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 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中出现。列出的机构是中心的机构，不一定是研究者的机构。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.）是一种黑色素革兰氏阴性厌氧细菌，是牙周炎发生和进展的主要病原体。细胞壁和附属结构，如脂多糖 (LPS) 和菌毛，在诱导先天免疫反应中发挥重要作用，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，实时 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，而不是细菌成分，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活 P.g. 差异诱导的独特信号通路。 粗菌毛在 C8 反相柱上纯化。 (1) 从健康供体和单核细胞样 THP-1 细胞中新鲜淘取的人 PBM（纯度为 95%）暴露于活 P.g、P.g。 LPS 和 P.g.菌毛。对差异产生的 PBM 和 THP-1 的细胞裂解物蛋白进行二维凝胶电泳 (2-DGE)。表达的蛋白质被切除并进行胰蛋白酶凝胶内消化。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鉴定。 P.g.当用 0.1% TFA 中的 CH3CN 梯度洗脱时，粗菌毛在 C8 柱上与其他组分能够很好地分离。通过使用带有 C8 柱的 RP-HPLC，P.g.菌毛可以快速有效地纯化。 当受到活 P.g. 挑战时，对 PBM 差异产生的细胞裂解物蛋白进行 2-DGE。与未受挑战的条件相比。 2-DGE 图谱鉴定了几个差异表达的蛋白质点。排除 P.g. 产生的蛋白质斑点。我们还对 P.g. 的蛋白质进行了 2-DGE。并将其用于实验凝胶。 P.g. 的差异表达蛋白。切除受攻击的 PBM，用胰蛋白酶进行凝胶内消化，并获得质谱来表征它们。迄今为止的结果表明，活体 P.g. 会诱导或下调特定蛋白质。 对于 LPS 和菌毛诱导的 PBM 也实施了类似的程序。对经活 P.g.、LPS 和菌毛攻击的 PBM 进行一维凝胶电泳。这种方法可以识别可能丰度较低的具有生物学意义的蛋白质。将分离的蛋白质条带进行凝胶内胰蛋白酶消化并获得MALDI-MS谱图。进行在线数据库搜索来鉴定蛋白质。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。提取缓冲液避免使用 SDS，否则会阻碍第一维蛋白质的分离。 2-DGE 图谱显示对照、活 P.g、LPS（图 3）和菌毛诱导的 THP-1 的蛋白质分离模式相似，使得通过直接可视化识别差异表达的蛋白质点变得更容易。 2-DGE 图谱分析确定了几个差异表达的蛋白质点。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 也将采取类似的方法。还将确定已鉴定蛋白质的翻译后修饰。 1. Sojar, H.T.、Hamada, N. 和 Genco, R.J.蛋白质表达和纯化。 9, 49-52 (1997)。