Isolation of differential bands in EspF / EspF-N / EspF-C groups from cell lysates
After transfecting pEGFP-EspF, pEGFP-EspF / N, and pEGFP-EspF / C-terminus encoding plasmids into 293T cells for 48 hours, the lysis proteins were added to Flag columns and IgG columns for co-immunoprecipitation (Fig. 1). pEGFP-EspF was about 58 kDa, pEGFP-EspF / N was 30 kDa, and pEGFP-EspF / C was 55 kDa. Compared with the IgG group, between 35−40 kDa, there were two differentially expressed bands in the pEGFP-EspF group. at 40 kDa, there was a differential band in the pEGFP-EspF / N group. At 130 kDa, there was a differential band in the pEGFP-EspF / C group. Besides these bands, the bands at the same position in the lanes of the respective IgG groups were also cut out. The bands were digested by mass spectrometry to detect the interacting proteins, and the Flag group-specific proteins (minus the IgG group proteins) were considered as the putative interacting proteins.
Prediction and analysis of interacting protein with EspF
A total of 708 proteins were identified in this work, 311, 192, and 205 proteins were detected in the pEGFP-EspF group, pEGFP-EspF / N group, and pEGFP-EspF / C group, respectively. We also performed functional annotation (including GO, Pathway, STRING analysis) to identify proteins. Through these analyses, we attempted to discover essential proteins.
All possible target proteins that interacted with EspF were loaded into the DAVID database for KEGG pathway annotation and GO enrichment. The threshold was set to ps0.05, and pathways or gene functions with higher counts were analyzed. The top 20 pathways were plotted with Graphpad Prism 6 (Table 1). Analysis of the differential bands at about 38 kDa in the pEGFP-EspF group by GO annotation analysis revealed that the interacting proteins were involved in 25 biological processes (BPs). Of these, the primary were intracellular processes (12.3%), metabolic processes (10.9%), biological regulation (8.8%), and immune stimulation (6.6%). Cell Components (CCs) enrichment mainly involved cellular anatomical entities (37.7%), intracellular (36.6%), and protein-containing complex (20.9%). Molecular Functions (MFs) mostly involved binding (53.6%) and catalytic activity (22.3%) (Fig. 2A). Pathway analysis showed that the interacting proteins involved 183 pathways, notably Metabolic pathways (32.6%), Carbon metabolism (19.6%), and Biosynthesis of amino acids (13.0%). (Fig. 2B).
Table 1
The top 20 proteins of the 38 kDa differential band between the EspF group and IgG group.
Rank | Protein symbol | Protein annotation |
1 | NP_312577.1 | T3SS secreted effector EspF [Escherichia coli O157:H7 str. Sakai] |
2 | sp|P21796|VDAC1_HUMAN | Voltage-dependent anion-selective channel protein 1 OS = Homo sapiens OX 9606 GN = VDAC1 PE = 1 SV = 2 |
3 | sp|O00165|HAX1_HUMAN | HCLS1-associated protein X-1 OS = Homo sapiens OX = 9606 GN = HAX1 PE = 1 SV = 2 |
4 | sp|P36542|ATPG_HUMAN | ATP synthase subunit gamma, mitochondrial OS = Homo sapiens OX = 9606 GN = ATP5F1C PE = 1 SV = 1 |
5 | sp|Q9Y623|MYH4_HUMAN | Myosin-4 OS = Homo sapiens OX = 9606 GN = MYH4 PE = 2 SV = 2 |
6 | sp|Q13347|EIF3I_HUMAN | Eukaryotic translation initiation factor 3 subunit I OS = Homo sapiens OX = 9606 GN = EIF3I PE = 1 SV = 1 |
7 | sp|Q15006|EMC2_HUMAN | ER membrane protein complex subunit 2 OS = Homo sapiens OX = 9606 GN = EMC2 PE = 1 SV = 1 |
8 | sp|Q01449|MLRA_HUMAN | Myosin regulatory light chain 2, atrial isoform OS = Homo sapiens OX = 9606 GN = MYL7 PE = 1 SV = 1 |
9 | sp|P17661|DESM_HUMAN | Desmin OS = Homo sapiens OX = 9606 GN = DES PE = 1 SV = 3 |
10 | sp|Q9P035|HACD3_HUMAN | Very-long-chain (3R)-3-hydroxyacyl-CoA dehydratase 3 OS = Homo sapiens OX = 9606 GN = HACD3 PE = 1 SV = 2 |
11 | sp|P60709|ACTB_HUMAN | Actin, cytoplasmic 1 OS = Homo sapiens OX = 9606 GN = ACTB PE = 1 SV = 1 |
12 | sp|P35613|BASI_HUMAN | Basigin OS = Homo sapiens OX = 9606 GN = BSG PE = 1 SV = 2 |
13 | sp|P60891|PRPS1_HUMAN | Ribose-phosphate pyrophosphokinase 1 OS = Homo sapiens OX = 9606 GN = PRPS1 PE = 1 SV = 2 |
14 | sp|Q07955|SRSF1_HUMAN | Serine/arginine-rich splicing factor 1 OS = Homo sapiens OX = 9606 GN = SRSF1 PE = 1 SV = 2 |
15 | sp|Q13505|MTX1_HUMAN | Metaxin-1 OS = Homo sapiens OX = 9606 GN = MTX1 PE = 1 SV = 3 |
16 | sp|P05976|MYL1_HUMAN | Myosin light chain 1/3, skeletal muscle isoform OS = Homo sapiens OX = 9606 GN = MYL1 PE = 2 SV = 3 |
17 | sp|Q96FZ7|CHMP6_HUMAN | Charged multivesicular body protein 6 OS = Homo sapiens OX = 9606 GN = CHMP6 PE = 1 SV = 3 |
18 | sp|P62424|RL7A_HUMAN | 60S ribosomal protein L7a OS = Homo sapiens OX = 9606 GN = RPL7A PE = 1 SV = 2 |
19 | sp|P35232|PHB_HUMAN | Prohibitin OS = Homo sapiens OX = 9606 GN = PHB PE = 1 SV = 1 |
20 | sp|P10599|THIO_HUMAN | Thioredoxin OS = Homo sapiens OX = 9606 GN = TXN PE = 1 SV = 3 |
We used STRING to analyze the target proteins with which EspF interacted, and found that RPS6, RPL14, and EIF2S1 had the highest connectivity (Fig. 2C). RPS6 plays an essential role in controlling cell growth and proliferation by selectively translating specific kinds of mRNAs [13]. RPL14 is a large ribosomal subunit component that plays a role in mRNA catabolism and translation [14]. EIF2S1 works in the early stages of protein synthesis by forming a ternary complex with GTP and initiator tRNA [15]. This analysis showed that in addition to gene transcription regulation and protein synthesis, EspF also plays a crucial role in cell proliferation, and catabolism.
The GO annotation analysis of the 36 kDa differential band showed that the interacting proteins were involved in 26 BPs, such as intracellular processes (12.5%), metabolic processes (10.2%), and biological regulation (9.0%). CCs and MFs results were consistent with the 38 kDa differential band results (Fig. 3A) Pathway analysis showed that the interacting proteins involved 150 pathways, including Metabolic pathways (23.0%), Protein processing in the endoplasmic reticulum (13.1%), and Parkinson disease (11.5%) (Fig. 3B). STRING analysis showed that the RPL7A, RPS20, and EIF2S1 had a high degree of connectivity, and we found that there was also a high degree of connectivity between the MDH2 and GOT2 (Fig. 3C). Among them, MDH2 plays a role in cell metabolism and amino acid acetylation [16]. GOT2 is necessary for metabolite exchange between mitochondria and cytoplasm and plays a crucial role in amino acid metabolism [17].
Prediction and analysis of interacting proteins with EspF-N or C-terminus
The GO annotation analysis of the differential bands at about 40 kDa in the pEGFP-EspF / N group showed that the interacting proteins involved 25 BPs, such as intracellular processes (12.3%), metabolic processes (10.8%), and biological regulation (9.2%). The CCs and MFs analyses were similar to EspF (pEGFP-EspF). Pathway analysis revealed 228 interacting proteins that were mainly involved in Metabolic pathways (27.1%), Protein processing in the endoplasmic reticulum (9.41%), and pathways in cancer (8.2%). (Fig. 4A and B). STRING analysis of target proteins interacting with EspF-N terminus also found that RPL8, RPS9, and EIF3I proteins had the highest degree of connectivity, indicating that EspF may use its N-terminus for ribosomes recognize binding sites (Fig. 4C).
GO annotation analysis of the pEGFP-EspF / C group in the 130 kDa differential band showed that BPs enrichment mostly involved translation (23.1%), oxidation-reduction process (15.4%), and intracellular protein transport (13.5%). CCs enrichment mainly involved intermediate filament (21.3%), ribosome (13.5%). MFs mostly involved protein binding (22.4%), and ATP binding (18.6%). COG analysis showed that interacting proteins mostly involved translation, ribosomal structure, biogenesis, posttranslational modification, protein turnover, and chaperones (Fig. 5A). KEGG showed that most of the cellular processes involved protein transport, and the signal generation that affected cell growth and apoptosis. Of the interacting proteins, 30.3% were localized in the cytoplasm, and 27.27% were localized in the nucleus (Fig. 5BC). The Venn diagram showed 93 proteins that were all involved in the above four pathways (Fig. 5E). Among these proteins, TUBB, and ANXA2 showed strong interaction with EspF-C. TUBB, a main component of tubulin, has GTPase activity, and plays a key role in the microtubule cytoskeleton organization [18]. Studies have shown that EspF interacts with SNX9 in the cytoplasm to induce the formation of membrane tubules and the host cell membrane change [19]. In addition to the SNX9 binding site, EspF can also activate N-WASP to induce actin polymerization, TJ disruption, and anti-phagocytosis [20]. Moreover, EspF also directly binds to cytokeratin 18, which contributes to the collapse of the cytoskeleton[21]. Microfilaments, microtubules, and intermediate fibers together make up the cytoskeletal system. Therefore, we speculated that EspF might also cause cytoskeleton rearrangement by binding TUBB .
In our previous research, ANXA6 and EspF were confirmed to interact[22]. This time we found ANXA2, a member of the ANXA protein family, which is a calcium-regulated membrane-bound protein. ANXA2 participates in the host heat stress response by interacting with Hsp90 [23]. Meanwhile, EspF may regulate calcium ion accumulation and calcium channel protein activity by interacting with ANXA2.
In addition, we also found the SMC family proteins. The interaction between EspF and SMC1 is also found in our previous research and involves multiple signaling pathways [22]. It is understood that SMC1 is an adhesion protein that plays a role in DNA replication and cohesion of sister chromatids. It involves in chromosome dynamics, cell cycle regulation, cell proliferation, and genome stability [24]. Furthermore, when cells were stimulated by DNA damage, SMC1 can be phosphorylated by ATM or ATR to participate in DNA repair and acted as a downstream effector in the ATM / NBS1 branch and the ATR / MSH2 branch to active S-phase checkpoint [25]. Hence, we hypothesized EspF could mediate cellular DNA damage repair by phosphorylating SMC1.
SMC1 was identified as a novel EspF-interacting protein, and its interaction was more robust with the EspF-C terminus
We are the first to study the mechanism of interaction between EspF and DNA damage repair proteins. Western blotting and confocal analysis further confirmed the interaction between EspF and SMC1. Co-immunoprecipitation results confirmed that EspF interacted with SMC1, and the interaction was stronger when with EspF-C terminus (Fig. 6A). Immunofluorescence analysis also showed that EspF and SMC1 were co-localized in the cytoplasm (Fig. 6B), and EspF relocated SMC1 more from the nucleolus into the cytoplasm, suggesting that SMC1 may not play its usual role in the nucleolus.
To investigate the EspF-SMC1 interplay, we measured SMC1 and its phosphorylation levels after Caco2 cells were transfected with EspF. Immunofluorescence showed that EspF could significantly increase the expression of p-SMC1, and p-SMC1 was distinctly localized in the cytoplasm and co-localized with EspF (Fig. 6C). Compared with the control, the level of SMC1 remained unchanged after transfection with EspF, but p-SMC1 significantly increased (Fig. 6D-E). We then verified the results by infecting HT-29 cells with the strain. The expression of p-SMC1 in cells transfected with the EDL 933 was higher than infected ΔespF strain, and infecting the complement ΔespF / pespF restored p-SMC1 expression. The above results verified that EspF could increase the expression level of p-SMC1.
In general, when cell DNA is damaged, cyclin-mediated cell cycle arrest will occur. During this period, DNA damage repair proteins are recruited to double side-band break (DSB) [26]. Therefore, we speculated that EspF might lead to DNA damage, which stimulates the S-phase detection point by increases p-SMC1 expression, thus mediating damage repair.
EspF may mediate DNA damage by modifying the histones
Mass spectrum results showed that EspF could modify multiple sites of various proteins (Table 2). Among the modification results, SFXN1, HAX1, EIF3I, ATG16L1, and DNA damage binding proteins had high scores. SFXN1 is a mitochondrial serine transporter that mediates serine into mitochondria and plays an essential role in the single-carbon metabolic pathway[27]. EspF can cause oxidative phosphorylation and methylation of SFXN1, which may mediate the metabolism required component for transport in and out of the mitochondria.
Table 2
EspF-mediated phosphorylation, acetylation, and methylation of the host proteins.
| Protein symbol | Score | Pep before | Pep_seq | Pep after | Pep mod | |
1 | sp|Q9H9B4|SFXN1_HUMAN | 270 | R | ILMAAPGMAIPPFIMNTLEK | K | 2 Oxidation (M); Phospho (ST); Methyl (K) |
2 | sp|P09651|ROA1_HUMAN | 267 | K | SESPKEPEQLR | K | Phospho (ST) |
3 | sp|P05388|RLA0_HUMAN | 187 | K | EDLTEIR | D | Phospho (ST) |
4 | sp|P05388|RLA0_HUMAN | 187 | R | GNVGFVFTK | E | Phospho (ST); Acetyl (K) |
5 | sp|P05388|RLA0_HUMAN | 187 | K | CFIVGADNVGSK | Q | Phospho (ST) |
6 | sp|A0A075B6P5|KV228_HUMAN | 172 | R | FSGSGSGTDFTLK | I | Phospho (ST) |
7 | sp|O00165|HAX1_HUMAN | 145 | K | ITKPDGIVEERR | T | Phospho (ST); Acetyl (K) |
8 | sp|Q13347|EIF3I_HUMAN | 115 | K | QLALLKTNSAVR | T | Phospho (ST); Acetyl (K) |
9 | sp|Q01449|MLRA_HUMAN | 112 | K | VSVPEEELDAMLQEGK | G | Phospho (ST); Methyl (K) |
10 | sp|P35613|BASI_HUMAN | 89 | K | GSDQAIITLRVR | S | 2 Phospho (ST); Methyl (R) |
11 | sp|P11908|PRPS2_HUMAN | 89 | K | IASSSRVTAVIPCFPYAR | Q | 3 Phospho (ST); Phospho (Y); 2 Methyl (R) |
12 | sp|P60891|PRPS1_HUMAN | 78 | K | IASASRVTAVIPCFPYAR | Q | Phospho (ST); Phospho (Y); Methyl (R) |
13 | sp|Q13505|MTX1_HUMAN | 72 | K | YNADYDLSAR | Q | Phospho (Y) |
14 | sp|P35232|PHB_HUMAN | 70 | R | SRPRNVPVITGSK | D | Phospho (ST); Methyl (R); Acetyl (K) |
15 | sp|Q99880|H2B1L_HUMAN | 65 | K | AVTKYTSSK | - | Phospho (ST); Phospho (Y); 2 Methyl (K) |
16 | sp|Q92522|H1X_HUMAN | 64 | K | AAKPSVPK | V | Phospho (ST); Methyl (K); Acetyl (K) |
17 | sp|Q86V81|THOC4_HUMAN | 58 | - | MADKMDMSLDDIIK | L | 2 Oxidation (M); Phospho (ST); Methyl (K) |
18 | sp|P0C0S8|H2A1_HUMAN | 48 | K | KTESHHK | A | Phospho (ST); Acetyl (K) |
19 | sp|P16104|H2AX_HUMAN | 48 | K | TSATVGPKAPSGGK | K | 2 Phospho (ST); Methyl (K) |
20 | sp|Q8IUE6|H2A2B_HUMAN | 48 | K | KTESHKPGK | N | Phospho (ST); Methyl (K); 2 Acetyl (K) |
21 | sp|Q9Y375|CIA30_HUMAN | 48 | R | KFSKPTSALYPFLGIR | F | Phospho (ST); Acetyl (K) |
22 | sp|O14983|AT2A1_HUMAN | 46 | K | EYEPEMGKVYR | A | Oxidation (M); Phospho (Y) |
23 | sp|Q9Y394|DHRS7_HUMAN | 46 | K | LGVSLVLSAR | R | Phospho (ST); Methyl (R) |
24 | sp|P08758|ANXA5_HUMAN | 45 | R | VMVSRSEIDLFNIR | K | Oxidation (M); Phospho (ST) |
25 | sp|Q5JTV8|TOIP1_HUMAN | 41 | K | TPQEWAPQTAR | I | 2 Phospho (ST) |
26 | sp|P10412|H14_HUMAN | 40 | K | ATGAATPK | K | Phospho (ST) |
27 | sp|P16402|H13_HUMAN | 40 | K | KAASGEGKPK | A | Phospho (ST) |
28 | sp|P22492|H1T_HUMAN | 40 | K | KPRATTPK | T | Phospho (ST); Acetyl (K) |
29 | sp|Q02539|H11_HUMAN | 40 | K | KPKTVKPK | K | Phospho (ST) |
30 | sp|P16403|H12_HUMAN | 40 | K | AGGTKPK | K | Phospho (ST); Methyl (K) |
31 | sp|Q9UFE4|CCD39_HUMAN | 37 | R | SPSHTSLSAR | S | Phospho (ST) |
32 | sp|Q9Y4L1|HYOU1_HUMAN | 37 | K | FTKPRPRPK | D | Phospho (ST); Methyl (K) |
33 | sp|P31944|CASPE_HUMAN | 36 | R | LALILCVTK | A | Phospho (ST); Acetyl (K) |
34 | sp|P07355|ANXA2_HUMAN | 36 | R | KGTDVPK | W | Phospho (ST); Acetyl (K) |
35 | sp|P07355|ANXA2_HUMAN | 36 | K | GTDVPKWISIMTER | S | Oxidation (M); Phospho (ST); Methyl (R); Acetyl (K) |
36 | sp|P07355|ANXA2_HUMAN | 36 | K | GTDVPKWISIMTER | S | Oxidation (M); 2 Phospho (ST); Methyl (R) |
37 | sp|P07355|ANXA2_HUMAN | 36 | K | LSLEGDHSTPPSAYGSVK | A | Phospho (ST); Methyl (K) |
38 | sp|Q92466|DDB2_HUMAN | 34 | K | VTHVALNPCCDWFLATASVDQTVK | I | 3 Phospho (ST) |
39 | sp|Q8WV16|DCAF4_HUMAN | 34 | R | MGFNASSMLRK | S | Phospho (ST); Methyl (K) |
40 | sp|Q676U5|A16L1_HUMAN | 34 | K | CGSDWTR | V | Phospho (ST) |
41 | sp|Q7L5D6|GET4_HUMAN | 31 | K | NKSSASVVFTTYTQK | H | Phospho (ST); Methyl (K) |
42 | sp|P84243|H33_HUMAN | 30 | K | SAPSTGGVKKPHR | Y | 2 Phospho (ST) |
HAX1 recruits the Arp2 / 3 complex to the cell cortex, and through its interaction with KCNC3, it reconstitutes the cortical actin cytoskeleton[28]. EspF may rearrange the cytoskeleton and modulate cell survival through phosphorylation and acetylation of the HAX1. EIF3I is a part of the ElF-3 complex and uses different RNA stem-loop binding modes to increase ERK translation[29]. EspF may act on the translation of mRNA involved in cell proliferation (including cell cycle, differentiation, and apoptosis) through phosphorylation and acetylation of EIF3I. EspF may also phosphorylate some autophagy-related proteins, including ATG16L1 and PHB, that mediate BPs such as cell autophagy and protein transport[30].
We also discovered that EspF could modify a series of histones, such as Histone H1.3, Histone H2A type 1, and Histone H1x, which are the core components of nucleosomes. Nucleosomes encapsulate DNA into chromatin, limiting DNA's entry into cellular mechanisms[31]. Therefore, histone modifications play a central role in transcription regulation, DNA damage repair, DNA replication, and chromosomal stability. DNA accessibility is also regulated by a complex set of posttranslational modifications of histones and nucleosome remodeling[32]. EspF may affect epigenetic changes in host cells. Importantly, we found that EspF could phosphorylate histone H2AX, a known marker of DNA damage[33], suggesting that EspF can cause DNA damage.
Next, we discovered that EspF also modified the damage-specific DNA-binding protein 2 (DDB2), a kind of DNA repair protein. DDB2 is originally identified as a DNA damage recognition factor that promotes genomic nucleotide excision repair (GG-NER) in human cells. DDB2 is also involved in other important BPs such as chromatin remodeling, gene transcription, cell cycle regulation, and protein decay. Recently, the potential of DDB2 in the development and progression of various cancers has been described. DDB2 activity occurs in several stages of canceration, including cancer cell proliferation, survival, invasion, and cancer stem cell formation[34]. Previous research showed that EspF could cause cell apoptosis by targeting mitochondria and releasing cytochrome c[4], but it is still unclear whether EspF can mediate DNA damage through nuclear modification of histones in the early period, which lays the foundation for our future research.