Liu Yang, Zhang Ji-zeng, Wang Su-min, Fu Yu, Zhang Zong-de
Department of Molecular Biology, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing 101149, China.
Zhonghua Jie He He Hu Xi Za Zhi. 2010 Jul;33(7):500-4.
To study the relationship between cAMP response element binding protein (CREB) and the interferon-γ (IFN-γ) proximal promoter in patients with tuberculosis.
CD₃(+) T cells were isolated from 25 pulmonary tuberculosis patients, who had been treated in Beijing Chest Hospital from January to December 2007, and 18 PPD-positive healthy donors. After extraction of nuclear proteins, electrophoretic mobility shift assay (EMSA) was performed to determine nuclear protein binding to the IFN-γ proximal promoter in vitro, and the specificity of binding complex was tested by competitive EMSA. Chromatin immunoprecipitation (ChIP) with anti-CREB Ab was used to determine whether CREB binded to the IFN-γ proximal promoter in vivo in live T cells exposed to microbial Ags. Western blotting with anti-CREB Ab was performed to compare the expression level of CREB in tuberculosis patients and PPD-positive healthy donors. Western blotting with Abs specific for serine 133-phosphorylated CREB was performed to determine whether M.tuberculosis Ags elicited phosphorylation of CREB.
The results of EMSA showed a low-mobility complex binding to the IFN-γ promoter, and the binding pattern observed was similar for T cells from all 18 PPD-positive healthy donors. However, for T cells from 18 of 25 tuberculosis patients, the low-mobility complex was absent. The results of competitive EMSA showed that these nuclear proteins specifically bound to the IFN-γ promoter region and contained CREB. The results of ChIP showed a 204 bp band yielded in CD₃(+) T cells from 10 PPD-positive healthy donors, but 12 tuberculosis patients didn't yield the band. CREB expression markedly decreased in tuberculosis patients compared with healthy donors detected by Western blotting. Furthermore, M. tuberculosis Ags also elicited phosphorylation of CREB in CD₃(+) T cells from PPD-positive healthy donors, but not in CD₃(+) T cells from tuberculosis patients.
CREB protein binding to IFN-γ proximal promoter was reduced in tuberculosis patients compared with healthy donors. Tuberculosis patients had diminished CREB protein levels, and reduced ability of binding to the IFN-γ promoter.
研究结核病患者中环磷酸腺苷反应元件结合蛋白（CREB）与干扰素-γ（IFN-γ）近端启动子之间的关系。
从2007年1月至12月在北京胸科医院接受治疗的25例肺结核患者以及18例结核菌素纯蛋白衍生物（PPD）阳性健康供者中分离CD₃(+) T细胞。提取核蛋白后，进行电泳迁移率变动分析（EMSA）以体外确定核蛋白与IFN-γ近端启动子的结合情况，并通过竞争性EMSA检测结合复合物的特异性。使用抗CREB抗体进行染色质免疫沉淀（ChIP），以确定在暴露于微生物抗原的活T细胞中CREB在体内是否与IFN-γ近端启动子结合。使用抗CREB抗体进行蛋白质免疫印迹法，以比较结核病患者和PPD阳性健康供者中CREB的表达水平。使用针对丝氨酸133磷酸化CREB的抗体进行蛋白质免疫印迹法，以确定结核分枝杆菌抗原是否引发CREB的磷酸化。
EMSA结果显示存在一种与IFN-γ启动子结合的低迁移率复合物，并且在所有18例PPD阳性健康供者的T细胞中观察到的结合模式相似。然而，在25例结核病患者中的18例患者的T细胞中，未观察到低迁移率复合物。竞争性EMSA结果显示，这些核蛋白特异性结合至IFN-γ启动子区域并含有CREB。ChIP结果显示，10例PPD阳性健康供者的CD₃(+) T细胞中产生了一条204 bp的条带，但12例结核病患者未产生该条带。蛋白质免疫印迹法检测显示，与健康供者相比，结核病患者中CREB表达明显降低。此外，结核分枝杆菌抗原也在PPD阳性健康供者的CD₃(+) T细胞中引发了CREB的磷酸化，但在结核病患者的CD₃(+) T细胞中未引发。
与健康供者相比，结核病患者中CREB蛋白与IFN-γ近端启动子的结合减少。结核病患者的CREB蛋白水平降低，且与IFN-γ启动子的结合能力下降。
