Comprehensive succinylome analyses reveal that hyperthermia upregulates lysine succinylation of annexin A2 by downregulating sirtuin7 in human keratinocytes

Cell culture and hyperthermia treatment

HaCaT cells were purchased from American Type Culture Collection (Maryland, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Biological Industries, Beit-Haemek, Israel), penicillin (100 U/mL), and streptomycin (100 μg/mL) in a 37°C, 5% CO₂ constant temperature incubator.

For hyperthermia treatment, cell culture plates were sealed with parafilm and placed carefully in a circulating water bath for 30 min at 37°C for controls or 44°C for hyperthermia treatment. After these treatments, the cells were returned to a 37°C incubator and cultured for 0–24 h.

Sample preparation

Cells in the control group (37°C) and the heat-treated group (44°C) were harvested and washed with icecold PBS twice. Samples were sonicated on ice using a highintensity ultrasonic processor (Scientz, Ningbo, China) in lysis buffer. The remaining debris was discarded by centrifugation at 12,000 × g at 4°C for 10 min. Finally, the protein concentration was determined with a BCA kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Finally, trypsin (Promega, York, USA) was added at a 1:50 trypsin-to-protein mass ratio for the first digestion overnight and a 1:100 trypsin-to-protein mass ratio for a second 4 h digestion.

High performance liquid chromatography fractionation and affinity enrichment

The peptides were fractionated into 60 fractions by high pH reverse-phase high performance liquid chromatography (HPLC) using a Ther mo Betasil C18 column (5 μm particles, 10 mm ID, 250 mm length). Then, the peptides were combined into 6 fractions and dried by vacuum centrifugation.

The peptides dissolved in NETN buffer (100 mmol/L NaCl, 1 mmol/L ethylene diamine tetraacetic acid (EDTA), 50 mmol/L Tris-HCl, and 0.5% NP-40, pH 8.0) were incubated with prewashed pan-antisuccinyl antibody beads (PTM Biolabs, Hangzhou, China) at 4°C overnight. Then, the beads were washed with NETN buffer and H2O. The bound peptides were eluted from the beads with 0.1% trifluoroacetic acid (TFA) and vacuum-dried.

Liquid chromatography-tandem mass spectrometry analysis

For liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, the resulting peptides were desalted with C18 ZipTips (Millipore, Mass, USA). The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) using Q ExactiveTM Plus (Thermo Fisher Scientific, Mass, USA) coupled online to the UPLC.

Database search

The resulting mass spectrometry data were processed using the MaxQuant search engine v.1.5.2.8 (Max Planck Institute of Biochemistry, Munich, Germany). Tandem mass spectra were searched against the SwissProt Human database concatenated with the reverse decoy database. Carbamidomethyl on Cys was specified as a fixed modification, and succinylation on Lys was specified as a variable modification.

Bioinformatics analysis

GO annotation was obtained from the UniProt-GOA database (www. http://www.ebi.ac.uk/GOA/), InterProScan database (http://www.ebi.ac.uk/interpro/), and DAVID database (https://david.ncifcrf.gov/). The subcellular localization was analyzed by WoLF PSORT software. For the functional descriptions of the protein domain, the InterProScan database was used. Protein pathways were annotated by the KEGG database and KEGG online service tools KEGG mapper. Motif-x was used to predict specific Ksucc motifs. Analysis of secondary structure was conducted by NetSurfP v1.0 (DTU Health Tech, Lyngby, Denmark).

Isolated tissue and hyperthermia treatment

Fresh condyloma acuminata (CA) tissues were obtained from patients undergoing surgical treatment at the First Hospital of China Medical University. The lesion was equally divided into two parts and separately placed in cell culture dishes containing DMEM plus 10% fetal bovine serum. For hyperthermia management, the tissue was treated in the same way as HaCaT cells described above. Subsequently, tissue samples were fixed with paraformaldehyde 4 h after hyperthermia treatment and then used for immunohistochemical assays. This part of the study was approved by the Ethics Committee of the First Hospital of China Medical University (Approval No. 2018-135-2), and informed consent was obtained from the patients.

Western blotting

Cultured cell samples were treated with RIPA lysis buffer (Beyotime, Shanghai, China) containing protease inhibitors and phosphatase inhibitors (Beyotime, Shanghai, China) and centrifuged at 15,000 × g at 4°C for 10 min. The supernatant was carefully aspirated, mixed with SDS–PAGE loading buffer after protein quantification by BCA assay, boiled at 100°C for 5 min, and then subjected to SDS–PAGE detection. After being transferred to a polyvinylidene difluoride membrane (PVDF), nonspecific binding was blocked with QuickBlock blocking solution (Beyotime, Shanghai, China) for 15 min. The blots were probed with the following primary antibodies: fatty acid synthase (FASN) antibody (Cell Signaling Technology, Mass, USA), KATA2 antibody (Cell Signaling Technology, Mass, USA), succinyl lysine antibody (PTM Biolabs, Hangzhou, China), SIRT5 antibody (Cell Signaling Technology, Mass, USA), SIRT3 antibody (Cell Signaling Technology, Mass, USA), SIRT7 antibody (Cell Signaling Technology, Mass, USA) and annexin A2 antibody (Proteintech, Wuhan, China).

Plasmid construction and transfection

SIRT7 overexpression plasmids were constructed by Shanghai Heyuan Biotechnology Co., Ltd. According to the protocols provided by the manufacturer, plasmid DNA was transfected with Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific, Mass, USA), and cells were harvested 48 h after transfection.

Co-immunoprecipitation

Cultured cells were lysed by immunoprecipitation (IP) buffer (0.25% sodium deoxycholate, 50 mmol/L Tris, 1 mmol/L EDTA, 1% Triton X-100, 1% NP-40, and 150 mmol/L NaCl) after washing twice with ice-cold PBS. The supernatants of digested cells were transferred into new EP tubes, mixed with primary antibody and magnetic beads (Bimake, Houston, USA), and incubated overnight on a rotator at 4°C. The next day, the magnetic bead particles were washed 5 times with cold IP buffer, and then protein loading buffer was added and boiled at 100°C for 5 min.

Immunohistochemical analysis

Immunohistochemical (IHC) staining was performed to detect the expression of SIRT7 in CA tissues after in vitro hyperthermia. Briefly, CA tissues (hyperthermia-treated group or controls) were fixed with 4% paraformaldehyde and then embedded in paraffin. Tissue sections were prepared for antigen retrieval with 0.01 mol/L sodium citrate antigen retrieval solution in a pressure cooker for 2 min. Endogenous peroxidase activity was inactivated using 3% H₂O₂. Then, nonspecific interactions were blocked with 10% goat serum. SIRT7 antibody at a dilution of 1:200 was incubated with the slides overnight at 4°C. The following steps were performed according to the protocols of the EliVision kit (Maxim, Fuzhou, China). The slides were examined using light microscopy (Olympus IX51, Tokyo, Japan).

Statistical analysis

Two-tailed Fisher’s exact tests were applied to GO, KEGG, and protein domain enrichment analyses. Categories with a corrected P value < 0.05 were considered significant.

Proteome-wide analysis of succinylation proteins and sites identified in HaCaT cells

Hyperthermia clears multiple remote cutaneous warts by targeting a single lesion through eliciting anti-HPV immune responses.^[24] The clinical treatment of children’s perianal warts showed that local hyperthermia removed multiple lesions by treating one place of lesions (Figure 1A). Lysine succinylation is a novel PTM and has already been confirmed to be involved in multiple cellular processes. Here, we conducted a comprehensive succinylome in HaCaT cells (both the control and hyperthermia-treated groups) and identified 387 proteins with 969 succinylated lysine sites altogether (Figure 1B), of which 119 proteins with 197 lysine sites were upregulated in response to hyperthermia treatment (44°C) (Figure 1C).

Figure 1

The clinic treatment of cutaneous warts and total profiling of lysine succinylome in HaCaT cells after response to hyperthermia. (A) Local hyperthermia treatment in children’s perianal warts. The red circle is therapeutic target for multiple verrucous lesions. (B) Experimental workflow for the identification of succinylated proteins and sites in HaCaT cells (with or without 44°C treatment). (C) Quantification of succinylated sites of HaCaT cells in response to hyperthermia. (D) Distribution of succinylation proteins based on their number of succinylated sites. Ksucc: lysine succinylation; MS/MS: mass spectrometry.

The peptides contained different numbers of succinylated sites varying from 1 to 21. Of all succinylation proteins identified in our data, 51.9% (201/387) had a single site, 18.6% (72/387) contained two succinylated sites, 10.6% (41/387) possessed three modified sites, and 3.6% (14/387) had 10 or more succinylation peptide sites (Figure 1D).

Among the 387 identified proteins, the most extensively succinylated was HSPD1, with 21 modified lysine sites. In addition, endoplasmin (HSP90B1), alpha-enolase (ENO-1), annexin A2, and serine beta-lactamase-like protein (LACTB) contained 14, 12, 10, and 7 succinylated sites, respectively.

Functional annotation and enrichment analysis of identified succinylated proteins

To investigate the potential roles of succinylated proteins in HaCaT cells, we annotated all the identified proteins (387) using gene ontology (GO) functional characterization based on biological process (BP), cellular component (CC), and molecular function (MF) (Figure 2A). In terms of the biological process analysis, the two largest categories were related to cellular process (345 proteins) and metabolic process (284 proteins). Notably, 155 and 46 succinyl proteins were involved in response to stimuli and immune system processes, indicating that succinylated proteins may play a role in the immune response to exogenous stimuli. Within the molecular function category, the two largest groups belonged to binding (230 proteins) and catalytic activity (207 proteins), suggesting that lysine succinylation may alter the structure and functional activity of modified proteins.

Figure 2

Functional enrichment analysis of upregulated Ksucc proteins and structural analysis of identified succinyl proteins in HaCaT cells in response to hyperthermia. (A) GO characterization of succinylated proteins based on BP, CC, and MF. (B) The subcellular location of succinylated proteins. The enrichment analysis of succinylproteins in terms of (C) KEGG pathway. (D) Number of succinylated peptides in each motif. (E) Heat map of the amino acid surrounding succinyl-lysine. Properties of modified lysine based on (F) secondary structure probability and (G) absolute surface accessibility. Helix: alpha helix; Strand: beta-strand; Coil: coiled coil; BP: biological process; CC: cellular component; MF: molecular function; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes.

The subcellular localization may provide a clue for predicting protein function.^[25] We found that most succinylated proteins were in the mitochondria (47.29%), followed by the cytoplasm (29.46%) and nucleus (8.01%) (Figure 2B). All the above results indicated that succinylated proteins were distributed extensively and affected various cellular processes in HaCaT cells.

The KEGG enrichment analysis showed that succinylated proteins were more likely to be enriched in the thermogenesis, carbon metabolism, oxidative phosphorylation, glycolysis/ gluconeogenesis, and fatty acid metabolism pathways (Figure 2C). It can be predicted that lysine succinylation of proteins may influence the thermal process and energy metabolism in HaCaT cells.

Motif analysis and structural properties of identified succinyl proteins

To identify the specific succinyl-peptide patterns, we characterized the frequencies of position-specific amino acids flanking succinyllysine (10 amino acids upstream and downstream of the centered Ksu site) by the Motif-X program. Two preferred motifs, I* Ksu and V* Ksu, were identified (* indicates a random amino acid residue and Ksu indicates a succinylated lysine site). Nearly 84 and 94 succinylated peptides possessed these two motifs, respectively (Figure 2D). Surrounding succinylated lysine (K) at sites –1 and –2, the basic amino acids arginine (Arg) and another K were less frequent. Four residues, alanine (Ala), aspartic acid (Asp), isoleucine (Ile), and valine (Val), were commonly preferred around the modified lysine (Figure 2E).

Furthermore, we analyzed the secondary structure of the identified succinylation proteins to investigate whether lysine succinylation had a tendency for specific structural regions. Approximately 34.8% succinylated sites were detected in the ordered secondary structure. Among them, 28.5% were found in the α-helix, and 6.3% were in the β-sheet. The remaining sites (65.2%) were located at the regions of random coils, indicating that succinylated peptides are more frequently occurred in the unstructured regions of the protein (Figure 2F). In addition, compared with all lysine residues, succinylated sites did not show moderate bias for the protein surface (Figure 2G).

Functional annotation of differentially succinylated proteins in response to hyperthermia treatment

To determine the potential role of lysine succinylation in HaCaT cells in response to hyperthermia treatment, all upregulated succinylated proteins (119 proteins with 197 sites) were submitted to GO analysis (Figure 3A). Based on the classification of MF, the most of succinylation proteins belonged to binding and catalytic activity. Within the CC category, most proteins were related to extracellular exosomes, mitochondria, and cytoplasm, showing that succinylated proteins existed broadly in HaCaT cells. In terms of BP, proteins connected with metabolic process (101 proteins) and response to stimulus (54 proteins) were more likely to be succinylated, which was consistent with the CC analysis that most proteins were in metabolism-related regions. In addition, 19 and 6 proteins were involved in the immune system process and viral transcription, respectively, indicating that the treatment effect of hyperthermia for HPV-infected skin warts could be achieved by regulating lysine succinylation on these proteins.

Figure 3

Characterization description of differentially succinylated proteins in response to hyperthermia in HaCaT cells. (A) Functional annotation of differentially succinylated proteins in response to hyperthermia treatment. (B) The integration of upregulated succinylated proteins in immune system process and viral transcription. (C) The Ksucc sites analysis of annexin A2 and LACTB. GO: Gene Ontology; LACTB: serine beta-lactamase-like protein.

Moreover, three succinylated proteins, annexin A2, caveolin 1 (CAV1), and HSPA8, were involved in both the immune system process and viral transcription (Figure 3B). Among them, annexin A2 possessed four upregulated succinylated lysine sites (K47, 81, 152, 227) (Figure 3C, Table 1).

We sorted the proteins according to the upregulation ratio of succinylation sites in response to hyperthermia treatment. Among them, LACTB, mitochondria ranked first, and multiple sites were upregulated (K243, 259, 266, 283, 284) by more than 3 times (Figure 3C, Table 2).

Succinylation of global proteins was affected by hyperthermia and showed an obvious increase in annexin A2 and LACTB proteins after heat treatment

To verify the results of proteome analysis, we examined the succinylation of global proteins at different time points in HaCaT cells after hyperthermia treatment. The western blot results showed that the succinylation level of global proteins increased at 30 min after hyperthermia and gradually recovered to untreated levels at 6 h (Figure 4A).

Figure 4

Hyperthermia increase the succinylation levels of total proteins in HaCaT cells. (A) Western blot analysis of the succinylation of total proteins in HaCaT cells in different time points after hyperthermia. (B) Western blot analysis of the expression of LACTB, FASN, and annexin A2 in HaCaT cells in response hyperthermia. (C) Co-immunoprecipitation analysis of the succinylation of LACTB, FASN, and annexin A2 before and after hyperthermia. Suc indicates the lysine succinylation. FASN: fatty acid synthase; LACTB: serine beta-lactamase-like protein; IgG: immunoglobulin G; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

According to proteome analysis, LACTB, annexin A2, and FASN were the most evident molecules in the upregulation ratio of succinylation. Therefore, their expression was also verified by WB. The detection of protein levels showed that LACTB and FASN were not significantly changed in HaCaT cells after hyperthermia, but annexin A2 was decreased (Figure 4B). The succinylation of LACTB, FASN, and annexin A2 was examined by coimmunoprecipitation prior and post to hyperthermia (after 4 h). The data showed that the succinylation of LACTB and annexin A2 was significantly increased after hyperthermia compared to that of the untreated group, which was consistent with the results of our succinylome analysis (Figure 4C). However, the succinylation of FASN was not detected, and it is possible that FASN did not undergo succinylation in HaCaT cells.

Hyperthermia affects succinylation by influencing the expression of SIRT7

First, we examined the expression levels of succinylated enzymes after 44°C hyperthermia treatment. The results indicated that KATA2 and SIRT7 were apparently decreased in expression compared with nonheated cells (Figure 5A). Specifically, KATA2 and SIRT7 were highly decreased at 1 h after heat treatment, gradually began to recover at 4 h, and finally returned to the base level at 12 h (Supplementary Figure 1).

Figure 5

Hyperthermia influencing the succinylation of global proteins by downregulating SIRT7 in HaCat cells. (A) Western blot analysis of the expression of succinylated enzymes after hyperthermia. (B) Western blot analysis of the succinylation of global proteins in overexpression SIRT7. (C) Co-immunoprecipitation analysis of the interaction of SIRT7 with LACTB, FASN, and annexin A2. (D) The immunohistochemical staining of SIRT7 in condyloma acuminatum tissues. Suc indicates the lysine succinylation. - means empty vector plasmid. + means SIRT7 plasmid. Original magnification: 200×. FASN: fatty acid synthase; SIRT7: sirtuin7; LACTB: serine beta-lactamase-like protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

To explore the possible reasons for hyperthermia influences on the succinylation of HaCaT cells, SIRT7 overexpression plasmids were constructed. We observed that SIRT7 affected the succinylation of global proteins in HaCaT cells, and succinylation was lowered in the SIRT7 overexpression group than that in the control group (transfected vector plasmid), and this difference was more significant in cells treated with hyperthermia (Figure 5B).

HPV was shown to interact with annexin A2 at both the extracellular plasma membrane and endocytic vesicles, indicating the role of annexin A2 as a receptor for HPV infection, especially in virus attachment, internalization, and trafficking.^[26,27,28] To investigate whether SIRT7 might be the regulatory enzyme of annexin A2, co-immunoprecipitation of SIRT7 with annexin A2 was performed, and the results showed that SIRT7 interacted with annexin A2 (Figure 5C).

Hyperthermia downregulates the expression of SIRT7 in CA tissues

Immunohistochemical staining of CA tissue samples showed that SIRT7 was reduced in heat-treated tissue samples as compared with untreated samples (Figure 5D). This result was consistent with the ex vivo experiments in HaCaT cells. In summary, our results implied that hyperthermia reduces the expression of SIRT7 in CA tissues, which in turn affects the succinylation levels of HPV infection-related molecules.