International Journal of Molecular Sciences Review The Biological Axis of Protein Arginine Methylation and Asymmetric Dimethylarginine Melody D. Fulton , Tyler Brown and Y. George Zheng * Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA 30602, USA * Correspondence: [email protected]; Tel.: +1-706-542-0277 Received: 17 June 2019; Accepted: 4 July 2019; Published: 6 July 2019 Abstract: Protein post-translational modifications (PTMs) in eukaryotic cells play important roles in the regulation of functionalities of the proteome and in the tempo-spatial control of cellular processes. Most PTMs enact their regulatory functions by affecting the biochemical properties of substrate proteins such as altering structural conformation, protein–protein interaction, and protein–nucleic acid interaction. Amid various PTMs, arginine methylation is widespread in all eukaryotic organisms, from yeasts to humans. Arginine methylation in many situations can drastically or subtly affect the interactions of substrate proteins with their partnering proteins or nucleic acids, thus impacting major cellular programs. Recently, arginine methylation has become an important regulator of the formation of membrane-less organelles inside cells, a phenomenon of liquid–liquid phase separation (LLPS), through altering π-cation interactions. Another unique feature of arginine methylation lies in its impact on cellular physiology through its downstream amino acid product, asymmetric dimethylarginine (ADMA). Accumulation of ADMA in cells and in the circulating bloodstream is connected with endothelial dysfunction and a variety of syndromes of cardiovascular diseases. Herein, we review the current knowledge and understanding of protein arginine methylation in regards to its canonical function in direct protein regulation, as well as the biological axis of protein arginine methylation and ADMA biology. Keywords: ADMA; PRMT; arginine methylation; nitric oxide; metabolism 1. Introduction Higher-order organisms (e.g., mammals) generally share conserved or similar chemical mechanisms of bimolecular synthesis and metabolism in comparison to lower-order organisms such as prokaryotes. However, a remarkable distinction in the cell biology of higher-order organisms is the complexity of regulation of diverse biological processes such as gene expression, cell cycle, central metabolism, and signal transduction. Sophisticated regulatory mechanisms and networks are present in eukaryotic cells to control or modulate cellular programs for development, differentiation, survival, and adaptation. Such complex mechanisms of regulation include gene fusion/translocation, epigenetics, RNA splicing, protein-protein interaction, protein-nucleic acid association, and post-translational modifications (PTMs) of proteins. Especially, a myriad of post-translational modifications (PTMs) (e.g., phosphorylation, glycosylation, acetylation, methylation) allow for ON/OFF-switching or fine tuning of protein-controlled cellular processes in response to intracellular and extracellular signals. Among the various PTMs, arginine methylation is a widespread PTM that is conserved in all eukaryotic organisms, from yeasts to humans [1–3]. The frequency of the arginine methylome is parallel to the corresponding phosphoproteome and ubiquitylome cells [2]. About 0.5–1% of the total arginine residues in the cellular proteome are methylated and have a slow turnover, likely to confer lasting functional properties to proteins [4,5]. Int. J. Mol. Sci. 2019, 20, 3322; doi:10.3390/ijms20133322 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 3322 2 of 18 Arginine residues can be methylated on the guanidinium nitrogen atoms in three different states—NG-monomethylarginine (Rme1), asymmetric NG,NG-dimethylarginine (Rme2a), G G and symmetricInt. J. Mol. NSci. 2019, N’, 20, -dimethylargininex FOR PEER REVIEW (Rme2s) (Figure1)[ 3,6–8]. Reported results2 of 18 on the stoichiometric abundance of different methylation states, although varying at different degrees, Arginine residues can be methylated on the guanidinium nitrogen atoms in three different generally support that monomethylated arginine is more abundant than dimethylated arginine states—NG-monomethylarginine (Rme1), asymmetric NG,NG-dimethylarginine (Rme2a), and sites in proteins.symmetric Dilworth NG, N’G-dimethylarginine and Barsyte-Lovejoy (Rme2s) analyzed (Figure 1) the [3,6–8]. number Reported of arginine resultsmethylation on the sites in the Phosphositestoichiometric database abundance by of methyl different state, methylatio andn found states, thatalthough arginine varying methylation at different degrees, predominantly exists in thegenerally Rme1 support form that (~84%), monomethylated and the restarginine (~16%) is more are abundant dimethylarginine than dimethylated (including arginine sites Rme2a and in proteins. Dilworth and Barsyte-Lovejoy analyzed the number of arginine methylation sites in the Rme2s) [9]. Guo et al. performed immunoprecipitation and proteomic analysis and found that Rme1 is Phosphosite database by methyl state, and found that arginine methylation predominantly exists in 3–4 fold morethe Rme1 abundant form (~84%), than and Rme2a the rest in (~16%) HCT116 are dimethylarginine cells [10]. In the (includingTrypanosoma Rme2a and brucei Rme2s), it was [9]. reported that methylarginineGuo et al. performed comprised immunoprecipitation approximately and 10% proteomic of the analysis proteome and found [11], that and Rme1 the is proteomic3–4 fold study revealed thatmore theabundant number than ofRme2a peptide in HCT116 fragments cells [10]. containing In the Trypanosoma Rme1 isbrucei 8.2-fold, it was of reported dimethylarginines, that methylarginine comprised approximately 10% of the proteome [11], and the proteomic study and the abundance of Rme2a is 2-fold of Rme2s. A chromatographic analysis of heart/kidney tissue revealed that the number of peptide fragments containing Rme1 is 8.2-fold of dimethylarginines, and hydrolysatesthe showedabundance that of Rme2a Rme2a is level2-fold isof higherRme2s. thanA chromatographic Rme2s by 5–6 analysis fold [of4 ].heart/kidney tissue hydrolysates showed that Rme2a level is higher than Rme2s by 5–6 fold [4]. Figure 1. TheFigure biological 1. The biological axis of axis protein of protein arginine argininemethylation methylation and and formation formation of methylarginine of methylarginine small small molecule metabolites.molecule metabolites. Type Type I, II, I, and II, and III III PRMTs PRMTs catalyzecatalyze the the initial initial monomethylation monomethylation of an arginine of an arginine residue at the terminal guanidinium nitrogen. Subsequently, type I and II can catalyze a second residue at the terminal guanidinium nitrogen. Subsequently, type I and II can catalyze a second arginine methylation reaction to form the asymmetric and symmetric dimethylated arginine residues, arginine methylationrespectively. Protein reaction degradat to formion theof arginine asymmetric methylated and proteins symmetric produces dimethylated the ADMA, L arginine-NMMA, residues, respectively.and ProteinSDMA metabolites. degradation of arginine methylated proteins produces the ADMA, l-NMMA, and SDMA metabolites. 2. PRMTs 2. PRMTs 2.1. PRMT Family in Mammalian Cells Arginine methylation in mammalian cells is catalyzed by the class I, S-adenosyl-l-methionine (SAM or AdoMet)-dependent methyltransferases named protein N-arginine methyltransferases (PRMTs) [12–16]. Mammalian cells have nine PRMTs [17], and all have a highly conserved catalytic Int. J. Mol. Sci. 2019, 20, 3322 3 of 18 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 of 18 core composed2.1. PRMT of aFamily Rossmann-like in Mammalian fold Cells domain that binds SAM and a β-barrel that supports substrate binding (FigureArginine2)[ 18 ].methylation Upon the in initial mammalian binding cells of is SAMcatalyzed and by substrate, the class I, PRMTsS-adenosyl- transferL-methionine the electrophilic methyl group(SAM fromor AdoMet)-dependent SAM to a terminal methyltransferases nitrogen on named the guanidinium protein N-arginine moiety methyltransferases of arginine residues to produce the(PRMTs) monomethylarginine [12–16]. Mammalian cells residue, have nine Rme1 PRMTs [3,6 [17],–8]. and All all PRMTs have a highly will catalyze conserved this catalytic initial methyl core composed of a Rossmann-like fold domain that binds SAM and a β-barrel that supports substrate transfer. Dependingbinding (Figure on 2) whether [18]. Upon or notthe theinitial PRMT binding catalyzes of SAM aand second substrate, methyl PRMTs transfer transfer reaction the defines the type ofelectrophilic PRMT. PRMT1, methyl group -2, -3, from -4 (CARM1), SAM to a terminal -6, and nitrogen -8 are on identified the guanidinium as type moiety I PRMTs, of arginine which produce Rme1 andresidues catalyze to produce a second the reactionmonomethylarginine to produce residu asymmetrice, Rme1 [3,6–8]. dimethylarginine All PRMTs will catalyze residue this (Rme2a) [7]. In contrast,initial PRMT5 methyl andtransfer. PRMT9 Depending produce on whether Rme1 or and not the symmetric PRMT catalyzes dimethylarginine a second methyl residuetransfer (Rme2s), reaction defines the type of PRMT. PRMT1, -2, -3, -4 (CARM1), -6, and -8 are identified as type I which is characteristicPRMTs, which ofproduce type II PRMTsRme1 and [15 ,catalyze19–21]. Whilea second previously reaction misidentifiedto produce asymmetric as a type II PRMT, PRMT7 isdimethylarginine the