Dataset Information

Deep Protein Arginine Asymmetric Di-Methylation Profiling of Breast Cancer Patient-derived Xenografts

ABSTRACT: Arginine methylation catalyzed by protein arginine methyltransferases (PRMTs) is a prevalent posttranslational modification that regulates diverse cellular processes. Aberrant expression of type I PRMTs that catalyze asymmetric arginine demethylation (ADMA) is often found in cancer; however, little is known about the ADMA status of substrate proteins in tumors. Using LC-MS/MS along with pan-specific ADMA antibodies, we performed global mapping of ADMA in five patient-derived xenograft (PDX) tumors representing different subtypes of human breast cancer and identified 415 methylated peptides from 213 proteins. Approximately 70% of the putative substrates were validated using peptide arrays in vitro methylated by PRMT1, PRMT4, and PRMT6, among which 48% of substrates varied from estrogen receptor (ER) positive and negative tumors. Comparing with our previously identified ADMA sites from breast cancer cell lines, 75 ADMA sites overlapped between cell lines and PDX tumors. Collectively, this study provides a useful resource to PRMT and breast cancer communities to exploit the functions of PRMT dysregulation during breast cancer progression.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Min Ma

LAB HEAD: lingjun Li

PROVIDER: PXD038203 | Pride | 2024-01-26

REPOSITORIES: Pride

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Publications

Proteome-wide Profiling of Asymmetric Dimethylated Arginine in Human Breast Tumors.

Ma Min M Liu Fabao F Miles Hannah N HN Kim Eui-Jun EJ Fields Lauren L Xu Wei W Li Lingjun L

Journal of the American Society for Mass Spectrometry 20230718 8

Arginine methylation catalyzed by protein arginine methyltransferases (PRMTs) is a prevalent post-translational modification (PTM) that regulates diverse cellular processes. Aberrant expression of type I PRMTs that catalyze asymmetric arginine dimethylation (ADMA) is often found in cancer, though little is known about the ADMA status of substrate proteins in tumors. Using LC-MS/MS along with pan-specific ADMA antibodies, we performed global mapping of ADMA in five patient-derived xenograft (PDX) ...[more]

PMID: 37463068

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Project description:Arginine methylation is essential for both cellular viability and development and is also dysregulated in cancer. PRMTs catalyze the post translational monomethylation (Rme1/MMA, catalyzed by Type I-III), asymmetric (Rme2a/ADMA, Type I enzymes)-, or symmetric (Rme2s/SDMA, Type II enzymes) dimethylation of arginine. Despite many studies, a thorough integration of PRMT enzyme substrate determination and proteomic and transcriptomic consequences of inhibiting Type I and II PRMTs is lacking. To characterize cellular substrates for Type I (Rme2a) and Type II (Rme2s) PRMTs, human A549 lung adenocarcinoma cells were treated with either Type I (MS023) or Type II (GSK591) inhibitors. Using total proteome hydrolysis, we developed a new mass spectrometry approach to analyze total arginine and lysine content. We showed that Rme1 was a minor population (~0.1% of total arginine), Rme2a was highly abundant (~1.1%), and Rme2s was intermediate (~0.4%). While Rme2s was mostly eliminated by GSK591 treatment, total Rme1 and Rme2a were more resistant to perturbation. To quantitatively characterize substrate preferences of the major enzymes PRMT1, PRMT4(CARM1), and PRMT5, we used oriented peptide array libraries (OPAL) in methyltransferase assays. We demonstrated that while PRMT5 tolerates aspartic acid residues in the substrate, PRMT1 does not. Importantly, PRMT4 methylated previously uncharacterized hydrophobic motifs. To integrate our studies, we performed PTMScan on PRMT-inhibited A549 cells and enriched for methylated arginine containing tryptic peptides. For detection of highly charged peptides, a method to analyze the samples using electron transfer dissociation was developed. Proteomic analysis revealed distinct methylated species enriched in nuclear function, RNA-binding, intrinsically disordered domains, and liquid-liquid phase separation. Parallel studies with proteomics and RNA-Seq revealed distinct, but ontologically overlapping, consequences to PRMT inhibition. Overall, we demonstrate a wider PRMT substrate diversity and methylarginine functional consequence than previously shown.

Project description:Protein arginine methylation, catalyzed by the protein arginine methyltransferase (PRMT) family, is recognized as a widespread post-translational modification (PTM) with implications in a plethora of biological processes in eukaryotes. PRMT proteins were classified into three types, type I, II and III, according to the final methyl-arginine products generated. Despite that thousands of substrates have been identified for Type I and II PRMTs, a full scope of arginine methylation catalyzed by the only type III PRMT, PRMT7, as well as its connection with that of type I and II PRMTs remain unknown, limiting our understanding of the network of arginine methylation and its functions in cells. In this study, global profiling of PRMT4 (type I), PRMT5 (type II) and PRMT7 (type III) substrates (also referred as methylome) revealed that PRMT7 methylated a GAR (glycine and arginine) motif similar as PRMT5, while PRMT4 uniquely methylated a motif in which proline was highly enriched. PRMT4, 5 and 7-methylome were all enriched with proteins functioning in mRNA splicing, and knockdown of PRMT4, 5 or 7 led to a global change of alternative splicing events, among which a cohort with implications in cancer development was co-regulated by all three PRMTs. PRMT4, 5 and 7-mediated arginine methylation on splicing factors such as hnRNPA1, though at different status, were shown to enhance RNA binding in general and participate in the regulation of PRMT4, 5 and 7 co-regulated alternative splicing events.The clinical significance of PRMT4, 5 and 7-mediated arginine methylation was underscored by the fact that these three PRMTs as well as hnRNPA1 arginine methylation were over-represented in multiple types of cancers, which were associated with aberrant gene alternative splicing. Consistently, silencing or pharmacological inhibition of PRMT4, 5 and 7 altered gene alternative splicing and suppressed cancer cell growth, and co-treatment exhibited synergistic effects. Taken together, our study provided an integrative view of substrates for the three types of PRMTs, revealing the important function of arginine methylation in RNA splicing and cancer.

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	HCI001_ADMA_R1.raw	Raw
	HCI001_ADMA_R2.raw	Raw
	HCI002_ADMA_R1.raw	Raw
	HCI002_ADMA_R2.raw	Raw
	HCI007_ADMA_R1.raw	Raw