Central Annals of Vascular Medicine & Research

Cite this article: Huang R, Khalil EM, Mackie BD, Mao Y (2016) Methyltransferases: Key Regulators in Cardiovascular Development and Disease. Ann Vasc Med Res 3(2): 1032.

*Corresponding authorRong Huang, Department of Medicinal Chemistry, Virginia Commonwealth University, 800 E Leigh St, Suite 212, Richmond, VA 23219, USA, Tel: 0018048285619; Email:

Submitted: 01 April 2016

Accepted: 01 June 2016

Published: 13 June 2016

ISSN: 2378-9344

Copyright© 2016 Huang et al.

OPEN ACCESS

Keywords•Protein methyltransferase•Homoceysteine•PRMT•SmyD•NNMT

Review Article

Methyltransferases: Key Regulators in Cardiovascular Development and DiseaseEhab M. Khalil, Brianna D. Mackie, Yunfei Mao, and Rong Huang* Department of Medicinal Chemistry, Virginia Commonwealth University, USA

Abstract

Methylation has emerged as an increasingly important chemical modification that is involved in the regulation of many biological processes. Consistent with the extensive and diverse set of biological functions regulated by methylation, transmethylation reactions are catalyzed by a large family of methyltransferases with a broad range of substrate specificity, including histone proteins involved in transcriptional regulation, tumor suppressor proteins such as p53 and Rb, DNA and RNA substrates, and small molecule substrates such as catecholamines, nicotinamide, and various xenobiotics. This review focuses on a small subset of this large family of methyltransferases and the role they play in cardiovascular development, function, and disease. Specifically, we highlight two protein methyltransferase (PMT) subfamilies that modify histone and non-histone protein substrates: protein arginine methyltransferases (PRMT) that methylate the side chain of arginines, and the SmyD protein family that N-methylate the side chain of lysines. We also discuss a potential role that nicotinamide N-methyltransferase (NNMT) plays in cardiovascular disease by creating a metabolic sink for homocysteine, an amino acid known for its adverse effects on the cardiovascular endothelium and smooth muscle cells.

ABBREVIATIONSPMT: Protein Methyltransferase; PRMT: Protein Arginine

Methyltransferase; PKMT: Protein Lysine Methyltransferase; SmyD: SET and MYND Domain-Containing Protein; SAH: S-5´-Adenosyl-L-Homocysteine; SAM: S-5´-Adenosyl-L-Methionine; Hcy: Homocysteine; SAHH: S-5´-Adenosyl-L-Homocysteine Hydrolase; SDMA: Symmetric Dimethylarginine; ADMA: Asymmetric Dimethylarginine

INTRODUCTION Methyltransferases are enzymes that methylate a broad

spectrum of substrates, such as protein, nucleic acids (DNA and RNA), and naturally-produced small molecules. Methylation is one of the most common and well-studied chemical modifications found inside cells. It increases the biochemical diversity by altering the charge states and hydrophobicity and modulating the function of the target substrates. Methylation on histone proteins and DNA is considered an important epigenetic marker that regulates gene expression during development and differentiation [1]. More recently, emerging evidence on the methylation of non-histone proteins underscores the importance of this modification in regulating diverse cellular and biological functions as well as potential involvement in the pathogenesis of

many diseases, including cancer, neurodegenerative, metabolic, pulmonary, and cardiovascular disease [2-6].

Protein methyltransferases (PMTs) that catalyze methylation on nitrogen can be broadly classified into three distinct families based on their substrate specificity: (1) protein arginine methyltransferases (PRMTs) that modify the guanidine group of arginine [5,7,8], (2) protein lysine methyltransferases (PKMTs) that methylate the epsilon-amine of lysine side chain [1,4], and (3) N-terminal methyltransferases (NTMTs) that methylate the alpha-N-terminus of substrate proteins [9,10]. Across all family members of PMTs, as well as small molecule methyltransferases such as nicotinamide N-methyltransferase (NNMT), the classical methyl donor is S-5´-adenosyl-L-methionine (SAM or AdoMet). After the methyl group is transferred, the cofactor SAM is converted to S-5´-adenosyl-L-homocysteine (SAH) (Figure 1). SAH can be further hydrolyzed by the enzyme S-adenosyl homocysteine hydrolase (SAHH) to generate homocysteine (Hcy) and adenosine. The fate of Hcy in the cell follows two distinct pathways. Under methionine-deficient conditions, Hcy is remethylated to L-methionine in a reaction catalyzed by methionine synthase and the cofactors 5-methyltetrahydrofolate and vitamin B12. When methionine levels are normal, Hcy can be converted into L-cysteine in a trans-sulfuration reaction

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catalyzed by Cystathionine-β-synthase [11,12]. This biochemical process, which regulates the SAM-to-SAH ratio, is part of the methionine-methylation cycle (Figure 2) [11,13].

Abnormally high levels of homocysteine in the blood can be caused by genetic or environmental factors and will result in a medical condition known as hyperhomocysteinemia [11,14]. It is well documented that hyperhomocysteinemia can lead to vascular endothelial dysfunction and may play an important role in the development of cardiovascular disease. Since genetic defects in enzymes involved in the biosynthetic pathway of Hcy may lead to dysregulated levels of Hcy [11], it is plausible that this may provide a link between dysregulated methyltransferase activity and the risk for developing cardiovascular disease. Although methyltransferases are potential therapeutic targets for many diseases, in this review, we limit our discussion on the biochemistry of several methyltransferases and highlight their involvement in cardiovascular development and diseases.

DISCUSSION

PRMTs and cardiovascular disease

Arginine methylation plays a critical role in genomic stability, gene expression, chromatin stability, cell mitosis and more. This specific arginine modification is mediated by a family of enzymes known as PRMTs [8,15]. Specifically, over expressed levels of arginine methylation have been regarded as diagnostic markers of vascular pathological conditions [16,17]. The PRMTs are classified as a type I, type II or type III according to the resulting methylated arginine products. Type I PRMTs convert arginine to monomethylarginine (MMA) and then to asymmetric dimethylarginine (ADMA) (Figure 3), where both methyl groups are installed on the same nitrogen of the arginine guanidino group. Type II PRMTs convert arginine to MMA and subsequently to symmetric dimethylarginine (SDMA), where two methyl groups are installed on two different guanidino nitrogen atoms. Finally, type III PRMT enzymes only convert arginine to MMA and do not dimethylate arginine residues. The PRMT family is involved in a variety of roles including DNA repair, signal transduction, transcriptional control and protein translocation. Mutations or abnormal expression of these enzymes has been found in cancer, ALS, diabetes and cardiovascular disease [8,15,18,19].

PRMT1 is a type I PRMT that accounts for 85% of all cellular

PRMT activity [8,20] and 50% of all ADMA development [20], and is directly related to cardiovascular disease. ADMA levels are often biomarkers for cardiovascular conditions due to their endogenous inhibition of the formation of nitric oxide (NO) [16,17]. Interestingly, ADMA competes with the natural substrate, L-arginine, at the active site of nitric oxide synthase (NOS), resulting in inhibition of NO production [16]. Endothelium-derived NO is a highly potent vasodilator, but also has anti-thrombotic, anti-atherogenic and anti-inflammatory properties [16,21]. One function of the endothelium is to maintain vascular homeostasis through the release of vasodilators, including NO, upon increased blood flow. Disruption of the NOS pathway is often the predecessor for a variety of vascular diseases; therefore, NO is arguably the most important regulator of vascular homeostasis [21-24]. Consequently, inhibition of NOS by PRMT1’s production of ADMA contributes to an increased risk of vascular diseases.

Hyperhomocysteinemia plays a critical role in impaired endothelial function and cardiovascular diseases. Given that ADMA contains two methyl groups, two equivalents of homocysteine are generated after each catalytic reaction [19,20,22]. Homocysteine has a primary role in inhibition of dimethylarginine dimethylaminohydrolase (DDAH), which is responsible for the breakdown of ADMA. Consequently, hyperhomocysteine leads to an increase of ADMA and decrease of NO production [22,25,26]. In addition, homocysteine contains a reactive sulfhydryl group that readily undergoes disulfide exchange reactions with other proteins. Formation of such disulfide bonds impairs the ability of DDAH to bind ADMA and leads to an accelerated DDAH degradation, which results in increased level of free ADMA [25,26]. Such connection has been verified in hyperhomocysteine-induced studies since there is a decrease of DDAH activity, an increase of ADMA, and a reduction of NO in endothelial cells [26].

Therefore, PRMT1 represents a promising therapeutic target for cardiovascular disease. Inhibition of PRMT1 restores the proper balance of NO by decreasing the production of ADMA and the homocysteine generated after the catalytic reaction. Further studies are underway to validate PRMTI as a target for treating various cardiovascular conditions.

PRMT1 Inhibition

Many efforts have been taken to develop inhibitors for PRMT1

Figure 1 Methylation of proteins on the side chain of arginine (R) or lysine (K) residues, Catalyzed by PMTs. Abbrevations: SAM: S-5´-adenosyl-L-methionine; SAH: S-5´-adenosyl-L-homocysteine.

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cystathionine-β-synthase

Cysteine Homocysteine MethionineSynthase,

Vit. B12SAHH

adenosine MethionineSAH

Methylation of Proteins, DNA, MAT, ATPRNA, and other biomolecules SAM

PMTs and otherMTs

Figure 2 Metabolic pathways for Homocysteins. Abbrevations: SAHH: S-adenosyl Homocysteine Hydrolase; MAT: Methionine Adenosyltransferase.

Figure 3 Methylation of the Arginine Side Chain by PRMTs.

to treat cardiovascular disease, diabetes and cancer. However, PRMT1 inhibitors have been deficient in selectivity and potency against other proteins within the PRMT family [3]. The first small molecule inhibitor of PRMT1, AMI-1, was discovered in 2004 through HTS in a 9000-compound library (Figure 4). Although this compound inhibited PRMT1 with an IC50 of 8.8 µM, the selectivity of AMI-1 was not reported [3,27]. In 2012, the PRMT1 inhibitor A36 was found through pharmacophore-based virtual screening with an IC50 of 12 ± 0.2 µM. A36 was highly selective for PRMT1 against many but not all PRMT family members [3,28]. The most recent PRMT1 inhibitor, Compound 1, was found in 2014 and had an IC50 value of 9.2 ± 1.1 µM. Compound 1 had significant selectivity for PRMT1 over all other PRMTs and was found to inhibit proliferation in multiple leukemia cell lines [3,29]. Although these inhibitors have not been tested against cardiovascular disease, there is vast potential for PRMT1 inhibitors to be an effective treatment option.

SmyD1 and smyD2 and its role in cardiovascular development and disease

SmyD proteins belong to a family of protein lysine methyltransferases PKMTs and share two highly conserved regions, the SET and MYND homology domains. There are currently five known SmyD protein members (SmyD1-5), all containing a catalytic SET domain that is split by an intervening MYND domain [4,30-32]. The SET domain is responsible for methylating lysine side chains on histone and non-histone substrates [31]. MYND is a zinc-finger domain that functions as a site for protein-protein interactions and corepressor complex recruitment. SmyD proteins are conserved across all vertebrates, underscoring their important biological function. The accumulating data show that SmyD proteins are involved in skeletal and cardiac muscle development and differentiation [30-33]. SmyD1 and SmyD2 are two members that have demonstrated

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Figure 4 Selected PRMT1 Inhibitors.

strong links to cardiovascular development and disease.

SmyD1 (previously known as m-Bop and KMT3D) is a muscle specific PKMT that is expressed in heart and skeletal muscle tissue of vertebrate embryos [34]. In mice, targeted deletion of SmyD1 disrupted maturation of ventricular cardiomyocytes and resulted in malformation of the right ventricle. In the same study, the SmyD1 null mouse embryo failed to form a right heart ventricle and died around 10 days [35]. Analogous results were also observed in zebrafish embryos with knockdown of SmyD1b, one of two homologous SmyD1 genes in zebrafish. The affected fish embryos showed lack of heart contraction and severe myofibril disorganization in cardiac muscle and skeletal muscle tissue [33,36,37]. These studies indicate that SmyD1 is essential for cardiomyocyte differentiation and cardiac morphogenesis. Myofibril disorganization and disruption of normal myofibrillogenesis are critical factors that can lead to skeletal and cardiac muscle disease [38-40]. In addition to the role SmyD1 plays in cardiac and skeletal muscle development, a more recent study with conditional knockout of SmyD1 during the myoblast stage supports a key function of SmyD1 in myoblast differentiation into myocytes [41].

The expression of SmyD1 in cardiac tissue has been shown to be regulated by transcription factors MEF2C and serum response factors (SRF) [42,43]. In skeletal muscle, SmyD1 expression is regulated by SRF and myogenic transcriptional factors of MyoD and Myogenin [42,43]. Analysis of the subcellular localization of SmyD1 in mouse C2C12 myoblasts and in murine heart tissue shows that SmyD1 is localized in the nucleus [37,44], while immunostaining and SmyD1-GFP fusion proteins indicate SmyD1 being primarily cytosolic in myoblasts and myotubes. Taken together, it is hypothesized that dynamic subcellular localization of SmyD1 may contribute to its biological function in multiple pathways [45]. This hypothesis is supported by the observation that SmyD1 undergoes translocation from the nucleus to the cytosol during myoblast to myotubes differentiation [44].

While the molecular mechanisms of SmyD1 in muscle cells are not fully understood, the emerging data suggest that diverse biological functions of SmyD1 are mediated through multiple pathways. To elucidate the underlying mechanism for the diverse biological functions of SmyD1, many studies have attempted to map the protein-protein interactions involving SmyD1 through yeast two-hybrid screening, Co-IP assays, and affinity capture luminescence [46,47]. This effort has identified over twenty

binding partners. Important members of the SmyD1 interactome include histone proteins such as H3, histone modifying enzymes such as HDACs, molecular chaperone HSP90 and co-chaperone Unc45b, and muscle specific proteins such as myosin and transcription factor skNAC [35,46]. Unc45b is a myosin chaperone and Hsp90 co-chaperone, required for proper folding of myosin and sarcomere/myofibril assembly [48–50]. In vitro studies indicate that methylation of histone H3 on specific lysines by SmyD1 and recruitment of repressors such as class I and class II HDACs may lead to chromatin remodeling and repression of transcription. Alternatively, localization in the cytosol and interaction with myosin, Hsp90 and Unc45b suggest that SmyD1 plays an important role in the cytoplasm by assisting with myosin folding, a critical step in myofibrillogenesis [51].

Like SmyD1, SmyD2 is highly expressed in the heart and skeletal muscle. SmyD2 can also methylate both histone and non-histone targets such Hsp90, p53, and RB. In contrast to SmyD1 which is localized at the M-band region of the sarcomere and binds to myosin, SmyD2 is localized at the I-band region and binds to the sarcomeric spring like protein, titin. This interaction is promoted by methylation of Hsp90 and subsequent binding of the SmyD2-methyl-Hsp90 complex with the tintin N2A-domain. The net result of this interaction is to protect sarcomeric-I region proteins and support myofilament organization [52]. How this underlying biochemistry translates to cardiac physiology is not yet fully defined. For example, SmyD2 knockdown in the zebrafish resulted in severely impaired cardiac function [52]. In contrast to these results, another SmyD2 conditional knockout showed no discernable impact on heart development in mice [53].

SmyD inhibition

The SmyD family offers a potential therapeutic target for cardiovascular and cancer related disease. In particular, over expression of SmyD1 has been shown to be associated with increased risk of heart failure due to repression of genes that are required for the proper functioning of ion channels in the heart [54]. Moreover, SmyD1 over expression was shown to be a key factor in patients with hypoplastic left heart syndrome (HLHS), a disorder characterized by an underdeveloped left ventricle [55]. To date, there are no known selective inhibitors of SmyD1. The development of small molecule selective inhibitors of SmyD1 would not only provide valuable tools to further our understanding of the biological function of SmyD1 and other SmyD family members, but also have the potential of becoming

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‘first in class’ therapeutics to treat a number of cardiovascular conditions. In the case of SmyD2, two small molecule inhibitors were recently identified from high throughput enzymatic screens. Both inhibitors, AZ-505 [56,57] and LLY-507 [39,40] possess unique scaffolds and are potent and selective for SmyD2 (Figure 5). In cell-free assays, AZ-505 and LLY-507 had an IC50 of 120 nM and < 15 nM, respectively. AZ-505 was reported to be competitive with respect to peptide and non-competitive with respect to SAM. LLY-507 was designed to be competitive with the peptide substrate; however, no direct data has confirmed this hypothesis [58]. Significantly, LLY-507 was found to be active in cells with an IC50 of 600 nM at inhibiting monomethylation of p53 K370. LLY-507 also inhibited the proliferation of several esophageal, liver and breast cancer cell lines in a dose-dependent manner [58]. These compounds have not yet been tested in cardiovascular disease models.

NNMT and its role in cardiovascular disease

Nicotinamide N-methyltransferase (NNMT) is a methyltransferase that also utilizes SAM to methylate nicotinamide (Figure 6). NNMT can be detected in many different tissues, although it is predominantly expressed in the liver [59]. It plays a vital role in catabolizing nicotinamide and other pyridine analogues and thus it is involved in the biotransformation of various medicines through methylation [59,60]. Upregulation of NNMT has been reported in various cancers and

neurodegenerative disease [61,62]. More importantly, NNMT was identified as a major determinant of plasma Hcy levels according to a genome-wide linkage analysis. Among 39 studied SAM-dependent methyltransferases, NNMT obtained the highest LOD (logarithm of odds) score of genes involved in Hcy metabolism and implied it has the strongest correlation with Hcy metabolism [63]. In another recent study, significantly higher levels of NNMT mRNA and protein compared to controls were observed in the adipose tissue of Wistar Ottawa Karlsburg W (WOKW) rat’s model, an animal model for metabolic syndrome. Patients suffering from metabolic syndrome have hyperhomocysteinemia and higher risks of cardiovascular disease [62]. Therefore, this result not only indicated there is a correlation between metabolic syndrome and high NNMT expression levels, but also corroborated NNMT’s contribution to plasma Hcy levels. Furthermore, NNMT polymorphism can enhance the risk of congenital heart defects (CHDs) among children who were exposed in utero to certain drugs like antibiotics, anticonvulsants, anti-inflammatory medicines, hormones, and antimycotics. It is hypothesized that NNMT polymorphism causes decreased enzyme activity which can lead to inefficient detoxification of such drugs [64]. Although current studies about the association between NNMT and cardiovascular disease are sparse and preliminary [65], the strong correlation between NNMT activity and Hcy plasma levels, suggest that NNMT is a potential target for cardiovascular diseases.

Figure 5 Known Inhibitors of SmyD2.

Figure 6 Methylation of Nicotinamide by NNMT.

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CONCLUSIONThere is strong evidence that points to the role of the PRMTs

and SmyD family members in cardiovascular biology and disease. We have only just begun our journey to understand the underlying mechanisms by which transmethylation of targeted proteins can regulate enzymatic activity, recruit other proteins, affect chromatin structure, and turn on or off genetic transcription. With nearly one hundred members of protein methyltransferases known, we predict emerging roles in cardiovascular diseases will be discovered for methyltransferases families. For example, α-N-terminal methyltransferase 1 (NTMT1) is a new addition to the methyltransferase family. NTMT1 methylates the N-terminal nitrogen of substrate proteins. Of the substrates identified thus far, NTMT1 is known to methylate myosin light chains 3 and 4, slow cardiac myosin regulatory light chain 2, fast skeletal myosin light chain, and smooth muscle/non-muscle myosin alkali light chain [9,66,67]. Although a direct link has not been established, these target proteins imply a potential involvement of NTMT1 in cardiac and skeletal muscle development and function. These and related questions will be addressed in future studies with� selective NTMT1 inhibitors [67,68].

From the standpoint of developing targeted therapeutics, a conserved SAM binding site imposes a big challenge. However, the substrate specificity and structural diversity of PMTs, offer an opportunity for rational design to achieve selectivity among the various family members [69,70]. Other strategies like bisubstrate analogs that covalently link a peptide substrate and SAM analog can serve as a starting point for specific and potent inhibitor design [67,68,71]. While this goal is still in its early stages, there is a significant effort underway to discover selective methyltransferase inhibitors. As was observed with inhibitors of other critical post-translational modification enzymes such as acetyltransferase and kinase inhibitors, methyltransferase selective inhibitors will prove valuable in dissecting the complex biology of protein methylation and will pave the way for novel therapeutics for heart disease, cancer, and other indications.

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