Overview of proteome and acetylome in epirubicin-induced early DNA damage
We profiled the proteome and acetylome of DNA repair proteins in HEK293T cells in 1 h after treated with epirubicin using LC-MS/MS (Figure 1a). In total, 6291 proteins were detected and 5526 proteins were quantified with a label-free strategy. Among these quantified proteins, 106 repair proteins associated with NER, BER, MMR, HR, and NHEJ pathways were identified (Figure 1b, Table S1, S3).
The acetylated proteins and their modification sites were identified using a label-free strategy and anti-acetyl antibody affinity enrichment followed by high-resolution LC-MS/MS. The length of most peptides was distributed between 7 and 20, which agreed with the property of tryptic peptides (Figure 1d; Table S5). Among 6789 Kac sites in 2400 proteins identified, 4457 Kac sites in 1778 proteins were quantified, including 190 Kac sites in 50 repair proteins (Figure 1b; Table S2, S4). The overall acetylated proteins contained different numbers of acetylation sites from 1 to 29. 1132 acetylated proteins (47.2%) contained only one acetylation site. The proportions of proteins with two, three, four or more modification sites were 18.4, 10.3, and 24.1%, respectively (Figure 1c; Table S6). Of these 50 repair proteins, the acetylation changes were identifed in 30 proteins. we next analyzing lysine sites on repair proteins, 42 acetylated and 24 deacetylated lysine sites were observed in 21 and 16 repair proteins, respectively, whereas, both acetylated and deacetylated lysine sites were detected in 7 repair proteins (Figure 1b, 2a). In 17 repair proteins, the epirubicin induced acetylation changes were identified for the first time in the present study. (Figure 5b). The number of Kac sites in repair proteins with acetylation modification ranged from 1 to 27 (Figure 1b; Table S7). Repair proteins with acetylated or deacetylated lysine sites were shown in Figure 2 b.
Sites that previously have identified to underwent acetylation modification in DNA damage and repair processes were also identified in our result, such as K120 and K164 in TP53 (17), K77 and K13 in PCNA (18). Of the 106 repair proteins analysed, 50 repair proteins were identifed to contain acetylation and deacetylation modification. Analysing the expression of 106 proteins, only RAD23A was significantly up-regulated (1.76 fold) in cells treated with epirubicin (Figure 2c). The proteome and acetylome results indicated that rapid acetylation or deacetylation of lysine in DNA repair proteins were responsible for manipulating their functions to coordinate the repair progress earlier than the alteration of expression levels in the early stage of DNA damage repair process.
KEGG pathway classification
The 50 repair proteins with acetylation were able to be classified into six major pathways including NER, BER, MMR, HR, NHEJ, DNA replication, and other pathways related to DNA repair process (Figure 3a, Table S8). The NER pathway ranked the first place containing 21 in 34 repair proteins. Epirubicin can induce inter-chain cross-linking and DNA adduct, inhibit the activity of topoisomerase II, and release oxygen-free radicals resulting in DNA lesions and activating several repair pathways, which was consistent with our results.
Functional analysis of the repair proteins with acetylated or deacetylated lysine sites
Of 50 repair proteins containing acetylation modifications, we analysed the cellular component, molecular function, and biological process of 30 proteins with acetylated or deacetylated changes (Figure 3b). These repair proteins were mainly distributed in the nuclear including nuclear lumen organelles (96.7%), nucleoplasm (93.3%), and chromosome (60%), respectively. The top three molecular functions of these proteins were DNA binding; catalytic activity acting on DNA and ATPase activity. The foremost biological process that these repair proteins were involved in DNA metabolic process and DNA repair, chromosome organization, and DNA recombination.
Protein to protein interaction between repair proteins
The protein interaction of acetylated repair proteins was conducted with PPI network analysis. The PPI sources were originated from the STRING database and visualized through the Cystoscope. Interaction information came from experiments and databases resources, and the minimum required interaction score was set to the high level (0.700) to ensure the reliability of the relationship. The relationship of the 50 repair proteins was illustrated in Figure 4a (Table S10). The PPI network revealed the interacting partners of the 17 proteins with new identified acetylation lysine sites, suggesting possible molecular functions related to the effect of acetylations (Figure 4a, 4b).
Acetylation sites located in the functional domains of repair proteins.
The PTM on the domains of proteins can significantly regulate the protein functions. We subsequently explored the relationship between the acetylation sites and functional domain of 17 repair proteins. Of the 32 acetylation sites analysed, 9 acetylation sites were located in the functional domains of 7 repair proteins (Figure 5a). Rad23B simultaneously have 2 acetylated and 1 deacetylated lysine sites that respectively are AcK67, AcK36 and DeacK45 which were all located in UBL domain. AcK64 and DeacK380 on PRP19 were positioned in the U box and WD40-repeat regions. AcK171 and AcK313 on RECQL were both located in the 2 RecA-like domains. The other four proteins (RFC5, RFC3, XAB2 and RAD17) contained 1 acetylation site located .in their main functional domain.
Analysis of Acetylated Lysine Motifs
To identify the possible specific motifs flanking acetylation lysine site, the amino acid sequence from the −10 to the +10 positions surrounding the 1090 acetylated peptides and 1047 deacetylated peptides were analyzed using the MeMe suite. Motifs K[Ac]Y, K[Ac]N, and K[Ac]T ranked the top three of acetylated-Lys motifs, and among deacetylated-Lys motifs, the top three motifs were GK[Ac], K[Ac]S, and K[Ac]Y (Figure 6a, 6b). The matching peptides accounted for all peptides respectively were 27.3% and 24.1%. Among the 30 repair protein with acetylation sites, motifs K[Ac]K, K[Ac]H, and K[Ac]F were most enriched (Figure 6c).