Dataset Information

Comprehensive analysis of symmetric arginine dimethylation in colorectal cancer tissues from patients using immuniaffinity enrichment followed by LC-MS/MS analysis

ABSTRACT: Post-translational arginine methylation is responsible for regulation of a variety of biological processes. The protein arginine methyltransferase 5 (PRMT5) is the major enzyme responsible for generating monomethyl- and symmetric dimethyl arginine (MMA and SDMA, respectively) in proteins. PRMT5 is essential for viability and normal development, and overexpressed in a wide variety of cancer types. In the present study, we found that the levels of PRMT5 and symmetric dimethylation in proteins from colorectal cancer (CRC) tissues are more highly maintained relative to adjacent normal tissues from patients. Using immunoaffinity enrichment of methylated peptides combined with high resolution mass spectrometry, a total of 147 SDMA sites from 94 proteins were identified. Most abundant methylated proteins identified from normal and CRC tissues are RNA processing proteins, transcriptional and translational regulators. Furthermore, when quantitative analysis of the two tissue types of samples was carried out, 77 SDMA sites exhibited statistically significant changes (>2.0-fold, p-value <0.05) between normal and CRC tissues. We confirmed KSRP, G3BP2 and TRIP6 which are more highly maintained in cancer tissues relative to adjacent normal tissues from same patients as well as the expression levels of the proteins. This study is the first comprehensive analysis of symmetric arginine dimethylation using clinical samples and suggests that the modification can be used in clinical application.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Colon

DISEASE(S): Colon Cancer

SUBMITTER: Yae Eun Park

LAB HEAD: Ji Eun Lee

PROVIDER: PXD015653 | Pride | 2020-06-01

REPOSITORIES: Pride

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A Comprehensive Analysis of Symmetric Arginine Dimethylation in Colorectal Cancer Tissues Using Immunoaffinity Enrichment and Mass Spectrometry.

Lim Yongchul Y Park Yae Eun YE Ha Sujin S Lee Ji Eun JE Kim Hee Cheol HC

Proteomics 20200702 15-16

Protein arginine methyltransferase 5 (PRMT5) is a major enzyme responsible for generating monomethyl and symmetric dimethyl arginine in proteins. PRMT5 is essential for cell viability and development, and its overexpression is observed in a variety of cancers. In the present study, it is found that levels of PRMT5 protein and symmetric arginine dimethylation in colorectal cancer (CRC) tissues are increased compared to those in adjacent noncancerous tissues. Using immunoaffinity enrichment of met ...[more]

PMID: 32468700

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Project description:PRMT5 is the major enzyme that catalyzes the symmetric dimethylation of arginine (sDMA) in mammalian cells. Its activity is crucial for many biological processes including transcriptional regulation, mRNA processing, as well as DNA damage and repair. However, the protein substrates identified for PRMT5 have yet been limited in numbers, hindering the understanding of PRMT5 functions in normal as well as diseased cells. Herein we developed an optimized strategy for proteomic profiling of cellular sDMA and apply it to the discovery of novel PRMT5 substrates. Our results show that a combination of proteolytic digestion by the metalloprotease ulilysin and using electron-transfer dissociation with supplemental activation as the mass spectrometry method significantly increased sDMA site identification and localization after immuno-affinity enrichment. By employing SILAC-based differential quantitative proteomic approach and a PRMT5 specific inhibitor, we identified 325 sDMA sites being regulated by PRMT5, 220 being novel sites, which greatly expanded the number of PRMT5 substrates. Biochemical studies confirmed the newly discovered PRMT5 substrate, Plasminogen activator inhibitor 1 RNA-binding protein (SERBP1) and revealed that the RGG/RG motif in the central region of SERBP1 contains both PRMT5-catalyzed sDMA and Type I PRMT-catalyzed asymmetric dimethylation of arginine (aDMA). Unexpectedly, using the optimized methylarginine profiling strategy, we also discovered arginine trimethylation, a previously unknown type of modification, on the central RGG/RG region of SERBP1. By mutational studies we demonstrated that the trimethyl modification on R172 of SERBP1 regulates its recruitment to stress granule (SG) under oxidative stress, indicating a functional role of arginine trimethylation in the cell. Overall, the optimized profiling strategy not only enabled the identification of extensive, non-overlapping sDMA sites and novel PRMT5 substrates, but also revealed a potentially important new PTM, arginine trimethylation. The work lays the foundation for further investigations on the functional interplay of different methylation modifications on arginines, especially in the heavily methylated RGG/RG motif of many RNA-binding proteins.

Project description:Protein methylation has been implicated in many important biological contexts including signaling, metabolism, and transcriptional control. Despite the importance of this post- translational modification, the global analysis of protein methylation by mass spectrometry-based proteomics has not been extensively studied due to the lack of robust, well-characterized techniques for methyl-peptidemethyl peptide enrichment. Here, to better investigate protein methylation, we optimized and compared two methods for methyl-peptidemethyl peptide enrichment: immunoaffinity purification (IAP) and high pH strong cation exchange (SCX). Comparison of these methods revealed that they are largely orthogonal for monomethyl arginine (MMA), suggesting that the usage of both techniques is required to provide a global view of protein methylation. Using both IAP and SCX, we investigated changes in protein methylation downstream of protein arginine methyltransferase 1 (PRMT1). Together, these techniques allowed us to quantify over ~1,000 arginine methylation sites on 407 proteins. Of these methylation sites, PRMT1 knockdown resulted in significant changes to 110 arginine methylation sites on 59 proteins. In contrast, zero lysine methylation sites were significantly changed upon PRMT1 knockdown. In PRMT1 knockdown cells, 24 MMA sites exhibited both significant downregulation (ie, putative PRMT1-mediated MMA targets) and 63 significant upregulation (ie, putative PRMT1-mediated ADMA targets). PRMT1 knockdown induced significant changes in asymmetric dimethyl arginine (ADMA), consistent with being PRMT1 targets. Additionally, We also observed that PRMT1 knockdown induced significant increases in symmetric dimethyl arginine (SDMA) methylation, suggesting that loss of PRMT1 activity allows scavenging of PRMT1 substrates by Type II PRMTs. Analysis of the subcellular localization and protein function of the significantly changing methyl-proteins revealed that the majority of PRMT1 substrates are localized to the nucleus and involved in RNA and DNA binding. Taken together, our results suggest that deep protein methylation profiling by mass spectrometry requires orthogonal enrichment techniques, identify novel PRMT1 methylation targets, and highlight in order to understand the dynamic interplay between methyltransferases in mammalian cells.

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