Identification of different proteins binding to Na, K-ATPase α1 in LPS-induced ARDS cell model by proteomic analysis

Abstract

Background Acute respiratory distress syndrome (ARDS) is characterized by refractory hypoxemia caused by accumulation of pulmonary fluid, which is related to inflammatory cell infiltration, impaired tight junction of pulmonary epithelium and impaired Na, K-ATPase function, especially Na, K-ATPase α1 subunit. Up until now, the pathogenic mechanism at the level of protein during lipopolysaccharide- (LPS-) induced ARDS remains unclear. Methods Using an unbiased, discovery and quantitative proteomic approach, we discovered the differentially expressed proteins binding to Na, K-ATPase α1 between LPS-A549 cells and Control-A549 cells. These Na, K-ATPase α1 interacting proteins were screened by co-immunoprecipitation (Co-IP) technology. Among them, some of the differentially expressed proteins with significant performance were identified and quantified by liquid chromatography-tandem mass spectrometry (LC–MS/MS). Data are available via ProteomeXchange with identifier PXD032209. The protein interaction network was constructed by the related Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Several differentially expressed proteins were validated by Western blot. Results Of identified 1598 proteins, 89 were differentially expressed proteins between LPS-A549 cells and Control-A549 cells. Intriguingly, protein–protein interaction network showed that there were 244 significantly enriched co-expression among 60 proteins in the group control-A549. while the group LPS-A549 showed 43 significant enriched interactions among 29 proteins. The related GO and KEGG analysis found evident phenomena of ubiquitination and deubiquitination, as well as the pathways related to autophagy. Among proteins with rich abundance, there were several intriguing ones, including the deubiquitinase (OTUB1), the tight junction protein zonula occludens-1 (ZO-1), the scaffold protein in CUL4B-RING ubiquitin ligase (CRL4B) complexes (CUL4B) and the autophagy-related protein sequestosome-1 (SQSTM1). Conclusions In conclusion, our proteomic approach revealed targets related to the occurrence and development of ARDS, being the first study to investigate significant differences in Na, K-ATPase α1 interacting proteins between LPS-induced ARDS cell model and control-A549 cell. These proteins may help the clinical diagnosis and facilitate the personalized treatment of ARDS. Graphical Abstract

Hypoxia induces a glycolytic complex in intestinal epithelial cells independent of HIF-1-driven glycolytic gene expression

“…S3 ). Several proteins previously identified to interact with PFKP [e.g., ATP1A1 ( 22 ) or MTHFD1 ( 23 )] or PKM2 [PSMD14/POH1 ( 24 )] were readily detected in our screens, thereby supporting our experimental approach. Comparisons of the PFKP and PKM2 interactomes from both hypoxic- and normoxic-exposed cells revealed numerous hypoxia-sensitive interactions between PFKP and PKM2 and proteins involved in glucose metabolism including GLUT1, GLUT3, and HK2, particularly following 24 h and 48 h of hypoxic exposure ( Fig.…”

Insulin reverses impaired alveolar fluid clearance in ARDS by inhibiting LPS-induced autophagy and inflammatory

“…The above evidence suggests that insulin is likely to upregulate P62 levels, inhibit the release of inflammatory factors, and reduce inflammatory cell infiltration and thus reduce pulmonary water production. Interestingly, in our previous study, we found that ATP1A1 interacts with P62 through Co-IP and western blot verification ( 24 ). It is thought-provoking that P62 may act as a key regulatory mediator in this, so we conjecture that insulin most likely regulates ATP1A1 and P62 expression through inhibition of autophagy and thereby plays some key role in the accumulation of alveolar epithelial fluid.…”

“…In addition, the novel mechanism of ATP1A1 as a mediator of signal transduction and autophagy during ischemia-reperfusion provides new ideas for the intervention of ischemic stroke ( 22 , 23 ). Our previous study revealed that the ATP1A1 regulatory mechanism may be related to the autophagy-lysosome pathway ( 24 ). Few studies have been conducted on the degradation of Na, K-ATPase via the autophagy-lysosome pathway, However, the relationship between them is worthy of extensive attention and discussions to affect the abundance and enzyme activity of ATP1A1, enhance the lung water clearance ability, and improve the prognosis of ARDS in the future.…”

Protein interactome analysis of ATP1B1 in alveolar epithelial cells using Co-Immunoprecipitation mass spectrometry and parallel reaction monitoring assay

“…Co-IP-MS technology is considered a powerful screening tool for the composition of protein complexes and has been widely used in studies of targeted protein interactions [ 16 , 17 ]. In our previous study, Co-IP-MS was conducted to investigate significant proteins interacting with Na, K-ATPase α1 in LPS-A549 cells and control-A549 cells [ 18 ]. Parallel reaction monitoring (PRM) is a novel targeted quantification method with high resolution and mass accuracy [ 19 ].…”