MAPPING THE STIMULUS-SPECIFIC SIGNALING PATHWAYS IN PERIODONTITIS BY PROTEOMICS

通过蛋白质组学绘制牙周炎中刺激特异性信号通路

基本信息

  • 批准号:
    7369293
  • 负责人:
  • 金额:
    $ 0.48万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2006
  • 资助国家:
    美国
  • 起止时间:
    2006-07-01 至 2007-06-30
  • 项目状态:
    已结题

项目摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Periodontitis is an inflammatory disease with host-parasite interactions which contribute to connective tissue destruction and alveolar bone resorption. Porphyromonas gingivalis (P.g.) a black-pigmented Gram-negative anaerobic bacterium, is a major pathogen in the development and progression of periodontitis. Cell wall and appendage structures, such as lipopolysaccharide (LPS) and fimbriae, play important roles in the induction of innate immune responses, including cytokine production by localized and circulating monocyte/macrophage. Based on preliminary data, live P.g. stimulates unique pro-inflammatory signal transduction pathways in human peripheral blood monocytes (PBM) as opposed to bacterial components, such as LPS or fimbriae. In these experiments we test at the level of protein expression that unique signaling pathways are differentially induced by live P.g. The crude fimbriae are purified on a C8 reversed-phase column. (1) Human PBM (95% pure) freshly elutriated from healthy donor and monocyte-like THP-1 cells are exposed to live P.g., P.g. LPS and P.g. fimbriae. 2-dimensional gel electrophoresis (2-DGE) are performed on the cell lysate proteins of PBM and THP-1 differentially produced. The expressed proteins are excised and subjected to tryptic in-gel digestion. Matrix-assisted laser desorption/ionization (MALDI) spectra are acquired and protein identification is performed using online protein database search. P.g. crude fimbriae are well resolved from other components on a C8 column when eluted with a gradient of CH3CN in 0.1% TFA . By using RP-HPLC with C8 column, the P.g. fimbriae can be purified rapidly and efficiently. 2-DGE was performed on cell lysate proteins differentially produced by PBM when challenged by live P.g. vs. unchallenged conditions. The 2-DGE profiles identified several differentially expressed protein spots. To rule out protein spots resulting from P.g. proteins we performed also 2-DGE of P.g. and used it against the experimental gel. The differentially expressed proteins for P.g. challenged PBM were excised, in-gel digested with trypsin and mass spectra were obtained to characterize them. The results so far show that specific proteins are induced or down-regulated by live P.g. Similar procedures were implemented for LPS and fimbriae induced PBM. 1-D gel electrophoresis was performed on the PBM challenged by live P.g., LPS and fimbriae. This approach allowed for identification of biologically significant proteins that might be in low abundance. The separated protein bands were subjected to in-gel tryptic digestion and MALDI-MS spectra were obtained. On-line data base search was undertaken to identify the proteins. To improve separation during isoelectric focusing stage of 2-DGE, novel buffer conditions were used for extraction of proteins from the cell lysates of PBM and THP-1. The extraction buffer avoids the use of SDS, which hampers separation of proteins in the first dimension. The 2-DGE profiles showed similar patterns of protein separation for control, live P.g., LPS (Figure 3) and fimbriae induced THP-1, making it easier for identification of differentially expressed protein spots by direct visualization. Analysis of the 2-DGE profiles identified several differentially expressed protein spots. MALDI spectra were acquired and protein identification was performed using online protein database search. Similar approach will be undertaken for human PBM in the future. Post-translational modifications of the identified proteins will also be determined. 1. Sojar, H.T., Hamada, N. and Genco, R.J. Protein Expression and Purif. 9, 49-52 (1997).
该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者(PI)可能从另一个NIH来源获得主要资金,因此可以在其他CRISP条目中表示。所列机构为中心,不一定是研究者所在机构。牙周炎是一种宿主-寄生虫相互作用的炎症性疾病,导致结缔组织破坏和牙槽骨吸收。牙龈卟啉单胞菌(P.g.)一种黑色的革兰氏阴性厌氧菌,是牙周炎发生和发展的主要病原体。细胞壁和附属结构,如脂多糖(LPS)和菌毛,在诱导先天性免疫应答中起重要作用,包括局部和循环单核细胞/巨噬细胞产生细胞因子。根据初步数据,现场P.G.刺激人外周血单核细胞(PBM)中独特的促炎信号转导途径,而不是细菌组分,如LPS或菌毛。在这些实验中,我们在蛋白质表达水平上测试了独特的信号传导途径被活的P.g. 在C8反相柱上纯化粗菌毛。(1)将从健康供体新鲜淘析的人PBM(95%纯)和单核细胞样THP-1细胞暴露于活的P.g.,P.G. LPS和P.g.菌毛2-对差异产生的PBM和THP-1的细胞裂解物蛋白进行双向凝胶电泳(2-DGE)。切下表达的蛋白质并进行胰蛋白酶凝胶内消化。获得基质辅助激光解吸/电离(MALDI)光谱,并使用在线蛋白质数据库搜索进行蛋白质鉴定。 P.G.当用0.1%TFA中的CH 3CN梯度洗脱时,在C8柱上将粗菌毛与其它组分很好地分离。采用反相高效液相色谱法,C_8柱,对P.g.菌毛可以快速有效地纯化。 2-DGE对PBM在活P.g. vs.不受挑战的条件2-DGE图谱鉴定了几个差异表达的蛋白质点。为了排除由P.g.蛋白质,我们也进行了2-DGE的P.g.并将其用于实验凝胶。P.g.切下攻击的PBM,用胰蛋白酶进行凝胶内消化,并获得质谱以表征它们。到目前为止的结果表明,特定的蛋白质被诱导或下调活P.g. 对LPS和菌毛诱导的PBM实施类似的程序。对经活P.g.攻击的PBM进行一维凝胶电泳,LPS和菌毛。这种方法允许鉴定可能丰度低的生物学显著蛋白质。将分离的蛋白条带进行胶内胰蛋白酶消化,获得MALDI-MS谱。进行在线数据库搜索以鉴定蛋白质。为了改善2-DGE等电聚焦阶段的分离,使用新的缓冲条件从PBM和THP-1的细胞裂解物中提取蛋白质。提取缓冲液避免了SDS的使用,SDS阻碍了蛋白质在第一维的分离。2-DGE图谱显示对照、活P.g. LPS(图3)和菌毛诱导THP-1,使得通过直接可视化更容易鉴定差异表达的蛋白质点。2-DGE图谱分析鉴定了几个差异表达的蛋白质点。获得MALDI光谱,并使用在线蛋白质数据库搜索进行蛋白质鉴定。将来将对人PBM采取类似的方法。还将确定鉴定的蛋白质的翻译后修饰。 1. Sojar,H. T.,Hamada,N.和Genco,R.J. Protein Expression and Purif. 9,49-52(1997)。

项目成果

期刊论文数量(0)
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Salomon Amar其他文献

Salomon Amar的其他文献

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{{ truncateString('Salomon Amar', 18)}}的其他基金

Role of LITAF in Inflammatory Disease
LITAF 在炎症性疾病中的作用
  • 批准号:
    9344787
  • 财政年份:
    2016
  • 资助金额:
    $ 0.48万
  • 项目类别:
Role of Obesity in Infection
肥胖在感染中的作用
  • 批准号:
    7809374
  • 财政年份:
    2009
  • 资助金额:
    $ 0.48万
  • 项目类别:
MAPPING THE STIMULUS-SPECIFIC SIGNALING PATHWAYS IN PERIODONTITIS BY PROTEOMICS
通过蛋白质组学绘制牙周炎中刺激特异性信号通路
  • 批准号:
    7723023
  • 财政年份:
    2008
  • 资助金额:
    $ 0.48万
  • 项目类别:
MAPPING THE STIMULUS-SPECIFIC SIGNALING PATHWAYS IN PERIODONTITIS BY PROTEOMICS
通过蛋白质组学绘制牙周炎中刺激特异性信号通路
  • 批准号:
    7602017
  • 财政年份:
    2007
  • 资助金额:
    $ 0.48万
  • 项目类别:
SYSTEMIC ENDOTHELIAL CONSEQUENCES OF PERIODONTAL DISEASE
牙周疾病的全身内皮后果
  • 批准号:
    7606251
  • 财政年份:
    2007
  • 资助金额:
    $ 0.48万
  • 项目类别:
Inflammation and Infection in Atherosclerosis
动脉粥样硬化的炎症和感染
  • 批准号:
    8308411
  • 财政年份:
    2005
  • 资助金额:
    $ 0.48万
  • 项目类别:
"Infection and Inflammation in Atherosclerosis"
“动脉粥样硬化中的感染和炎症”
  • 批准号:
    9273596
  • 财政年份:
    2005
  • 资助金额:
    $ 0.48万
  • 项目类别:
Infection and Inflammation in Atherosclerosis
动脉粥样硬化中的感染和炎症
  • 批准号:
    8888634
  • 财政年份:
    2005
  • 资助金额:
    $ 0.48万
  • 项目类别:
Inflammation and Infection in Atherosclerosis
动脉粥样硬化的炎症和感染
  • 批准号:
    7005828
  • 财政年份:
    2005
  • 资助金额:
    $ 0.48万
  • 项目类别:
SYSTEMIC ENDOTHELIAL CONSEQUENCES OF PERIODONTAL DISEASE
牙周疾病的全身内皮后果
  • 批准号:
    7379508
  • 财政年份:
    2005
  • 资助金额:
    $ 0.48万
  • 项目类别:

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