Differential expression of lysine acetylation proteins in gastric cancer treated with a new antitumor agent bioactive peptide chelate selenium

Abstract

The method of anticancer bioactive peptide (ACBP) functionalized selenium particle (Se), which has enhanced anticancer activity, inhibited the growth of gastric cancer (GC) cells, and increased the ability of apoptosis in vitro, has been reported in previous studies. We used tandem mass spectrometry (TMT) labeling to construct a complete atlas of the acetylation-modified proteome in GC MKN-45 cells treated with ACBP-Se. The proteomics data database was searched and analyzed by bioinformatics: Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), functional enrichment, and protein-protein interaction network. Finally, we conducted a quantitative PRM analysis of the selected target-modified peptides. We identified 4,958 acetylation sites from 1,926 proteins in this research. Among these, 4,467 acetylation sites corresponding to 1,777 proteins were quantified. Based on the above data and standards, we found that in the ACBP-Se group vs. the control group, 297 sites were upregulated, and 665 sites were downregulated. We systematically assessed the proteins containing quantitative information sites, including protein annotation, functional classification, and functional enrichment, cluster analysis supported by functional enrichment, domain structures, and protein interaction networks. Finally, we evaluated differentially expressed lysine acetylation sites. We revealed that SHMT2 K200 and PGK1 K97 were the most critical acetylated non-histone proteins, which may have an essential role in ACBP-Se treatment. Here, we identified and quantified the lysine acetylation proteins in GC cells treated with ACBP-Se. The characterization of acetylation indicates that acetylated proteins might be pivotal in the biological process, molecular binding, and metabolic pathways of ACBP-Se treatment progress. Our findings provide a broad understanding of acetylation ACBP-Se treatment of GC, suggesting a potential application for molecular targeted therapy.

Keywords: Acetylation; Anticancer bioactive peptides; Gastric cancer; Selenium.

Figure 1. The entire experimental procedure.

Experimental design for the quantitative proteomic analysis, the experiment was divided into two groups (control, ACBP-Se), and proteins were extracted from three independent biological replicates per treatment. Extracted proteins were prepared labeled with TMT regents. The labeled peptides were separated by HPLC fractionation, and fractions were analyzed by reversed-phase LC-MS/MS.

Figure 2. Quantification overview of acetylated sites and proteins.

The results identified 4,958 acetylation sites from 1,926 proteins, among which 4,467 acetylation sites corresponding to 1,777 proteins were quantified. The results showed that 655 acetylated sites of 499 proteins were downregulated in ACBP-Se group compared to that in control, while 297 acetylated sites of 238 proteins were unregulated.

Figure 3. GO functional enrichment of locations.

The cell component of acetylated proteins was analyzed by GO enrichment distributed in various kinds of locations, mainly in cytoplasm (37.12%), nucleus (30.5%), and mitochondria (13.09%).

Figure 4. GO analyses of low-expression or over-expression of acetylated proteins in ACBP-Se group compared to control group.

(A) The cell component of acetylated proteins also primarily in extracellular matrix, cell cortex, endoplasmic reticulum chaperone complex, brush border, pore complex, cluster of actin-based cell projections, sarcomere, contractile fiber. (B) The molecular function analysis showed that of 499 acetylated proteins mainly in actin filament binding, rRNA binding, enhancer binding, chemoattractant activity, anion channel activity, voltage-gated ion channel activity, voltage-gated channel activity, carbon–carbon lyase activity, et al. (C) The biological process are allocated in cell morphogenesis, cell morphogenesis involved in differentiation, positive chemotaxis, response to unfolded protein, skeletal system development, hair cycle process, bone development, limb development, regulation of gene expression, epigenetic, skin development, blood coagulation, heart development, telomere organization, tube morphogenesis, et al.

Figure 5. KEGG enrichment of ACBP-Se group compare to the control group.

KEGG pathways enrichment analysis of ACBP-Se compare to the control group showed that acetylated proteins were widely involved in signaling pathways, including focal adhesion, human papillomavirus infection, prostate cancer, synthesis and degradation of ketone bodies, human T-cell leukemia virus 1 infection, TGF-beta signaling pathway, thyroid hormone synthesis, arrhythmogenic right ventricular cardiomyopathy (ARVC), butanoate metabolism, N-Glycan biosynthesis, Th17 cell differentiation, mTOR signaling pathway, adherens junction.

Figure 6. Clustering analysis of the Lys acetylation.

Lys acetylation sites with different modification divide into four parts according to their different modification multiples, as Q1 to Q4.

Figure 7. GO analyses Lys acetylation data sets enrichment in ACBP-Se group compare to the control group.

(A) Biological process of Lys acetylation data sets enrichment in ACBP-Se group compare to the control group, processes associated with regulation of response to protein activation cascade and cytoplasmic sequestering of protein are significantly enriched in up-regulated protein cluster. In contrast, meiotic cell cycle process and cell morphogenesis involved in differentiation are enriched in downregulated proteins. (B) The cellular component enrichment results showed that Lys acetylation proteins sets are enriched mainly in the extracellular matrix and cell surface in the up-regulated proteins while extracellular matrix and nucleoid in the downregulated proteins. (C) The molecular function enrichment results showed that Lys acetylation proteins sets are mainly in single-stranded DNA binding and nucleic acid binding transcription factor activity, while actin filament binding and histone threonine kinase activity in down-regulated protein cluster.

Figure 8. Domain enrichment analysis of Lys acetylation data sets in ACBP-Se group compare to the control group and KEGG pathway clustering identify cellular pathways playing role in ACBP-Se treatments.

(A) The results of domain enrichment analysis showed that protein domains involved in Concanavalin A-like lectin/glucanase domain, Thiolase-like, Band 7 domain, translation protein, beta-barrel domain, B30.2/SPRY domain, SPRY domain, heat shock protein 70kD, peptide-binding domain, heat shock protein 70kD, C-terminal domain, High mobility group box domain. (B) KEGG pathway clustering identify cellular pathways playing role in ACBP-Se treatments. The KEGG pathway clustering analysis results showed that protein processing in endoplasmic reticulum and complement and coagulation cascades enrichment in up-regulation proteins, while leucine and isoleucine degradation and Th17 cell differentiation in down-regulated protein cluster.

Figure 9. Protein interaction networks of Lys acetylation proteome.

Using the MCODE tool, through an intergroup comparison between the ACBP-Se and control groups demonstrated that PCBP1 (Poly (rC)-binding protein 1), SHMT2, Integrin beta-4 (ITGB4), PGK1, PDIA3 exhibited a high degree of connectivity, which located at the center of the network.