Protein arginine methylation is an important post-translational modification, but its impact remains rather mysterious. Approximately 7% of all arginine residues in the human proteome are modified by mono- or di-methylation (which is a similar order of magnitude to the 9% of serine residues that are phosphorylated and the 7% of lysine residues that are ubiquitinated).¹ But whereas phosphorylation and ubiquitination have relatively well-characterized biological roles, the function of arginine methylation is less understood.

Knocking out protein arginine methyltransferase 4 (PRMT4), also known as coactivator-associate arginine methyltransferase 1 (CARM1), is lethal in mammals. Both CARM1 knockout and enzyme-inactive CARM1 knock-in mice die at birth and have defects in T-cell development and adipocyte differentiation, indicating that the protein holds an essential role.2,3

CARM1 / PRMT4 Has a Role in a Variety of Diseases

CARM1 Target for Lung CancerThere are also many indications that CARM1 is involved in disease states, most notably cancer. Recent research has found that cancer of the lung,4 breast,5,6 pancreas,7 colon and rectum8 are all influenced by the action of CARM1. The enzyme is also implicated in spinal muscle atrophy,9 a disease characterized by degeneration of spinal cord alpha motor neurons, resulting in muscle weakness, paralysis and death during early childhood. Additional disease states that CARM1 may be involved in include emphysema,10 diabetic11 retinopathy, cardiovascular disease,12 endometriosis and infertility.13

Given this tantalizing body of literature on CARM1, it is surprising how little we know about its substrates, partners, and inhibitors. Two known substrates, NCOA314 and BAF155,15 have provided some insight into its role as a co-activator and modulator of tumor progression, but only give a glimpse into the totality of CARM1 function. There are no inhibitors of CARM1 that have been shown to be effective in animals, and its crystal structure remains unsolved.

New Substrates for CARM1

A recent study by researchers at the University of Wisconsin-Madison has changed this situation considerably.  By using high-resolution mass spectrometry (MS) and a newly developed antibody to ω-NG,NG-asymmetric dimethylarginine (ADMA), the team was able to globally profile CARM1 substrates in two human breast cancer cell lines.16 They identified more than 130 CARM1 protein substrates, and validated more than 90% of them with biochemical assays.16

This “jackpot” of methylation sites was obtained by an innovative approach. Researchers in the Coon and Xu teams immunoprecipitated ADMA-containing peptides using ADMA antibodies in two parental and CARM1 KO paired cell lines, MCF7 and MDA-MB-231, and mapped the sites of arginine methylation using nano-liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) in biological triplicates.16 They leveraged tandem mass tag techniques to simultaneously enrich and analyze ADMA-modified peptides from wild-type and knockout samples. In all, this first step identified over 300 unique ADMA sites on 138 different proteins that are candidates for modification by CARM1 in vivo; over 90% of these had not been previously reported as substrates of the enzyme.

Then, researchers proceeded with the validation of CARM1 substrates using protein arrays. The first array they designed had 200 peptides, each 15 amino acids in length, with 75% of the peptides centered on the putative CARM1 methylation sites identified by immunoprecipitation-MS. A total of 75% of the peptides also included putative proline-rich recognition sequences that bioinformatics analyses had detected among the candidate substrates. Purified recombinant CARM1 methylated over 90% of the putative sites and over 85% of the proline-rich motifs. Importantly, methylation activity was specific to CARM1, as neither PRMT1 or PRMT5 showed methylation activity, and PRMT6 only showed weak activity. A second peptide array further investigated specificity and the proximal amino acid environment of methylation sites.

The team also performed structure-function studies on CARM1.16 The results supported a hypothesis that the N-terminus of CARM1 is involved in substrate recognition.

Overall, this study was a powerful step forward for understanding the biological function of protein arginine methylation. The results indicate that CARM1 methylation sites and recognition sequence are vulnerable to mutation in cancers, providing an important strategy for developing new therapeutic approaches. Other substrates identified in the study indicate key roles of CARM1 in chromatin organization, mRNA-processing pathways, and lipid metabolism.16

Will this breakthrough be followed by rapid progress in identifying CARM1 inhibitors? Time will tell, but the power of HTS screens to find methylation sites is clear. And it is likely to be matched by HTS approaches for drug discovery—perhaps using advanced screening methods such as the new AptaFluor® SAH Methyltransferase assay.

– Robyn M. Perrin, PhD

References

[1] Larsen SC, Sylvestersen KB, Mund A, Lyon D, Mullari M, Madsen MV, Daniel JA, Jensen LJ, Nielsen ML. 2016. Proteome-wide analysis of arginine monomethylation reveals widespread occurrence in human cells. Sci Signal. 9(443):rs9.

[2] Yadav N, Lee J, Kim J, Shen J, Hu MC, Aldaz CM, Bedford MT. 2003. Specific protein methylation defects and gene expression perturbations in coactivator-associated arginine methyltransferase 1-deficient mice. PNAS USA 100(11):6464-8.

[3] Kim D, Lee J, Cheng D, Li J, Carter C, Richie E, Bedford MT. 2010. Enzymatic activity is required for the in vivo functions of CARM1. J Biol Chem. 285(2):1147-52.

[4] Elakoum R, Gauchotte G, Oussalah A, Wissler MP, Clément-Duchêne C, Vignaud JM, Guéant JL, Namour F. 2014. CARM1 and PRMT1 are dysregulated in lung cancer without hierarchical features. Biochimie. 97:210-8.

[5] Cheng H, Qin Y, Fan H, Su P, Zhang X, Zhang H, Zhou G. 2013. Overexpression of CARM1 in breast cancer is correlated with poorly characterized clinicopathologic parameters and molecular subtypes. Diagn Pathol. 8:129.

[6] Sharma P, Bhattacharyya DK, Kalita J. 2017. Disease biomarker identification from gene network modules for metastasized breast cancer. Sci Rep. 7(1):1072.

[7] Wang YP, Zhou W, Wang J, Huang X, Zuo Y, Wang TS, Gao X, Xu YY, Zou SW, Liu YB, Cheng JK, Lei QY. 2016. Arginine Methylation of MDH1 by CARM1 Inhibits Glutamine Metabolism and Suppresses Pancreatic Cancer. Mol Cell. 64(4):673-687.

[8] associated arginine methyltransferase 1 promotes cell growth and is targeted by microRNA-195-5p in human colorectal cancer. Tumour Biol. 39(3):1010428317694305.

[9] Sanchez G, Bondy-Chorney E, Laframboise J, Paris G, Didillon A, Jasmin BJ, Côté J. 2016. A novel role for CARM1 in promoting nonsense-mediated mRNA decay: potential implications for spinal muscular atrophy. Nucleic Acids Res. 44(6):2661-76.

[10] Sarker RS, John-Schuster G, Bohla A, Mutze K, Burgstaller G, Bedford MT, Königshoff M, Eickelberg O, Yildirim AÖ. 2015. Coactivator-Associated Arginine Methyltransferase-1 Function in Alveolar Epithelial Senescence and Elastase-Induced Emphysema Susceptibility. Am J Respir Cell Mol Biol. 53(6):769-81.

[11] Kim DI, Park MJ, Lim SK, Choi JH, Kim JC, Han HJ, Kundu TK, Park JI, Yoon KC, Park SW, Park JS, Heo YR, Park SH. 2014. High-glucose-induced CARM1 expression regulates apoptosis of human retinal pigment epithelial cells via histone 3 arginine 17 dimethylation: role in diabetic retinopathy. Arch Biochem Biophys. 560:36-43.

[12] Liu X, Wang L, Li H, Lu X, Hu Y, Yang X, Huang C, Gu D. 2014. Coactivator-associated arginine methyltransferase 1 targeted by miR-15a regulates inflammation in acute coronary syndrome. Atherosclerosis. 233(2):349-56.

[13] Baumann C, Olson M, Wang K, Fazleabas A, De La Fuente R. 2015. Arginine methyltransferases mediate an epigenetic ovarian response to endometriosis. Reproduction. 150(4):297-310.

[14] Feng Q, Yi P, Wong J, O’Malley BW. 2006. Signaling within a coactivator complex: methylation of SRC-3/AIB1 is a molecular switch for complex disassembly. Mol Cell Biol. 26(21):7846-57.

[15] Wang L, Zhao Z, Meyer MB, Saha S, Yu M, Guo A, Wisinski K5, Huang W, Cai W, Pike JW, Yuan M, Ahlquist P, Xu W. 2014. CARM1 methylates chromatin remodeling factor BAF155 to enhance tumor progression and metastasis. Cancer Cell. 25(1):21-36.

[16] Shishkova E, Zeng H, Liu F, Kwiecien NW, Hebert AS, Coon JJ, Xu W. 2017. Global mapping of CARM1 substrates defines enzyme specificity and substrate recognition. Nat Commun. 8:15571.

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