A Potent, Selective, and Cell-Active Inhibitor of Human Type I Protein Arginine Methyltransferases
Mohammad S. EramYudao ShenMagdalena M. SzewczykHong WuGuillermo SenisterraFengling LiKyle V. ButlerH. Ümit KanıskanBrandon A. SpeedCarlo dela SeñaAiping DongHong ZengMatthieu SchapiraPeter J. BrownC.H. ArrowsmithDalia Baršytė-LovejoyJing LiuMasoud VedadiJian Jin
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Protein arginine methyltransferases (PRMTs) play a crucial role in a variety of biological processes. Overexpression of PRMTs has been implicated in various human diseases including cancer. Consequently, selective small-molecule inhibitors of PRMTs have been pursued by both academia and the pharmaceutical industry as chemical tools for testing biological and therapeutic hypotheses. PRMTs are divided into three categories: type I PRMTs which catalyze mono- and asymmetric dimethylation of arginine residues, type II PRMTs which catalyze mono- and symmetric dimethylation of arginine residues, and type III PRMT which catalyzes only monomethylation of arginine residues. Here, we report the discovery of a potent, selective, and cell-active inhibitor of human type I PRMTs, MS023, and characterization of this inhibitor in a battery of biochemical, biophysical, and cellular assays. MS023 displayed high potency for type I PRMTs including PRMT1, -3, -4, -6, and -8 but was completely inactive against type II and type III PRMTs, protein lysine methyltransferases and DNA methyltransferases. A crystal structure of PRMT6 in complex with MS023 revealed that MS023 binds the substrate binding site. MS023 potently decreased cellular levels of histone arginine asymmetric dimethylation. It also reduced global levels of arginine asymmetric dimethylation and concurrently increased levels of arginine monomethylation and symmetric dimethylation in cells. We also developed MS094, a close analog of MS023, which was inactive in biochemical and cellular assays, as a negative control for chemical biology studies. MS023 and MS094 are useful chemical tools for investigating the role of type I PRMTs in health and disease.Keywords:
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Abstract Protein Arginine (R) methylation is the most common post-translational methylation in mammalian cells. Protein Arginine Methyltransferases (PRMT) 1 and 5 dimethylate their substrates on R residues, asymmetrically and symmetrically, respectively. They are ubiquitously expressed and play fundamental roles in tumour malignancies, including glioblastoma multiforme (GBM) which presents largely deregulated Myc activity. Previously, we demonstrated that PRMT5 associates with Myc in GBM cells, modulating, at least in part, its transcriptional properties. Here we show that Myc/PRMT5 protein complex includes PRMT1, in both HEK293T and glioblastoma stem cells (GSCs). We demonstrate that Myc is both asymmetrically and symmetrically dimethylated by PRMT1 and PRMT5, respectively, and that these modifications differentially regulate its stability. Moreover, we show that the ratio between symmetrically and asymmetrically dimethylated Myc changes in GSCs grown in stem versus differentiating conditions. Finally, both PRMT1 and PRMT5 activity modulate Myc binding at its specific target promoters. To our knowledge, this is the first work reporting R asymmetrical and symmetrical dimethylation as novel Myc post-translational modifications, with different functional properties. This opens a completely unexplored field of investigation in Myc biology and suggests symmetrically dimethylated Myc species as novel diagnostic and prognostic markers and druggable therapeutic targets for GBM.
HEK 293 cells
Proto-Oncogene Proteins c-myc
Protein methylation
Temozolomide
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Autophagy is a self-degradative process that is important for protecting cells and organisms against stress and plays a protective role in the heart. However, the underlying mechanism by which autophagy protects the heart against stress is poorly understood. ATG proteins are required for autophagy and modulated by post-translational modifications (PTMs) such as phosphorylation, ubiquitination, and acetylation. The level of arginine methylation, another form of PTM, is altered in the heart in response to stress. Our hypothesis is that arginine methylation plays an important role in the regulation of autophagy in the heart during stress. To this end, we examined the level of arginine methylation in response to energy stress in vivo . We observed a time-dependent increase in arginine methylation in the mouse heart under starvation conditions for 24 to 48 hours. Arginine methylation was increased (2.32-fold, p<0.01) after 48 hours of fasting compared to in those without fasting. Expression of protein arginine methyltransferase 5 (PRMT5) was increased at both the mRNA and protein level (1.6- and 1.72-fold, p<0.05) in the mouse heart after 48 hours of starvation. In order to investigate the role of endogenous PRMT5, we knocked down PRMT5 with adenovirus harboring sh-RNA. LC3-II, a marker of autophagy activity, was increased during glucose deprivation in sh-scramble-treated cardiomyocytes (CMs) (2.14-fold, p<0.01), but not in sh-PRMT5-treated CMs. Arginine methylation was reduced in response to glucose deprivation in CMs when PRMT5 was downregulated. Mice were also injected intraperitoneally with a PRMT5-specific inhibitor (EPZ015666, 2 mg/kg) and hearts were harvested after 48 hours of starvation. Both arginine methylation levels and autophagy activity in the heart were decreased in the presence of EPZ015666 compared to in non-treated mice. Increased arginine methylation was also observed in the mouse heart in response to 1 hour of myocardial ischemia (1.87-fold, p<0.01), which was accompanied by upregulation of PRMT5 (1.54-fold, p<0.05). Taken together, these data suggest that arginine methylation mediated through PRMT5 may play a key role in mediating autophagy during energy stress conditions such as starvation and ischemia.
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Abstract 1. The arginine and lysine requirements for maximum growth of the 3‐week‐old turkey, determined in a factorial manner, were 1.75% arginine and 1.55% lysine. 2. It is demonstrated that the arginine required to support a growth rate of about 20 g/d is similar in turkeys and chicks, suggesting that the efficiency with which dietary arginine was utilised for growth is similar in both species.
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Histone lysine methyltransferases (KMTs) play an important role in epigenetic gene regulation and have emerged as promising targets for drug discovery. However, the scope and limitation of KMT catalysis on substrates possessing substituted lysine side chains remain insufficiently explored. Here, we identify new unnatural lysine analogues as substrates for human methyltransferases SETD7, SETD8, G9a and GLP. Two synthetic amino acids that possess a subtle modification on the lysine side chain, namely oxygen at the γ position (KO, oxalysine) and nitrogen at the γ position (KN, azalysine) were incorporated into histone peptides and tested as KMTs substrates. Our results demonstrate that these lysine analogues are mono-, di-, and trimethylated to a different extent by trimethyltransferases G9a and GLP. In contrast to monomethyltransferase SETD7, SETD8 exhibits high specificity for both lysine analogues. These findings are important to understand the substrate scope of KMTs and to develop new chemical probes for biomedical applications.
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Both lysine and arginine methyltransferases are thought to be promising therapeutic targets for malignant tumors, yet how these methyltransferases function in malignant tumors, especially hepatocellular carcinoma (HCC), has not been fully elucidated. Here, we reported that SMYD4, a lysine methyltransferase, acts as an oncogene in HCC. SMYD4 was highly upregulated in HCC and promoted HCC cell proliferation and metastasis. Mechanistically, PRMT5, a well-known arginine methyltransferase, was identified as a SMYD4-binding protein. SMYD4 monomethylated PRMT5 and enhanced the interaction between PRMT5 and MEP50, thereby promoting the symmetrical dimethylation of H3R2 and H4R3 on the PRMT5 target gene promoter and subsequently activating DVL3 expression and inhibiting expression of E-cadherin, RBL2, and miR-29b-1-5p. Moreover, miR-29b-1-5p was found to inversely regulate SMYD4 expression in HCC cells, thus forming a positive feedback loop. Furthermore, we found that the oncogenic effect of SMYD4 could be effectively suppressed by PRMT5 inhibitor in vitro and in vivo. Clinically, high coexpression of SMYD4 and PRMT5 was associated with poor prognosis of HCC patients. In summary, our study provides a model of crosstalk between lysine and arginine methyltransferases in HCC and highlights the SMYD4-PRMT5 axis as a potential therapeutic target for the treatment of HCC.
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The role of arginine methylation in Drosophila melanogaster is unknown. We identified a family of nine PRMTs (protein arginine methyltransferases) by sequence homology with mammalian arginine methyltransferases, which we have named DART1 to DART9 ( Drosophila arginine methyltransferases 1-9). In keeping with the mammalian PRMT nomenclature, DART1, DART4, DART5 and DART7 are the putative homologues of PRMT1, PRMT4, PRMT5 and PRMT7. Other DART family members have a closer resemblance to PRMT1, but do not have identifiable homologues. All nine genes are expressed in Drosophila at various developmental stages. DART1 and DART4 have arginine methyltransferase activity towards substrates, including histones and RNA-binding proteins. Amino acid analysis of the methylated arginine residues confirmed that both DART1 and DART4 catalyse the formation of asymmetrical dimethylated arginine residues and they are type I arginine methyltransferases. The presence of PRMTs in D. melanogaster suggest that flies are a suitable genetic system to study arginine methylation.
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Protein Arginine (R) methylation is the most common post-translational methylation in mammalian cells. Protein Arginine Methyltransferases (PRMT) 1 and 5 dimethylate their substrates on R residues, asymmetrically and symmetrically, respectively. They are ubiquitously expressed and play fundamental roles in tumour malignancies, including glioblastoma multiforme (GBM) which presents largely deregulated Myc activity. Previously, we demonstrated that PRMT5 associates with Myc in GBM cells, modulating, at least in part, its transcriptional properties. Here we show that Myc/PRMT5 protein complex includes PRMT1, in both HEK293T and glioblastoma stem cells (GSCs). We demonstrate that Myc is both asymmetrically and symmetrically dimethylated by PRMT1 and PRMT5, respectively, and that these modifications differentially regulate its stability. Moreover, we show that the ratio between symmetrically and asymmetrically dimethylated Myc changes in GSCs grown in stem versus differentiating conditions. Finally, both PRMT1 and PRMT5 activity modulate Myc binding at its specific target promoters. To our knowledge, this is the first work reporting R asymmetrical and symmetrical dimethylation as novel Myc post-translational modifications, with different functional properties. This opens a completely unexplored field of investigation in Myc biology and suggests symmetrically dimethylated Myc species as novel diagnostic and prognostic markers and druggable therapeutic targets for GBM.
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Proto-Oncogene Proteins c-myc
Temozolomide
Druggability
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A crystalline amino acid diet was modified to be limiting in arginine to study the effects of excess dietary lysine on the utilization of and requirement for arginine by the chick. At the same level of feed and arginine consumption, chicks fed a diet containing 0.95% lysine gained faster than those fed a diet containing 1.95% lysine. Chicks responded to supplemental arginine when fed either level of lysine, but the growth response was considerably greater when chicks were fed ad libitum than when they were equal fed. The results indicate that excess dietary lysine not only reduces feed intake but also impairs arginine utilization. These effects of excess lysine are reflected by a 51% increase in the dietary arginine requirement of chicks fed approximately 1% excess dietary lysine. The magnitude of increase in the arginine requirement is close to that predicted from slope-ratio assays reported in a previous study.
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