Proteomic analysis of macrophages stimulated by lipopolysaccharide: Lipopolysaccharide inhibits the cleavage of nucleophosmin

Abstract

Lipopolysaccharide (LPS) is a complex glycolipid composed of a hydrophilic polysaccharide and a hydrophobic domain that is responsible for the biological activity of LPS. There are many reports about LPS stimulation, and many activated proteins have been detected after LPS stimulation in various cell types. Furthermore, most of the LPS signaling pathways are clear. However, we were interested in examining the changes of LPS-induced total cytosolic proteins expression and the LPS signaling pathway by the proteomics technique during LPS-induced macrophage activation. Our study employed two-dimensional gel electrophoresis and mass spectrometry to analyze the proteins involved in LPS-induced activation in RAW 264.7 cells. We found 11 protein spots whose expression was different between untreated cells and LPS-treated cells. Ten protein spots were identified, seven of which, tubulin beta-4 chain (49.6 kDa, pI 4.78), nucleophosmin (32.6 kDa, pI 4.62, two spots), 40S ribosomal protein SA (P40) (32.7 kDa, pI 4.74), transforming protein RhoA (21.8 kDa, pI 5.83), nucleolin (76.6 kDa, pI 4.69), and T-complex protein 1 zeta subunit (58 kDa, pI 6.63) were down-regulated, and three of which, nucleophosmin (32.6 kDa, pI 4.62, two spots) and proteosome subunit alpha type-1 (29.5 kDa, pI 6.00), were up-regulated. The suppression of the proteolytic degradation of nucleophosmin was associated with LPS-induced RAW 264.7 cell activation. Cleaved caspase-3 decreased, thus it might be involved in proteolysis of nucleophosmin in LPS-induced macrophage activation. Our study also demonstrated that there was no change of the expression of nucleophosmin at the mRNA level.

Clinical microfluidics for neutrophil genomics and proteomics

“…Several examples are provided. Nucleolin (NCL), a protein involved in the synthesis and maturation of ribosomes, is 2.4 fold increased at mRNA level but 29 times decreased at protein level, which corresponds to a trend observed previously 30, suggesting a selective degradation of this protein after LPS stimulation. In addition, the endogenous amount of lipocalin 2 (LCN2), a secretive protein important to antibacterial innate immune response 31 and known to be induced by LPS 32, was decreased by 3-fold, despite of a 6 fold increase of mRNA.…”

Rapid Temporal Dynamics of Transcription, Protein Synthesis, and Secretion during Macrophage Activation

“…This led to the identification of a larger number of changing proteins in this model system than in previous studies (11,(15)(16)(17)(18)39). One exception is a study by Du et al (39), who identified Ͼ700 differentially expressed proteins after 10 min of LPS stimulation among 2144 detected proteins.…”

The impact of tyrosine kinase 2 (Tyk2) on the proteome of murine macrophages and their response to lipopolysaccharide (LPS)

“…Proteomics studies on the LPS response of murine macrophages have been performed in a few older studies [22,23,41], but at that time 2-DE patterns were characterised solely by physicochemical properties of the obtained spots (pI, M r ). More recent studies including identification of intracellular LPS responsive proteins employed cell lines rather than primary cells and, in addition, used different comparative 2-DE approaches [42][43][44]. Accordingly, the sets of proteins identified show generally little overlap with ours.…”