Publications (12)41.46 Total impact
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Article: Analysis of Streptococcus pneumoniae secreted antigens by immuno-proteomic approach.
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ABSTRACT: Streptococcus pneumoniae is an important human pathogen that causes a variety of diseases in both adults and children, such as pneumonia, bacteremia, meningitis, otitis media, and sinusitis. Despite their clinical importance, to date, there have been few proteomic studies of these strains for screening of virulence factors or diagnostic markers. In the present study, secreted proteins (secretome) of Streptococcus pneumoniae strains were enriched using ammonium sulfate precipitation and identified by the shotgun proteomic method using 1-dimensional electrophoresis liquid chromatography-mass spectrometry/mass spectrometry analysis. Characterization of the identified proteins revealed that 17.8% (42) of the secreted proteins possessed signal peptides. Twenty-one secreted proteins belonged to the extracellular group, and 4 secreted proteins belonged to the cell wall group. Well-known virulence factors (PrtA, PspC, PsaA, PbpA, PhtD, AmiA, ZmpB, Eno, and Ply) were included in the secreted protein fraction. Western blotting using antiserum against secreted protein mixtures showed that Gsp-781, Sphtra, NagA, PhtD, ZmpB, and Eno were strongly immunogenic. Our data suggest that the immuno-proteomic approach is a useful method for high-throughput identification of secreted proteins and screening of candidate vaccine antigens or diagnostic markers. Gsp-781 is introduced as a novel secreted antigen of Streptococcus pneumoniae.Diagnostic microbiology and infectious disease 02/2012; 72(4):318-27. · 2.45 Impact Factor -
Article: Analysis of the endoplasmic reticulum subproteome in the livers of type 2 diabetic mice.
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ABSTRACT: Type 2 diabetes is a chronic metabolic disease that results from insulin resistance in the liver, muscle, and adipose tissue and relative insulin deficiency. The endoplasmic reticulum (ER) plays a crucial role in the regulation of the cellular response to insulin. Recently, ER stress has been known to reduce the insulin sensitivity of the liver and lead to type 2 diabetes. However, detailed mechanisms of ER stress response that leads to type 2 diabetes remains unknown. To obtain a global view of ER function in type 2 diabetic liver and identify proteins that may be responsible for hepatic ER stress and insulin resistance, we performed proteomics analysis of mouse liver ER using nano UPLC-MSE. A total of 1584 proteins were identified in control C57 and type 2 diabetic db/db mice livers. Comparison of the rER and sER proteomes from normal mice showed that proteins involved in protein synthesis and metabolic process were enriched in the rER, while those associated with transport and cellular homeostasis were localized to the sER. In addition, proteins involved in protein folding and ER stress were found only in the rER. In the livers of db/db mice, however, the functions of the rER and sER were severely disrupted, including the capacity to resolve ER stress. These results provide new insight into the research on hepatic insulin resistance and type 2 diabetes and are suggestive of the potential use of the differentially expressed hepatic ER proteins as biomarkers for hepatic insulin resistance and type 2 diabetes.International Journal of Molecular Sciences 01/2012; 13(12):17230-43. · 2.60 Impact Factor -
Article: Comparative proteomic analysis of human somatic cells, induced pluripotent stem cells, and embryonic stem cells.
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ABSTRACT: Induced pluripotent stem cells (iPSCs) are somatic cells that have been reprogrammed to a pluripotent state via introduction of defined transcription factors. iPSCs are a valuable resource for regenerative medicine, but whether iPSCs are identical to embryonic stem cells (ESCs) remains unclear. In this study, we performed comparative proteomic analyses of human somatic cells [human newborn foreskin fibroblasts (hFFs)], human iPSCs (hiPSCs) derived from hFFs, and H9 human ESCs (hESCs). We reprogrammed hFFs to a pluripotent state using 4 core transcription factors: Oct4 (O), Sox2 (S), Klf4 (K), and c-Myc (M). The proteome of hiPSCs induced by 4 core transcription factors was relatively similar to that of hESCs. However, several proteins, including dUTPase, GAPDH, and FUSE binding protein 3, were differentially expressed between hESCs and hiPSCs, implying that hiPSCs are not identical to hESCs at the proteomic level. The proteomes of iPSCs induced by introducing 3, 5, or 6 transcription factors were also analyzed. Our proteomic profiles provide valuable insight into the factors that contribute to the similarities and differences between hESCs and hiPSCs and the mechanisms of reprogramming.Stem cells and development 07/2011; 21(8):1272-86. · 4.15 Impact Factor -
Article: Enrichment and proteome analysis of a hyperthermostable protein set of archaeon Thermococcus onnurineus NA1.
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ABSTRACT: Thermococcus onnurineus NA1 is a hyperthermophilic archaeon that can be used for the screening of thermophilic enzymes. Previously, we characterized the metabolic enzymes of the cytosolic proteome by two-dimensional electrophoresis/tandem mass spectrometry (2-DE/MS-MS). In this study, we identified a subset of hyperthermostable proteins in the cytosolic proteome using enrichment by in vitro heat treatment and protein identification. After heat treatment at 100°C for 2 h, 13 and 149 proteins were identified from the soluble proteome subset by 2-DE/MS-MS and 1-DE/MS-MS analysis, respectively. Representative proteins included intracellular protease I, thioredoxin reductase, triosephosphate isomerase, putative hydroperoxide reductase, proteasome, and translation initiation factors. Intracellular protease, deblocking aminopeptidases, and fructose-1,6-bisphosphatase were overexpressed in Escherichia coli and biological activity above 85°C was confirmed. The folding transition temperature (Tm) of identified proteins was analyzed using the in silico prediction program TargetStar. The proteins enriched with the heat treatment have higher Tm than the homologous proteins from mesophilic strains. These results suggested that the heat-stable protein set of hyperthermophilic T. onnurineus NA1 can be effectively fractionated and enriched by in vitro heat treatment.Extremophiles 07/2011; 15(4):451-61. · 2.94 Impact Factor -
Article: Proteomic characterization of influenza H5N1 virus-like particles and their protective immunogenicity.
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ABSTRACT: Recombinant virus-like particles (VLPs) have been shown to induce protective immunity. Despite their potential significance as promising vaccine candidates, the protein composition of VLPs produced in insect cells has not been well characterized. Here we report a proteomic analysis of influenza VLPs containing hemagglutinin (HA) and matrix M1 proteins from a human isolate of avian influenza H5N1 virus (H5 VLPs) produced in insect cells using the recombinant baculovirus expression system. Comprehensive proteomic analysis of purified H5 VLPs identified viral proteins and 37 additional host-derived proteins, many of which are known to be present in other enveloped viruses. Proteins involved in different cellular structures and functions were found to be present in H5 VLPs including those from the cytoskeleton, translation, chaperone, and metabolism. Immunization with purified H5 VLPs induced protective immunity, which was comparable to the inactivated whole virus containing all viral components. Unpurified H5 VLPs containing excess amounts of noninfluenza soluble proteins also conferred 100% protection against lethal challenge although lower immune responses were induced. These results provide important implications consistent with the idea that VLP production in insect cells may involve similar cellular machinery as other RNA enveloped viruses during synthesis, assembly, trafficking, and budding processes.Journal of Proteome Research 06/2011; 10(8):3450-9. · 5.11 Impact Factor -
Article: Proteome analysis of Thermococcus onnurineus NA1 reveals the expression of hydrogen gene cluster under carboxydotrophic growth.
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ABSTRACT: Thermococcus onnurineus NA1 is a typical sulfur-reducing hyperthermophilic archaeon. Genome sequence analysis has shown that T. onnurineus NA1 retains the metabolic pathways necessary not only for organotrophic, but also for carboxydotrophic, growth. T. onnurineus NA1 carboxydotrophic growth may result in hydrogen production, as this archaeon produces hydrogen during oxidizing formate. In this study, we profiled the proteome of T. onnurineus NA1 cultured under carboxydotrophic conditions using CO as an electron donor by the SDS-PAGE/LC-MS/MS method. A total of 1395 proteins were identified by two independent proteomic analyses, which corresponds to ~71% of the total predicted open reading frames. To our knowledge, this level of identification coverage exceeds those of other global proteome profiling studies in Archaea. Furthermore, the biological functions of the identified proteins were predicted and cognate enzymes were mapped to the appropriate metabolic pathways. More than 90% of the genes belonging to hydrogenase gene clusters such as Mbx, Sulf-I, Mbh, Hyg4-I, Hyg4-II, and Hyf4-III were expressed during CO culture. This means that hydrogenases induced under carboxydotrophic conditions surpass those induced under an organotrophic condition (yeast extract-peptone-sulfur). Our data suggest that hydrogen production is caused by the expression and functional assembly of T. onnurineus NA1 hydrogenase genes during culture in the presence of CO.Journal of proteomics 05/2011; 74(10):1926-33. · 5.07 Impact Factor -
Article: Proteomic characterization of the Pseudomonas putida KT2440 global response to a monocyclic aromatic compound by iTRAQ analysis and 1DE-MudPIT.
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ABSTRACT: Pseudomonas putida KT2440 is a metabolically versatile soil bacterium. To examine the effects of an aromatic compound on the proteome of this bacterium, cytosolic proteins induced by the presence of benzoate and succinate were analyzed using two liquid chromatography (LC)-based proteomic approaches: an isobaric tag for relative and absolute quantitation (iTRAQ) for quantitative analysis and one-dimensional gel electrophoresis/multidimensional protein identification technology (1-DE MudPIT) for protein identification. In total, 1286 proteins were identified by 1-DE MudPIT; this represents around 23.3% of the total proteome. In contrast, 570 proteins were identified and quantified by iTRAQ analysis. Of these, 55 and 52 proteins were up- and down-regulated, respectively, in the presence of benzoate. The proteins up-regulated included benzoate degradation enzymes, chemotaxis-related proteins, and ABC transporters. Enzymes related to nitrogen metabolism and pyruvate metabolism were down-regulated. These data suggest that a combination of 1-DE MudPIT and iTRAQ is an appropriate method for comprehensive proteomic analysis of biodegradative bacteria.Journal of proteomics 02/2011; 74(5):620-8. · 5.07 Impact Factor -
Article: Acinetobacter baumannii secretes cytotoxic outer membrane protein A via outer membrane vesicles.
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ABSTRACT: Acinetobacter baumannii is an important nosocomial pathogen that causes a high morbidity and mortality rate in infected patients, but pathogenic mechanisms of this microorganism regarding the secretion and delivery of virulence factors to host cells have not been characterized. Gram-negative bacteria naturally secrete outer membrane vesicles (OMVs) that play a role in the delivery of virulence factors to host cells. A. baumannii has been shown to secrete OMVs when cultured in vitro, but the role of OMVs in A. baumannii pathogenesis is not well elucidated. In the present study, we evaluated the secretion and delivery of virulence factors of A. baumannii to host cells via the OMVs and assessed the cytotoxic activity of outer membrane protein A (AbOmpA) packaged in the OMVs. A. baumannii ATCC 19606(T) secreted OMVs during in vivo infection as well as in vitro cultures. Potential virulence factors, including AbOmpA and tissue-degrading enzymes, were associated with A. baumannii OMVs. A. baumannii OMVs interacted with lipid rafts in the plasma membranes and then delivered virulence factors to host cells. The OMVs from A. baumannii ATCC 19606(T) induced apoptosis of host cells, whereas this effect was not detected in the OMVs from the ΔompA mutant, thereby reflecting AbOmpA-dependent host cell death. The N-terminal region of AbOmpA(22-170) was responsible for host cell death. In conclusion, the OMV-mediated delivery of virulence factors to host cells may well contribute to pathogenesis during A. baumannii infection.PLoS ONE 01/2011; 6(2):e17027. · 4.09 Impact Factor -
Article: Analysis of cytoplasmic membrane proteome of Streptococcus pneumoniae by shotgun proteomic approach.
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ABSTRACT: In this study, cytoplasmic membrane proteins of S. pneumoniae strain R6 (ATCC BBA-255) were effectively separated from cell wall or extracellular proteins by sodium carbonate precipitation (SCP) and ultracentrifugation. Forty seven proteins were analyzed as cytoplasmic membrane proteins from the 260 proteins identified by the shotgun proteomic method using SDS-PAGE/LC/MS-MS. ABC transporters for metabolites such as metals, oligopeptides, phosphate, sugar, and amino acids, and membrane proteins involved in phosphotransferase systems, were identified as the predominant and abundant, cytoplasmic membrane proteins that would be essential for nutrient uptake, antibiotic resistance and virulence mechanisms. Our result supports that gel-based shotgun proteomics combined with sodium carbonate precipitation and ultracentrifugation is an effective method for analysis of cytoplasmic membrane proteins of S. pneumoniae.The Journal of Microbiology 12/2010; 48(6):872-6. · 1.10 Impact Factor -
Article: Quantitative proteomic analysis of cell wall and plasma membrane fractions from multidrug-resistant Acinetobacter baumannii.
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ABSTRACT: Acinetobacter baumannii is a Gram-negative, nonmotile aerobic bacterium that has emerged as an important nosocomial pathogen. Multidrug-resistant (MDR) A. baumannii is difficult to treat with antibiotics, and treatment failure in infected patients is of great concern in clinical settings. To investigate proteome regulation in A. baumannii under antibiotic stress conditions, quantitative membrane proteomic analyses of a clinical MDR A. baumannii strain cultured in subminimal inhibitory concentrations of tetracycline and imipenem were performed using a combination of label-free (one-dimensional electrophoresis-liquid chromatography-tandem mass spectrometry) and label (isobaric tag for relative and absolute quantitation) approaches. In total, 484 proteins were identified, and 302 were classified as outer membrane, periplasmic, or plasma membrane proteins. The clinical A. baumannii strain DU202 responded specifically and induced different cell wall and membrane protein sets that provided resistance to the antibiotics. The induction of resistance-nodulation-cell division transporters and protein kinases, and the repression of outer membrane proteins were common responses in the presence of tetracycline and imipenem. Induction of a tetracycline resistant pump, ribosomal proteins, and iron-uptake transporters appeared to be dependent on tetracycline conditions, whereas β-lactamase and penicillin-binding proteins appeared to be dependent on imipenem conditions. These results suggest that combined liquid chromatography-based proteomic approaches can be used to identify cell wall and membrane proteins involved in the antibiotic resistance of A. baumannii.Journal of Proteome Research 11/2010; 10(2):459-69. · 5.11 Impact Factor -
Article: Proteomic analysis of porcine pancreas development.
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ABSTRACT: Porcine pancreas development is not well studied at the molecular level despite being a therapeutic resource for diabetic patients. In this study, we investigated expression of lineage markers and performed proteomic analysis. Expression of the early lineage markers Pdx1 and Ptf1a was developmentally conserved between mice and pigs, whereas expression of the islet differentiation marker Pax4 was delayed in porcine compared with murine pancreas development. Proteomic analysis found that expression levels of chymotrypsinogen were downregulated during porcine pancreas development while those of digestive enzymes like lipases, elastase and serine protease were up-regulated. In addition, specific isoforms of protein folding assistants such as protein disulfide isomerase and prefoldin were expressed at specific stages during the maturation of digestive enzymes. Taken together, these results show that development of the porcine pancreas is regulated by a concerted interplay of pancreas lineage marker proteins and other specified proteins, resulting in a functional endocrine and exocrine organ.BMB reports 10/2009; 42(10):661-6. · 1.72 Impact Factor -
Article: Proteome analysis of outer membrane vesicles from a clinical Acinetobacter baumannii isolate.
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ABSTRACT: The secretion of outer membrane vesicles (OMVs) is one of the major mechanisms by which Gram-negative bacteria deliver effector molecules to host cells. Acinetobacter baumannii is an important opportunistic pathogen in hospital-acquired infections, but the secretion system for effector molecules to induce host cell damage has not been characterized. In the present study, we investigated the secretion of OMVs from a clinical A. baumannii isolate and analyzed the comprehensive proteome of A. baumannii-derived OMVs. Acinetobacter baumannii secreted OMVs into the extracellular milieu during in vitro growth. Using 1-DE and LC-MS/MS protein identification and assignment analysis, 132 different proteins associated with OMVs were identified. These proteins were derived from outer membranes (n=26), periplasmic space (n=6), inner membranes (n=8), cytoplasm (n=43), and unknown localization or multiple localization sites (n=49) according to the cell location prediction programs. Among the proteins associated with OMVs, a potent cytotoxic molecule, outer membrane protein A, was highly enriched and several putative virulence-associated proteins were also identified. These results suggest that OMVs from A. baumannii are an important vehicle designed to deliver effector molecules to host cells.FEMS Microbiology Letters 07/2009; 297(2):150-6. · 2.04 Impact Factor
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2009–2012
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Korea Basic Science Institute KBSI
Seoul, Seoul, South Korea
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