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: 7369293
• 负责人: Salomon Amar
• 金额: $ 0.48万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/7369293
• 关键词: MAPPING; STIMULUS; SPECIFIC; SIGNALING; PATHWAYS

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。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构适用于该中心，这不一定是调查员的机构。牙周炎是一种炎症性疾病，具有宿主 - 寄生虫相互作用，导致结缔组织破坏和肺泡骨吸收。牙龈卟啉单胞菌（P.G.）是一种黑色色素的革兰氏阴性厌氧菌细菌，是牙周炎发育和进展的主要病原体。细胞壁和附属结构，例如脂多糖（LPS）和纤维化，在诱导先天免疫反应的诱导中起着重要作用，包括通过局部和循环的单核细胞/巨噬细胞产生细胞因子。基于初步数据，Live P.G.与细菌成分（例如LPS或fimbriae）相反，刺激人外周血单核细胞（PBM）中独特的促炎信号转导途径。在这些实验中，我们以蛋白质表达水平进行测试，即通过Live P.G.诱导独特的信号通路。 在C8反相柱上纯化了粗膜。 （1）从健康的供体和单核细胞样THP-1细胞中新鲜出生的人类PBM（纯纯）（95％）暴露于P.G.，P.G. LPS和P.G. fimbriae。在PBM的细胞裂解物蛋白和Thp-1差异产生的细胞裂解物蛋白上进行二维凝胶电泳（2-DGE）。切除表达的蛋白质并进行胰蛋白酶内消化。获取基质辅助激光解吸/电离（MALDI）光谱，并使用在线蛋白质数据库搜索进行蛋白质识别。 P.G.当用0.1％TFA中的CH3CN梯度洗脱时，粗膜从C8柱上的其他组件中得到很好的解析。通过将RP-HPLC与C8列一起使用P.G.可以快速有效地纯化叶片。 当由Live P.G.挑战时，对PBM差异化产生的细胞裂解物蛋白进行了2-DGE。与无挑战的条件。 2-DGE轮廓鉴定出几个差异表达的蛋白质斑点。排除P.G.产生的蛋白质斑点蛋白质我们也进行了2 dge的P.G.并使用它来对抗实验凝胶。 P.G.差异表达的蛋白质切除挑战的PBM，用胰蛋白酶消化凝胶，并获得质谱以表征它们。迄今为止的结果表明，Live P.G.诱导或下调特定的蛋白质。 实施了LPS和Fimbriae诱导的PBM的类似程序。 1-D凝胶电泳是对Live P.G.，LPS和Fimbriae挑战的PBM进行的。这种方法允许鉴定可能含量低的生物学意义蛋白质。分离的蛋白质带进行了凝胶胰蛋白酶消化，并获得了MALDI-MS光谱。进行了在线数据库搜索以识别蛋白质。为了改善2-DGE的等电聚焦期间的分离，使用新型缓冲液条件用于从PBM和THP-1的细胞裂解物中提取蛋白质。提取缓冲液避免使用SD，从而阻碍了第一维中蛋白质的分离。 2-DGE曲线显示了对照，Live P.G.，LPS（图3）和Fimbriae诱导的THP-1的蛋白质分离模式相似，从而使通过直接可视化更容易鉴定差异表达的蛋白质斑点。对2-DGE轮廓的分析确定了几个差异表达的蛋白斑。获取MALDI光谱，并使用在线蛋白质数据库搜索进行蛋白质鉴定。未来人类PBM将采取类似的方法。还将确定已鉴定蛋白的翻译后修饰。 1。Sojar，H.T.，Hamada，N。和R.J.蛋白质表达和纯净。 9，49-52（1997）。