti-Bacterial Agents/pharmacology , Bacteria , Carbon-Nitrogen Ligases , Crizotinib/pharmacology , DNA , Gram-Negative Bacteria , Mice , Microbial Sensitivity Tests , Pyrimidines/pharmacologyABSTRACT
Sirtuin-1 (SIRT1) is a critical nuclear deacetylase that participates in a wide range of biological processes. We hereby employed quantitative acetyl-proteomics to globally reveal the landscape of SIRT1-dependent acetylation in colorectal cancer (CRC) cells stimulated by specific SIRT1 inhibitor Inauhzin (INZ). We strikingly observed that SIRT1 inhibition enhances protein acetylation levels, with the multisite-acetylated proteins (acetyl sites >4/protein) mainly enriched in mitochondria. INZ treatment increases mitochondrial fission and depolarization in CRC cells. The acetylation of mitochondrial proteins promoted by SIRT1 inhibition prevents the recruitment of ubiquitin and LC3 for mitophagic degradation. We then found that, SIRT1 inhibition increases the acetylation of mitochondrial calcium uniporter (MCU) at residue K332, resulting in mitochondrial Ca2+ overload and depolarization, and ultimately CRC apoptosis. Arginine substitution of the K332 (K332R) dramatically decreases the mitochondrial Ca2+ influx, mitochondrial membrane potential loss and ROS burst induced by INZ. This finding uncovers a non-canonical role of SIRT1 in regulating mitochondrial function and implicates a possible way for anticancer intervention through SIRT1 inhibition.
Subject(s)
Calcium , Sirtuin 1 , Acetylation , Calcium/metabolism , Cell Death , Mitochondria/metabolism , Sirtuin 1/genetics , Sirtuin 1/metabolismABSTRACT
Colorectal cancer (CRC) is one of the most common malignancies worldwide, and effective therapy remains a challenge. In this study, we take advantage of a drug repurposing strategy to screen small molecules with novel anticancer activities in a small-molecule library consisting of 1056 FDA-approved drugs. We show, for the first time, that lomitapide, a lipid-lowering agent, exhibits antitumor properties in vitro and in vivo. Activated autophagy is characterized as a key biological process in lomitapide-induced CRC repression. Mechanistically, lomitapide stimulated mitochondrial dysfunction-mediated AMPK activation, resulting in increased AMPK phosphorylation and enhanced Beclin1/Atg14/Vps34 interactions, provoking autophagy induction. Autophagy inhibition or AMPK silencing significantly abrogated lomitapide-induced cell death, indicating the significance of AMPK-regulated autophagy in the antitumor activities of lomitapide. More importantly, PP2A was identified as a direct target of lomitapide by limited proteolysis-mass spectrometry (LiP-SMap), and the bioactivity of lomitapide was attenuated in PP2A-deficient cells, suggesting that the anticancer effect of lomitapide occurs in a PP2A-dependent manner. Taken together, the results of the study reveal that lomitapide can be repositioned as a potential therapeutic drug for CRC treatment.
ABSTRACT
Trans-Cinnamaldehyde (TC) is a widely used food additive, known for its sterilization, disinfection, and antiseptic properties. However, its antibacterial mechanism is not completely understood. In this study, quantitative proteomics was performed to investigate differentially expressed proteins (DEPs) in Escherichia coli in response to TC treatment. Bioinformatics analysis suggested aldehyde toxicity, acid stress, oxidative stress, interference of carbohydrate metabolism, energy metabolism, and protein translation as the bactericidal mechanism. E. coli BW25113ΔyqhD, ΔgldA, ΔbetB, ΔtktB, ΔgadA, ΔgadB, ΔgadC, and Δrmf were used to investigate the functions of DEPs through biochemical methods. The present study revealed that TC exerts its antibacterial effects by inducing the toxicity of its aldehyde group producing acid stress. These findings will contribute to the application of TC in the antibacterial field.
Subject(s)
Escherichia coli Proteins , Escherichia coli , Acrolein/analogs & derivatives , Acrolein/pharmacology , Anti-Bacterial Agents/pharmacology , Bacterial Outer Membrane Proteins , Escherichia coli/genetics , Escherichia coli Proteins/genetics , ProteomicsABSTRACT
This study aimed to screen novel anticancer strategies from FDA-approved non-cancer drugs and identify potential biomarkers and therapeutic targets for colorectal cancer (CRC). Methods: A library consisting of 1056 FDA-approved drugs was screened for anticancer agents. WST-1, colony-formation, flow cytometry, and tumor xenograft assays were used to determine the anticancer effect of azelastine. Quantitative proteomics, confocal imaging, Western blotting and JC-1 assays were performed to examine the effects on mitochondrial pathways. The target protein of azelastine was analyzed and confirmed by DARTS, WST-1, Biacore and tumor xenograft assays. Immunohistochemistry, gain- and loss-of-function experiments, WST-1, colony-formation, immunoprecipitation, and tumor xenograft assays were used to examine the functional and clinical significance of ARF1 in colon tumorigenesis. Results: Azelastine, a current anti-allergic drug, was found to exert a significant inhibitory effect on CRC cell proliferation in vitro and in vivo, but not on ARF1-deficient or ARF1-T48S mutant cells. ARF1 was identified as a direct target of azelastine. High ARF1 expression was associated with advanced stages and poor survival of CRC. ARF1 promoted colon tumorigenesis through its interaction with IQGAP1 and subsequent activation of ERK signaling and mitochondrial fission by enhancing the interaction of IQGAP1 with MEK and ERK. Mechanistically, azelastine bound to Thr-48 in ARF1 and repressed its activity, decreasing Drp1 phosphorylation. This, in turn, inhibited mitochondrial fission and suppressed colon tumorigenesis by blocking IQGAP1-ERK signaling. Conclusions: This study provides the first evidence that azelastine may be novel therapeutics for CRC treatment. ARF1 promotes colon tumorigenesis, representing a promising biomarker and therapeutic target in CRC.
Subject(s)
ADP-Ribosylation Factor 1/metabolism , Colonic Neoplasms/drug therapy , Dynamins/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Mitochondrial Dynamics/drug effects , Phthalazines/pharmacology , ras GTPase-Activating Proteins/metabolism , ADP-Ribosylation Factor 1/genetics , Animals , Anti-Allergic Agents/pharmacology , Apoptosis , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Cell Proliferation , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Dynamins/genetics , Extracellular Signal-Regulated MAP Kinases/genetics , Female , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Middle Aged , Prognosis , Survival Rate , Tumor Cells, Cultured , Xenograft Model Antitumor Assays , ras GTPase-Activating Proteins/geneticsABSTRACT
Lung cancer is the leading cause of cancer-related deaths worldwide, but effective therapeutics is limited. This study aims to identify novel anticancer strategy from a Food and Drug Administration (FDA)-approved drug library consisting of 528 compounds. Benzethonium Chloride (BZN), a FDA-approved drug for anti-infective, was found to markedly induce apoptosis and inhibit proliferation and colony formation ability of lung cancer cells in dose- and time-dependent manners. BZN also enhanced the sensitivity of lung cancer cells to gefitinib, the first-line treatment strategy for selected lung cancer patients. Furthermore, BZN significantly delayed the growth of tumor xenografts in nude mice by increasing apoptosis and decreasing Ki-67 proliferation index, without obvious toxic effects to the vital organs of animals. Mechanistically, quantitative proteomics coupled with bioinformatics analyses and a series of functional assays demonstrated that BZN induced cell cycle arrest at G1 phase, and this was associated with an increase in p38-mediated phosphorylation at threonine 286 (T286) and accelerated degradation of cyclin D1. Our findings provide the first evidence that BZN could be a promising therapeutic agent in lung cancer treatment.
ABSTRACT
Odoroside A (OA) is an active ingredient extracted from the leaves of Nerium oleander Linn. (Apocynaceae). This study aims to examine the anticancer bioactivity of OA against CRC cells and to investigate the action mechanisms involved. As a result, OA can significantly inhibit cellular ability and induce apoptosis of CRC cells in a concentration-dependent manner without any obvious cytotoxicity in normal colorectal epithelial cells. Then, quantitative proteomics combined with bioinformatics is adopted to investigate the alterations of proteins and signaling pathways in response to OA treatment. As suggested by the proteomic analysis, flow cytometry and Western blotting analyses validate that exposure of CRC cells to OA causes cell cycle arrest and apoptosis, accompanied with the activation of the ROS/p53 signaling pathway. This observation demonstrates that OA, as a natural product, can induce oxidative stress to suppress tumor cell growth, implicating a novel therapeutic agent against CRC without obvious side effects.
Subject(s)
Cardenolides/pharmacology , G2 Phase Cell Cycle Checkpoints/drug effects , Proteomics/methods , Reactive Oxygen Species/metabolism , Tumor Suppressor Protein p53/metabolism , Apoptosis/drug effects , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Computational Biology , HT29 Cells , Humans , Signal Transduction/drug effectsABSTRACT
SCOPE: Colorectal cancer (CRC) is one of the most common cancers worldwide with poor survival and limited therapeutic options, and there is an urgent need to develop novel therapeutic agents with good treatment efficiency and low toxicity. This study aims to examine the anticancer bioactivity of liensinine, a constituent of Nelumbo nucifera Gaertn, in CRC and investigate the action mechanisms involved. METHODS AND RESULTS: Liensinine was found to induce apoptosis and exert a significant inhibitory effect on the proliferation and colony-forming ability of CRC cells in a dose-dependent manner without any observed cytotoxicity on normal colorectal epithelial cells. Mechanistically, our data from quantitative proteomics, western blot analysis and flow cytometry analyses demonstrated that exposure of CRC cells to liensinine caused cell cycle arrest, mitochondrial dysfunction and apoptosis, accompanied by the activation of the JNK signaling pathway. Furthermore, animal experiments showed that liensinine markedly suppressed the growth of CRC tumor xenografts in nude mice by reducing the Ki-67 proliferation index, but did not damage the vital organs of the animals. CONCLUSION: This study demonstrated for the first time that liensinine, a food-source natural product, could be a novel therapeutic strategy for treating CRC without obvious side effects.
Subject(s)
Apoptosis/drug effects , Carcinogenesis/drug effects , Isoquinolines/pharmacology , Mitochondria/drug effects , Perchlorates/pharmacology , Phenols/pharmacology , Animals , Cell Cycle Checkpoints/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Colorectal Neoplasms/drug therapy , Female , HT29 Cells , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Mitochondria/pathology , Reactive Oxygen Species/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Protein lysine acetylation is a well-known modification with vital regulatory roles in various biological processes. Currently, the acetylated proteome in Streptococcus pneumoniae (S. pneumoniae) is not yet clear. Combining immune-affinity enrichment with mass spectrometry-based proteomics, we identified the first lysine acetylome of S. pneumoniae. In total, 653 lysine acetylated sites on 392 proteins were identified, which are involved in diverse important biological pathways, including gene expression and central metabolism. S. pneumoniae has a relatively high acetylation level, implying its prominent and diverse roles in the regulation of biological processes. In the acetylome of S. pneumoniae, the most frequently occurring motifs of acetylation are KacK, KacR, KacxK, KacxxK and KacH. Compared with the reported acetylation motifs in various bacterial species, the motif unique to S. pneumoniae is KacT, indicating that species-specific characteristics, regulations and molecular mechanisms of acetylation may exist in this bacterium. Notably, many proteins directly or indirectly contributing to virulence are prevalently acetylated, suggesting that acetylation may coordinate bacterial virulence. This work presented here provides the first system-wide analysis of lysine acetylation in Streptococcus species, which may facilitate a deeper understanding on the regulatory roles of acetylation in the bacteria. BIOLOGICAL SIGNIFICANCE: S. pneumoniae causes a series of serious human diseases. Protein acetylation regulates many important biological pathways in bacteria. In this study, the first lysine acetylome of S. pneumoniae was identified and comprehensively analyzed with bioinformatics methods. One unique acetylated motif (KacT) was identified, suggesting that specific characteristics of lysine acetylation reaction may exist in S. pneumoniae. Besides, our data suggest that lysine acetylation closely regulates bacterial virulence. Further study focusing on the biological functions of these acetylproteins may provide important clues for the therapy of S. pneumoniae infection.
Subject(s)
Lysine/metabolism , Protein Processing, Post-Translational/physiology , Proteomics/methods , Streptococcus pneumoniae/pathogenicity , Acetylation , Computational Biology/methods , Humans , Species Specificity , Tandem Mass Spectrometry , VirulenceABSTRACT
Isodeoxyelephantopin (ESI), isolated from Elephantopus scaber L. has been reported to exert anticancer effects. In this study, we aimed to investigate whether and how cancer cells exert protective responses against ESI treatment. Confocal fluorescence microscopy showed that ESI significantly induced autophagy flux in the lung cancer cells expressing mCherry-EGFP-LC3 reporter. Treatment of the cells with ESI increased the expression levels of the autophagy markers including LC3-II, ATG3 and Beclin1 in a dose-dependent manner. Pretreatment with autophagy inhibitor 3-methyladenine (3-MA) not only attenuated the effects of ESI on autophagy, but also enhanced the effects of ESI on cell viability and apoptosis. Mechanistically, the SILAC quantitative proteomics coupled with bioinformatics analysis revealed that the ESI-regulated proteins were mainly involved in Nrf2-mediated oxidative stress response. We found that ESI induced the nuclear translocation of Nrf2 for activating the downstream target genes including HO-1 and p62 (SQSTM1). More importantly, ESI-induced p62 could competitively bind with Keap1, and releases Nrf2 to activate downstream target gene p62 as a positive feedback loop, therefore promoting autophagy. Furthermore, knockdown of Nrf2 or p62 could abrogate the ESI-induced autophagy and significantly enhanced the anticancer effect of ESI. Taken together, we demonstrated that ESI can sustain cell survival by activating protective autophagy through Nrf2-p62-keap1 feedback loop, whereas targeting this regulatory axis combined with ESI treatment may be a promising strategy for anticancer therapy.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Autophagy , Kelch-Like ECH-Associated Protein 1/metabolism , Lactones/pharmacology , Lung Neoplasms/drug therapy , Membrane Proteins/metabolism , NF-E2-Related Factor 2/metabolism , Plant Extracts/pharmacology , Sesquiterpenes/pharmacology , A549 Cells , Active Transport, Cell Nucleus , Apoptosis , Asteraceae/chemistry , Beclin-1/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Computational Biology , Humans , Microscopy, Confocal , Oxidative Stress , Proteomics , Sequestosome-1 Protein/metabolism , Signal TransductionABSTRACT
Sodium new houttuyfonate (SNH), an addition product of active ingredient houttuynin from the plant Houttuynia cordata Thunb., inhibits a variety of bacteria, yet the mechanism by which it induces cell death has not been fully understood. In the present study, we utilized iTRAQ-based quantitative proteomics to analyze the protein alterations in Streptococcus pneumoniae in response to SNH treatment. Numerous proteins related to the production of reactive oxygen species (ROS) were found to be up-regulated by SNH, suggesting that ROS pathways may be involved as analyzed via bioinformatics. As reported recently, cellular reactions stimulated by ROS including superoxide anion (O2(â¢-)), hydrogen peroxide (H2O2), and hydroxyl radicals (OH(â¢)) have been implicated as mechanisms whereby bactericidal antibiotics kill bacteria. We then validated that SNH killed S. pneumoniae in a dose-dependent manner accompanied by the increasing level of H2O2. On the other hand, the addition of catalase, which can neutralize H2O2 in cells, showed a significant recovery in bacterial survival. These results indicate that SNH indeed induced H2O2 formation to contribute to the cell lethality, providing new insights into the bactericidal mechanism of SNH and expanding our understanding of the common mechanism of killing induced by bactericidal agents.
Subject(s)
Anti-Bacterial Agents/pharmacology , Drugs, Chinese Herbal/pharmacology , Houttuynia/chemistry , Streptococcus pneumoniae/drug effects , Anti-Bacterial Agents/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Drugs, Chinese Herbal/chemistry , Humans , Mass Spectrometry , Oxidative Stress/drug effects , Pneumococcal Infections/microbiology , Proteomics , Reactive Oxygen Species/metabolism , Streptococcus pneumoniae/chemistry , Streptococcus pneumoniae/genetics , Streptococcus pneumoniae/metabolism , Superoxides/metabolismABSTRACT
Amplification of the activated Cdc42-associated kinase 1 (ACK1) gene is frequent in gastric cancer (GC). However, little is known about the clinical roles and molecular mechanisms of ACK1 abnormalities in GC. Here, we found that the ACK1 protein level and ACK1 phosphorylation at Tyr 284 were frequently elevated in GC and associated with poor patient survival. Ectopic ACK1 expression in GC cells induced epithelial-mesenchymal transition (EMT) and promoted migration and invasion in vitro, and metastasis in vivo; the depletion of ACK1 induced the opposite effects. We utilized SILAC quantitative proteomics to discover that the level of the cell cycle-related protein ecdysoneless homologue (ECD) was markedly altered by ACK1. Overexpression of ECD promoted EMT, migration, and invasion in GC, similar to the effects of ACK1 overexpression. Silencing of ECD completely blocked the augmentation of ACK1 overexpression-induced EMT, migration, and invasion. Mechanistically, ACK1 phosphorylated AKT at Thr 308 and Ser 473 and activated the AKT pathway to up-regulate the transcription factor POU2F1, which directly bound to the promoter region of its novel target gene ECD and thus regulated ECD expression in GC cells. Furthermore, the phosphorylation levels of AKT at Thr 308 and Ser 473 and POU2F1 and ECD levels were positively associated with ACK1 levels in clinical GC specimens. Collectively, we have demonstrated that ACK1 promotes EMT, migration, and invasion by activating AKT-POU2F1-ECD signalling in GC cells. ACK1 may be employed as a new prognostic factor and therapeutic target for GC.
Subject(s)
Carrier Proteins/metabolism , Epithelial-Mesenchymal Transition/physiology , Octamer Transcription Factor-1/metabolism , Protein-Tyrosine Kinases/physiology , Proto-Oncogene Proteins c-akt/metabolism , Stomach Neoplasms/physiopathology , Adult , Aged , Animals , Cell Communication/physiology , Cell Line, Tumor , Female , Gene Knockdown Techniques , Heterografts , Humans , Male , Mice, Inbred NOD , Mice, SCID , Middle Aged , Neoplasm Invasiveness , Neoplasm Metastasis , Neoplasm Transplantation , Prognosis , Protein-Tyrosine Kinases/metabolism , Signal Transduction/physiology , Stomach Neoplasms/metabolism , Up-RegulationABSTRACT
Glycogen synthase kinase-3 beta (GSK3ß) was initially identified as a key protein in glucose metabolism. GSK3ß might be involved in cell growth, motility and apoptosis. Systematic identification of GSK3ß-associated proteins is crucial for the exhaustive understanding of its functions. Using GST pull-down experiment and LCMS/MS analysis coupled to bioinformatics tools, we have identified 114 proteins that interacted with GSK3ß1 in hepatocellular carcinoma HepG2 cells. Most of the identified proteins are implicated in metabolic process, whereas other proteins are important for cell proliferation or migration, and have been associated with cancer development and metastasis. Several representative proteins, such as hnRNPK, PCNA, Ezrin and STAT1, have been confirmed to interact with GSK-3ß1 by co-immunoprecipitation in HepG2 cells. Further studies of these interactions may discover the precise roles and the underlying mechanisms of GSK-3ß1 in tumour growth and metastasis.
Subject(s)
Glycogen Synthase Kinase 3/metabolism , Cell Movement , Cell Proliferation , Cytoskeletal Proteins/metabolism , Glycogen Synthase Kinase 3 beta , HEK293 Cells , HeLa Cells , Hep G2 Cells , Heterogeneous-Nuclear Ribonucleoprotein K , Humans , Immunoprecipitation , Isoenzymes/metabolism , Molecular Sequence Annotation , Proliferating Cell Nuclear Antigen/metabolism , Protein Interaction Mapping , Protein Interaction Maps , Proteome/metabolism , Ribonucleoproteins/metabolism , STAT1 Transcription Factor/metabolismABSTRACT
Isolated from soybeans, genistein is an isoflavonoid that exhibits anti-carcinogenic effects. Genistein could induce G2/M arrest and apoptosis of various cancer cells in vivo and in vitro. Although ERK1/2, AKT, p90RSK and NFκB were previously found to be regulated by genistein, most of signaling components in genistein-inhibited signaling pathways were still unknown. Here, we used SILAC quantitative phosphoproteomics to globally identify the phosphoproteins and their regulatory sites in signaling pathways mediated by genistein. We detected 1177 phosphorylation sites on 635 unique proteins; among them, 320 phosphorylation sites on 222 unique phosphopeptides representing 215 non-redundant proteins were modulated at least 1.5-folds by genistein. Apart from ERK1/2, PI3K, p90RSK, Bad and topoisomerase that are known genistein-regulated effectors, many novel phosphoproteins were identified for the first time to be involved in genistein-regulated signal transduction networks. They mainly include 9 receptors, 5 signal adaptors, 13 protein kinases, 2 protein phosphatase regulatory subunits, and 14 transcription regulators. Several of these phosphoproteins have been proven to be involved in G2/M arrest or apoptosis such as GPCRs, DCC, NCK1, TNK2, BTK, TP53BP1, BCLAF, MAX and MAG. This dataset provides valuable insights into the cancer-related phosphorylation signaling pathways regulated by genistein.
Subject(s)
Genistein/pharmacology , Proteomics/methods , Signal Transduction/physiology , Cell Line, Tumor , Humans , Isotope Labeling/methods , Phosphopeptides/metabolism , Phosphoproteins/metabolism , Phosphorylation , Signal Transduction/drug effectsABSTRACT
Abstract Streptococcus pneumoniae is a Gram-positive human pathogen that causes a variety of serious mucosal and invasive diseases in human. Bacterial membrane proteins play crucial roles in host-pathogen interactions and bacterial pathogenesis, and thus are potential drug targets or vaccine candidates. In this study, membranes from Streptococcus pneumoniae D39 were enriched by mechanical grinding and ultracentrifugation, and then the membrane proteins were extracted with trifluroethanol and chloroform. Around 60% of the extracted proteins were identified to be membrane proteins with 2-DE coupled with MALDI-MS/MS and 2D-LC-ESI-MS/MS. These identified membrane proteins can be functionally categorized into various groups involved in nutriment transport, signal transduction, protein folding or secretion, oxidation, carbohydrate metabolism, and other physiological processes. A protein interaction network was constructed for understanding the regulation relationship of the membrane proteins. This study represents the first global characterization of membrane proteome from Gram-positive streptococcus species of bacteria, providing valuable clues for further investigation aiming at identifying drug/vaccine targets for the bacterial infection.
Subject(s)
Bacterial Proteins/metabolism , Membrane Proteins/metabolism , Streptococcus pneumoniae/metabolism , ATP-Binding Cassette Transporters/chemistry , ATP-Binding Cassette Transporters/isolation & purification , ATP-Binding Cassette Transporters/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Electrophoresis, Gel, Two-Dimensional , Isoelectric Focusing , Membrane Proteins/chemistry , Membrane Proteins/isolation & purification , Peptide Fragments/chemistry , Protein Interaction Mapping , Proteolysis , Proteomics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tandem Mass SpectrometryABSTRACT
Streptococcus pneumoniae is a major human respiratory pathogen causing considerable morbidity and mortality worldwide. In order to better understand the pathogenesis of S. pneumoniae, we employed SDS-PAGE combined with LC-MS/MS analysis and in-solution digestion coupled with 2D-LC-MS/MS to obtain the whole-cell proteome of the bacterium. Among the identified 1,210 proteins, 345 proteins were annotated for cellular components, 613 for biological processes, and 421 for molecular functions. Important virulence-associated surface proteins such as Eno, ZmpB, and PrtA were identified. Classification analysis and protein-protein interaction map revealed that these identified proteins are involved in many biological processes including protein biosynthesis, protein folding and proteolysis, cell cycle, or regulation and carbohydrate metabolism. These data represent a comprehensive reference map of S. pneumoniae proteome, providing a useful source for further analysis of the virulence factors and the regulatory network involved in the pathogenesis of the bacterium.
Subject(s)
Bacterial Proteins/metabolism , Chromatography, Liquid/methods , Proteome/metabolism , Streptococcus pneumoniae/metabolism , Tandem Mass Spectrometry/methods , Electrophoresis, Polyacrylamide GelABSTRACT
Phosphorylation dysregulation has been implicated in various diseases including cancer. The phosphorylation change of proteins in secretome may be a novel source for the discovery of biomarkers and drug targets. In this study, the phosphoproteins in cancer secretome (phosphosecretome) were globally analyzed for the first time by phosphoproteomics. One hundred forty-two phosphorylation sites on 62 unique phosphopeptides representing 49 nonredundant proteins were identified, several of which are known as secreted proteins involved in carcinogenesis, invasion, and metastasis. Most of them were first found as secreted proteins with no previously known function. Protein sublocation analysis showed that 33 proteins were found to be secreted as phosphoproteins, in which 27 (81.81%) were secreted by a nonclassic, ER/Golgi-independent pathway, suggesting that the phosphorylation modification of these proteins might play an important role in their nonconventional secretion processes. Their protein kinases and regulatory phosphosites involved in the secretion regulation of these phosphoproteins, such as stanniocalcin 2, annexin A2, and HSP90 alpha, were first identified. The phosphosecretome data enriched the secretome database and phosphoproteome database, and will help us to discover cancer biomarkers and drug targets, illustrating the mystery of the nonclassic protein secretion pathway.
Subject(s)
Neoplasm Proteins/metabolism , Phosphoproteins/metabolism , Proteome , Stomach Neoplasms/metabolism , Chromatography, Liquid , Humans , Phosphorylation , Tandem Mass SpectrometryABSTRACT
As an in vitro model for type II human lung cancer, A549 cells resist cytotoxicity via phosphorylation of proteins as demonstrated by many studies. However, to date, no large-scale phosphoproteome investigation has been conducted on A549. Here, we performed a systematical analysis of the phosphoproteome of A549 by using mass spectrometry (MS)-based strategies. This investigation led to the identification of 337 phosphorylation sites on 181 phosphoproteins. Among them, 67 phosphoproteins and 230 phosphorylation sites identified appeared to be novel with no previous characterization in lung cancer. Based on their known functions as reported in the literature, these phosphoproteins were functionally organized into highly interconnected networks. Western blotting and immunohistochemistry analyses were performed to validate the expression of a bottleneck phosphoprotein YAP1 in cancer cell lines and tissues. This dataset provides a valuable resource for further studies on phosphorylation and lung carcinogenesis.
Subject(s)
Lung Neoplasms/metabolism , Neoplasm Proteins/metabolism , Phosphoproteins/metabolism , Proteome , Blotting, Western , Cell Line, Tumor , Chromatography, Liquid , Humans , Immunohistochemistry , Lung Neoplasms/pathology , Peptide Mapping , Phosphorylation , Tandem Mass SpectrometryABSTRACT
Multiple myeloma (MM) is a malignant disorder of differentiated B cells. Clonal expansion of the tumor results in the excessive production of monoclonal immunoglobulin (Ig) which is a diagnostic feature of this disease. Previous investigations have demonstrated the alteration of the ERK, jun kinase, STAT, and AKT kinase signaling cascades in MM cells, suggesting that deregulated phosphorylation may contribute to MM pathogenesis. However, systematic analysis of the phosphoproteome in MM cells has not been reported. Here, we described a large-scale phosphorylation analysis of primary MM cells. Using a separation strategy involving immunomagnetic bead-positive selection of MM cells, preparative SDS-PAGE for prefractionation, in-gel digestion with trypsin, and titanium dioxide enrichment of phosphopeptides, followed by LC-MS/MS analysis employing a hybrid LTQ-Orbitrap mass spectrometer, we were able to catalog a substantial portion of the phosphoproteins present in primary MM cells. This analysis led to the identification of 530 phosphorylation sites from 325 unique phosphopeptides corresponding to 260 proteins at false positive rate (FPR) of 1.3%. This dataset provides an important resource for future studies on phosphorylation and carcinogenesis analysis of multiple myeloma.