Proteome-wide identification of arginine methylation in colorectal cancer tissues from patients

Abstract

Background: Protein arginine methylation reaction is catalyzed by protein arginine methyltransferase (PRMT) and the modification is implicated in various diseases including cancer. Currently, thousands of arginine methylation sites have been identified using high-resolution mass spectrometry-based proteomics technology. However, identification of arginine methylation using clinical samples at proteome level has not been reported yet. The objective of the present study was to identify, monomethyl-arginine (MMA) and asymmetric dimethyl-arginine (ADMA) sites in colorectal cancer (CRC) tissues at proteome level. Methods: Pooled CRC tissue samples from 10 patients with stage II and III were digested by trypsin and these digests were further processed and lyophilized. Using monomethyl-or asymmetric dimethyl arginine (MMA or ADMA, respectively) motif kits, methylarginine-containing peptides were enriched and subsequently analyzed by high-resolution LC-MS/MS. DLD1 and HCT116 colon cancer cells were treated with type I PRMTs inhibitor (MS023) alone or combined with SN-38, and the effect of the drugs on CRC cell proliferation and apoptosis was measured by water-soluble tetrazolium salt (WST-1) assay and FACS analysis, respectively. Results: In the present study, 455 MMA sites of 272 proteins and 314 ADMA sites of 155 proteins were identified from CRC tissues acquired from patients. In addition, 216 methylation sites and 75 substrates for PRMTs were newly identified. These results reveal the significant presence of MMA and ADMA sites on nucleic acid binding proteins and protein complexes involved in transcription. To investigate the effect of protein arginine methylation in CRC proliferation and apoptosis, MS023 was treated to two CRC cell lines. After 48 h treatment with various concentrations of MS023, CRC cell proliferation was significantly suppressed, with concomitant apoptosis induction. Furthermore, MS023 treatment significantly enhanced the inhibitory effect of SN-38 on CRC cell proliferation. Conclusion: This work reports the first comprehensive analysis of arginine methylation with clinical sample and suggests that type I PRMTs are potential therapeutic targets for drug discovery in CRC.

Independent transcriptomic and proteomic regulation by type I and II protein arginine methyltransferases

“…Lastly, to further expand our knowledge of methylarginine containing proteins and the residues which are specifically modified, we compiled previously published publicly available methylarginine datasets ( Table S5 ) ( Fedoriw et al., 2019 ; Fong et al., 2019 ; Guo et al., 2014 ; Hornbeck et al., 2015 ; Larsen et al., 2016 ; Li et al., 2021 ; Lim et al., 2020 ; Musiani et al., 2019 ; Wei et al., 2020 ). Together, these data sets—including our own—contained 5,255 unique methylarginine-modified proteins with 15,386 independent methylarginine residues ( Figures S7 A and S7B).…”

“…Lastly, we compiled all publicly available data for the human arginine methylome ( Fedoriw et al., 2019 ; Fong et al., 2019 ; Guo et al., 2014 ; Hornbeck et al., 2015 ; Larsen et al., 2016 ; Li et al., 2021 ; Lim et al., 2020 ; Musiani et al., 2019 ; Wei et al., 2020 ) ( Table S5 ). Together these data exemplify the diverse array of PRMT substrates.…”

Systematic Analysis of the Impact of R-Methylation on RBPs-RNA Interactions: A Proteomic Approach

“…In recent years, proteome-wide strategies to study R-methylated proteins have been optimized, thanks to the implementation of efficient biochemical protocols for methyl-peptide enrichment, coupled to off-line high pH (HpH) chromatographic fractionation and high-resolution mass spectrometry (MS) analysis. Recently published evidence ( Fedoriw et al, 2019 ; Fong et al, 2019 ; Lim et al, 2020 ; Szewczyk et al, 2020 ), together with MS-proteomics analyses carried out in our group ( Musiani et al, 2019 ; Musiani et al, 2020 ; Spadotto et al, 2020 ), has shown that pharmacological and genetic inhibition of PRMTs coupled with quantitative MS-based analysis are powerful approaches to expand the knowledge about the extent of this modification, its dynamics upon different perturbation and its involvement in different cellular pathways. One interesting piece of information emerging from these studies is that RNA-binding proteins (RBPs) are over-represented among experimentally-annotated R-methylated proteins.…”

Arginine methylation: the promise of a ‘silver bullet’ for brain tumours?

“…Both types of post-translational methylation contribute to the exquisite control of gene expression through modification of histone proteins. Lys and Arg methylation also regulate a range of non-histone proteins including proteins involved in transcription and RNA-binding, translation, chaperone, cytoskeletal and membrane proteins and adaptor/scaffold proteins (Guo et al 2014 ; Lim et al 2020 ). However, there are several and important differences between the methylation of lysines and arginines.…”