Ye Shuang, Pang Hai, Gu Yue-ying, Shen Nan, Chen Shun-le, Chen Xing-guo, Hua Jing, Qian Jie, Rao Zi-he
Department of Rheumatology, Renji Hospital, Clinical Center of Rheumatic Diseases and Institute of Rheumatology, Shanghai 2nd Medical University, Shanghai 200001, China.
Zhonghua Yi Xue Za Zhi. 2003 May 10;83(9):770-3.
To investigate the protein-to-protein interaction of interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), a newly discovered systemic lupus erythematosus (SLE) related up-regulated gene, and its possible function.
Peripheral blood of 40 SLE patients was obtained to extract total RNA and synthesized cDNA. Real-time PCR was used to determine the IFIT1 expression at transcript level. Peripheral blood of another 10 SLE patients was extracted to obtain specimens of white blood cell lysate. Molecular cloning and a modified gluthion S-transferase (GST)-pull down assay were used to capture the protein interacting with IFIT1 in the specimens of white blood cell lysate. MALDI-TOF mass spectrometry (MS) was preformed to identify the captured protein that could interact with IFIT1. Twenty-nine sex and age-matched healthy persons were used as controls.
By real-time PCR showed that the IFIT1Delta Ct value (x +/- s) was 2.344 +/- 1.200 in the SLE patients and was 3.734 +/- 1.274 in the controls (P < 0.001), showing a significant up-regulation in SLE patients. IFIT1 was cloned and GST-IFIT1 fusion protein was expressed in Escherichia coli. GST-IFIT1 fusion protein was further purified using Glutathione Sepharose 4B column, and was treated as bait to capture prey from peripheral white blood cell lysate of SLE patients. MALDI-TOF MS detected protein interaction between Rho/Rac guanine nucleotide exchange factor and IFIT1.
IFIT1 may interact with Rho/Rac guanine nucleotide exchange factor, and regulate the activation of Rho/Rac proteins, thus being involved in the pathogenesis of SLE.
研究干扰素诱导的含四肽重复序列蛋白1（IFIT1）——一个新发现的与系统性红斑狼疮（SLE）相关的上调基因——的蛋白质-蛋白质相互作用及其可能的功能。
采集40例SLE患者的外周血以提取总RNA并合成cDNA。采用实时荧光定量PCR检测转录水平的IFIT1表达。采集另外10例SLE患者的外周血以获取白细胞裂解液标本。采用分子克隆和改良的谷胱甘肽S-转移酶（GST）下拉实验从白细胞裂解液标本中捕获与IFIT1相互作用的蛋白质。采用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）鉴定能与IFIT1相互作用的捕获蛋白。选取29例年龄和性别匹配的健康人作为对照。
实时荧光定量PCR显示，SLE患者的IFIT1ΔCt值（x±s）为2.344±1.200，对照组为3.734±1.274（P<0.001），表明SLE患者中IFIT1显著上调。克隆IFIT1并在大肠杆菌中表达GST-IFIT1融合蛋白。使用谷胱甘肽琼脂糖4B柱进一步纯化GST-IFIT1融合蛋白，并将其作为诱饵从SLE患者外周白细胞裂解液中捕获猎物。MALDI-TOF MS检测到Rho/Rac鸟嘌呤核苷酸交换因子与IFIT1之间存在蛋白质相互作用。
IFIT1可能与Rho/Rac鸟嘌呤核苷酸交换因子相互作用，并调节Rho/Rac蛋白的激活，从而参与SLE的发病机制。
