MAPPING THE STIMULUS-SPECIFIC SIGNALING PATHWAYS IN PERIODONTITIS BY PROTEOMICS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。牙周炎是一种宿主和寄生虫相互作用的炎症性疾病，可导致结缔组织破坏和牙槽骨吸收。牙龈卟啉单胞菌(P.G.)黑色素革兰氏阴性厌氧菌是牙周炎发生发展的主要病原菌。细胞壁和附属物结构，如脂多糖(LPS)和菌毛，在诱导先天性免疫反应中发挥重要作用，包括由局部和循环中的单核/巨噬细胞产生细胞因子。根据初步数据，实况P.G.刺激人外周血单核细胞(PBM)中独特的促炎信号转导通路，而不是细菌成分，如内毒素或菌毛。在这些实验中，我们在蛋白表达水平上测试了活体P.G.对独特的信号通路进行了差异诱导。粗菌毛在C8反相柱上纯化。(1)新鲜从健康供者和单核细胞样THP-1细胞中洗脱出的人PBM(95%纯度)暴露于活体P.G.、P.G.LP和P.G.菌毛。对PBM和THP-1细胞裂解产物进行双向凝胶电泳(2-DGE)。表达的蛋白质被切下并进行胰酶凝胶内消化。获得基质辅助激光解吸/电离(MALDI)光谱，并通过在线蛋白质数据库搜索进行蛋白质鉴定。P.G.粗菌毛在C8柱上用0.1%TFA中的CH3CN梯度洗脱时可以很好地从其他组分中分离出来。采用反相高效液相色谱法，以C8为色谱柱，分离得到的有效成分为P.G.菌毛可以快速、高效地得到纯化。对PBM分化产生的细胞裂解蛋白进行2-DGE实验。与未受挑战的条件相比。2-DGE图谱确定了几个差异表达的蛋白质点。以排除P.G.引起的蛋白质斑点。我们还对P.G.的蛋白质进行了2-DGE。并用它来对抗实验凝胶。P.G.的差异表达蛋白取受攻击的PBM，用胰酶凝胶内消化，获得质谱图。到目前为止的结果表明，特定的蛋白质被活的P.G.诱导或下调。对于内毒素和菌毛诱导的PBM，也执行了类似的程序。在活菌、内毒素和菌毛作用下，对PBM进行一维凝胶电泳法。这种方法可以识别可能丰度较低的具有生物意义的蛋白质。对分离出的蛋白质带进行凝胶内胰酶消化，得到MALDI-MS图谱。通过在线数据库检索进行蛋白质鉴定。为了改进2-DGE等电聚焦阶段的分离，采用了新的缓冲条件从PBM和THP-1的细胞裂解产物中提取蛋白质。提取缓冲液避免了十二烷基硫酸钠的使用，这阻碍了第一维蛋白质的分离。2-DGE图谱显示了对照、活P.G.、内毒素(图3)和THP-1诱导的菌毛的相似的蛋白质分离模式，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。对2-DGE图谱的分析确定了几个差异表达的蛋白质点。获取MALDI光谱，并通过在线蛋白质数据库搜索进行蛋白质鉴定。未来将对人类PBM采取类似的方法。识别出的蛋白质的翻译后修饰也将被确定。1.Sojar，H.T.，Hamada，N.和Genco，R.J.Protein Expression and Purif.9、49-52(1997)。

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).