Na, K-ATPase α1 antibody successfully pulled down the binding proteins of Na, K-ATPase α1 by Co-IP

To verify whether there are proteins that can bind to Na, K-ATPase α1, the total proteins were isolated from cell lysates and then detected by Western blot. A549 cells were divided into two groups: the control-A549 and LPS-A549 groups. The protein expression of Na, K-ATPase α1 was detected in both groups (both p < 0.001) (Fig. 3). Thus, we concluded that there were proteins that could interact with and bind to Na, K-ATPase α1 in both groups, from which we could start related proteomic analysis and identify exactly related proteins of Na, K-ATPase α1 in the future work.

Fig. 3

The binding proteins of Na, K-ATPase α1 in A549 group and A549 + LPS group. The binding proteins of Na, K-ATPase α1 was detected in both groups using Co-IP assay followed by Western blot (P < 0.001). Proteins in whole-cell lysate were used as a positive control (input). ATP1A1: the gene name of Na, K-ATPase α1

Identification and quantification of different proteins

To identify and quantify the significant proteins, in this study, we performed that the total number of identified and quantitated proteins in this assay was 1598, and among them, there are 738 proteins after filtration and 89 differentially expressed proteins in the group LPS-A549 compared with control-A549 (Table S2). Six hundred ninety-eight significant proteins in the group Control-A549 compared with IgG-A549 and 478 significant proteins in the group LPS-A549 compared with IgG-LPS (Table S3).

Respectively, Venn diagrams were then drawn to confirm the different proteins in the three comparisons, namely, LPS-A549 vs. control-A549 (Fig. 4), LPS-A549 vs. IgG-LPS (Fig S1) and control-A549 vs. IgG-A549 (Fig S2). In the discovery phase, we identified that 29 proteins were enriched in the LPS-A549 group, 60 proteins were enriched in the Control-A549 group, and 649 proteins were co-enriched in both groups (Fig. 4). We ranked the proteins according to fold change in expression levels and listed the top 10 candidates of significantly up-or down-regulated proteins (Table 1). Also, we found that 738 proteins contained E3 ubiquitin ligase or its complex components: TRIP12, RNF21 and CUL4B, deubiquitinases UCHL1, EIF3F and OTUB1, tight junction protein TJP1, and multifunctional protein SQSTM1. Among them, UCHL1 and RNF213 only enriched in the control group, while CUL4B, TRIP12, EIF3F, TJP1, SQSTM1 and OTUB1 enriched in both groups, indicating that they are strongly bound to Na, K-ATPase α1. These analysis results suggested that the difference in proteomic profiling is reliable. In future work, we can select the proteins we are interested in to carry out some relevant verification.

Fig. 4

Venn diagram of the different proteins in LPS-A549 vs. control-A549. The green part represents proteins enriched in LPS-A549; The light blue represents proteins enriched in A549; The middle part is the protein identified by both of them

Table 1 Top 10 up-or down-regulated proteins ranking by FC in LPS-A549 group vs. control-A549 groupGO & KEGG enrichment analysis of proteins interacting with Na, K-ATPase α1

To search for shared functions among genes, a common way is to incorporate the biological knowledge, by GO and KEGG analysis, to identify predominant biological themes of a collection of genes.

GO enrichment

GO analysis (http://www.geneontology.org/) was applied to search for significantly enriched GO terms in areas of biological process (BP), cellular component (CC), and molecular function (MF). Prediction terms with P-value less than 0.05 were selected and ranked by gene count ((Count/Pop. Hits)/(List. Total/Pop. Total)) or enrichment score (log10(adjust p-value)).

According to the results of group Control-A549 vs. IgG-A549, 750 BP terms, 229 CC terms, and 204 MF terms were found enriched in class-specific test enriched (T) sample compared with control enriched (C) sample. Similarly, in line with the results of group LPS-A549 vs. IgG-LPS, 697 BP terms, 202 CC terms, and 177 MF terms were found enriched in T sample. These generally changed GO terms in T sample and classified by BP, CC, MF, and ranked by gene count and enrichment score (Fig. 5A and B) (p < 1.0 × 10−7 in all terms).

Fig. 5

A Enriched GO items of < T > in Control-A549 vs. IgG-A549. Top axis is log10 (adjust p-value), bottom axis is gene count. B Enriched GO items of < T > in LPS-A549 vs. IgG-LPS

Nevertheless, for what we are concerned about the results of group LPS-A549 vs. Control-A549, only 2 BP terms, 0 CC terms, and 40 MF terms were found enriched in T sample compared with C sample (namely, 1 BP terms, 20 CC terms, and 15 MF terms were found enriched) (Fig. 6A and B). Intriguingly, almost all of the most enriched and meaningful BP terms were related to biosynthetic process in the LPS-A549 group, for instance, “thioester biosynthetic process (GO:0,035,384),” “acyl-CoA biosynthetic process (GO:0,071,616),” and only “ribosome biogenesis (GO:0,042,254)” in the Control-A549 group. Some more detailed data can be found in (Fig S3 and S4).

Fig. 6

A Enriched GO items of < T > in LPS-A549 vs. Control-A549. B Enriched GO items of < C > in LPS-A549 vs. Control-A549

The most enriched CC terms were primarily about the cell in only group control-A549 such as “90S pre-ribosome (GO:0,030,686),” “pre-ribosome (GO:0,030,684),” “melanosome (GO:0,042,470),” “pigment granule (GO:0,048,770),” and “small-subunit processsome (GO:0,032,040).”

As for GO MF terms ranked by either gene count or enrichment score, the mainly enriched terms were closely related to enzymatic activity and protein binding. Represented terms were “misfolded protein binding (GO:0,051,787),” “double-strand/single-strand DNA junction binding (GO:0,000,406),” “formate-tetrahydrofolate ligase activity (GO:0,004,329),” “SUMO activating enzyme activity (GO:0,019,948),” in the LPS-A549 group, and “ATP-dependent RNA helicase activity (GO:0,004,004),” and “ATPase activity (GO:0,016,887).” “ATPase activity, coupled (GO:0,042,623),” in the Control-A549 group.

KEGG pathway

We selected differentially expressed proteins for KEGG enrichment analysis, and the results demonstrated that the KEGG pathway was significantly enriched (p.adjust < 0.05). Pathways (p.adjust < 0.05) were selected and ranked by gene counts. Overall, in the group Control-A549 vs. IgG-A549, 689 differentially expressed proteins were involved in 23 KEGG pathways, like Ribosome, Spliceosome and Carbon metabolism. And in the group LPS-A549 vs. IgG-LPS, 478 differentially expressed proteins were involved in 29 KEGG pathways. The proteins were primarily enriched in RNA transport and Fatty acid metabolism (Fig S5 and S6). And top 20 pathways were listed for up-regulated genes, respectively (Table S4).

STRINGdb protein–protein interaction (PPI) analysis

To further examine the comprehensive information obtain from the identified protein data, the PPI network was analyzed. The network model was generated using the STRING website. A merged network is shown in (Fig S7 and S8), and, significant proteins annotation (show 50 if available) are shown in (Table S5 and S6). Seven hundred thirty-eight proteins after filter were screened into the PPI network complex, which showed that there were 244 significant enriched interactions among 60 proteins in the group control-A549 (Fig. 7A). Moreover, in the group LPS-A549, it contained 43 significant enriched interactions among 29 proteins (Fig. 7B). Some explanations of protein–protein interaction links are shown (Fig. 7C).

Fig. 7

STRING protein–protein interaction analysis. A Protein interaction analysis of control-A549 specifically enriched proteins by STRINGdb showed that there were 244 significant enriched interactions among 60 proteins. (p-value: 6.73572309040082e-13). B Protein interaction analysis of LPS-A549 specifically enriched proteins by STRINGdb showed that there were 43 significant enriched interactions among 29 proteins. (p-value: 0.0488221145460772). C Edge color legends. The explanation of protein–protein interaction links. It is divided into two parts: known interactions and predicted interactions. D The binding of Na, K-ATPase α1 to SQSTM1 and CUL4B were verified by endogenous Co-IP. Proteins in whole-cell lysate were used as a positive control (Input). IP: Na, K-ATPase murine monoclonal antibody, abcam, ab2872. Na, K-ATPase α1 group, WB: 1:1000, 100KD. Second antibody: Goat anti-Mouse IgG (Light Chain Specific), HRP Conjugated, S003, 1:5000. SQSTM1 group, WB: SQSTM1 rabbit polyclonal antibody, Proteintech, 18,420–1, 1:10,000, 62KD; Second antibody: Mouse anti-Rabbit IgG (Light Chain specific), HRP Conjugated, S006, 1:5000. CUL4B group, WB: CUL4B rabbit polyclonal antibody, Immunoway, YM5188, 1:1000, 110KD; Second antibody: Mouse anti-Rabbit IgG (Light Chain specific), HRP Conjugated, S006, 1:5000.62KD

Ubiquitination and de-ubiquitination enrichment related to OTUB1

In the analysis of protein mass spectrometry, OTUB1(known as a deubiquitinases) is of particular interest. OTUB1 belongs to the ovarian cancer proteases family. In this study, we found that Na, K-ATPase α 1 can bind to the deubiquitinase OTUB1 by protein mass spectrometry. Also, in the A549 cell group, GO analysis of Na, K-ATPase α1 interacting proteins showed significant enrichment of ubiquitination and deubiquitination, both were related to OTUB1. The enrichment items of ubiquitination and deubiquitination enrichment items are shown in (Table 2).

Na, K-ATPase α 1 interacts with SQSTM1 and CUL4B through Co-IP and western blot verification

Protein ubiquitination is a key step in the ubquitin-proteasome degradation pathway, and autophagy plays an indispensable role in maintaining cell homeostasis, clearing excess proteins and organelles, apoptosis, metabolism and senescence. We next selected the autophagy-related protein (SQSTM1) and the scaffold protein in CUL4B-RING ubiquitin ligase (CRL4B) complexes (CUL4B) from the significantly differentially expressed proteins for verification. Western blot assay followed after the Co-IP by Na, K-ATPase α1 antibody showed that both of SQSTM1 and CUL4B were positive (Fig. 7D), which indicated that there was a close relationship between Na, K-ATPase α1 and proteins related to autophagy and ubiquitination pathway. In all, further studies are needed to verify these results.