应用亲和磷酸蛋白质组学筛选氧化应激重塑型LPS通路的侯选信号分子

Screen of novel candidate regulators involved in oxidative stress-reprogrammed LPS signaling pathway by comparative phosphoprotein-affinity profiling

目的:研究低剂量LPS刺激的Thp-1细胞在有或无H2O2预处理时磷酸化蛋白组的差异,以初步筛选氧化应激重塑型LPS通路中的新信号分子。方法:使用PMA刺激Thp-1单核细胞36h使之分化成为成熟的巨噬细胞,静息36h后,先以等体积的培养基或100μmol/L H2O2刺激1h,再以培养基或10μg/L LPS刺激30min。采用PMAC(phosphoprotein metal affinity column)富集各组细胞的磷酸化蛋白质,经超滤除盐后进行二维凝胶电泳,比较LPS组和H2O2+LPS组的磷酸化蛋白图谱的差异,并挑选部分斑点进行质谱鉴定。结果:相对于LPS组(仅用LPS刺激的细胞),在H2O2+LPS组(LPS刺激前经H2O2提前处理的细胞)中,有29个磷酸化蛋白斑点在双向电泳图谱中表现出可重复的差异,其中,17个斑点发生了上调(包括新出现的斑点),12个发生了下调(包括消失的斑点)。我们已经鉴定出其中5个差异蛋白,这些蛋白参与多种多样的细胞过程例如蛋白质降解、信号转导和蛋白质折叠等。其中,在H2O2+LPS组中显著下调的proteasome beta-4亚基与LPS信号传递关系密切。结论:亲和磷酸蛋白组学有效减少了高丰度的结构蛋白的干扰并增加了信号分子检出的可能性。上述5个蛋白尤其是proteasome beta-4亚基可能是氧化应激重塑型LPS通路的重要调节分子。

AIM; To determine the differences in phosphoproteome between LPS stimulated THP - 1 cells with and without previous oxidative stress for screening of more potential regulators. METHODS： Differentiation of THP - 1 cells into macrophages was induced by treatment with 100 μg/L PMA for 36 h. Differentiated cells were rested for additional 36 h without PMA treatment, then treated with 100 μmol/L H2O2 or medium for 1 h followed by LPS or medium treatment for 30 min. After desalted, phosphoproteins were enriched by phosphoprote.in metal affinity column, and were run on 2 -D electrophoresis, then the spots were analyzed to show the difference between LPS group （cells treated with LPS alone） and H2O2 ＋ LPS group （LPS stimulated cells also pretreated with H2O2 ）. Finally, some of these spots were identified by MS and subsequent bioinformatic analysis was also conducted. RESULTS： Compared to LPS group, 29 reproducibly changed spots on the 2 -D map in H2O2 ＋ LPS group were visualized and selected for MS analysis. Among these, 12 down- regulated spots （include those disappeared）, 17 up- regulated spots （include those newly emerged） were selected. Up to now, 5 of these were identified, which were shown to be involved in various cellular processes such as proteolysis, signal transduction and protein folding. Among these, proteasome beta -4 subunit, which was dramatically down - regulated in H2O2 ＋ LPS group, was a major component of the proteasome complex and might participate in LPS signalling through various ways. CONCLUSION： With comparative phosphoprotein - affinity profiling, the interference brought by highly abundant house -keeping proteins is minimized, rendering us to detect less abundant signalling molecules. Aforementioned 5 proteins, especially proteasome beta -4 subunit, might be involved in LPS pathway reprogrammed by oxidative stress.