Screen of novel candidate regulators involved in oxidative stress-reprogrammed LPS signaling pathway by comparative phosphoprotein-affinity profiling
- VernacularTitle: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
- Author:
Zhipeng ZOU
;
Ting PAN
;
Yusheng LI
;
Wei LIU
;
Zhenyu ZHU
;
Yong JIANG
- Publication Type:Journal Article
- Keywords:
Lipopolysaccharides;
Electrophoresis;
Oxidative stress;
Phosphoproteomics
- From:
Chinese Journal of Pathophysiology
1989;0(06):-
- CountryChina
- Language:Chinese
-
Abstract:
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 ?g/L PMA for 36 h. Differentiated cells were rested for additional 36 h without PMA treatment, then treated with 100 ?mol/L H2O2 or medium for 1 h followed by LPS or medium treatment for 30 min. After desalted, phosphoproteins were enriched by phosphoprotein 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.
