Protein arginine methyltransferase 6 is a novel substrate of protein arginine methyltransferase 1.

BACKGROUND: Post-translational modifications play key roles in various biological processes. Protein arginine methyltransferases (PRMTs) transfer the methyl group to specific arginine residues. Both PRMT1 and PRMT6 have emerges as crucial factors in the development and progression of multiple cancer types. We posit that PRMT1 and PRMT6 might interplay directly or in-directly in multiple ways accounting for shared disease phenotypes. AIM: To investigate the mechanism of the interaction between PRMT1 and PRMT6. METHODS: Gel electrophoresis autoradiography was performed to test the methyltranferase activity of PRMTs and characterize the kinetics parameters of PRMTs. Liquid chromatography-tandem mass spectrometryanalysis was performed to detect the PRMT6 methylation sites. RESULTS: In this study we investigated the interaction between PRMT1 and PRMT6, and PRMT6 was shown to be a novel substrate of PRMT1. We identified specific arginine residues of PRMT6 that are methylated by PRMT1, with R106 being the major methylation site. Combined biochemical and cellular data showed that PRMT1 downregulates the enzymatic activity of PRMT6 in histone H3 methylation. CONCLUSION: PRMT6 is methylated by PRMT1 and R106 is a major methylation site induced by PRMT1. PRMT1 methylation suppresses the activity of PRMT6.

Chemical probes and methods for the study of protein arginine methylation.

Protein arginine methylation is a widespread post-translational modification (PTM) in eukaryotic cells. This chemical modification in proteins functionally modulates diverse cellular processes from signal transduction, gene expression, and DNA damage repair to RNA splicing. The chemistry of arginine methylation entails the transfer of the methyl group from S-adenosyl-l-methionine (AdoMet, SAM) onto a guanidino nitrogen atom of an arginine residue of a target protein. This reaction is catalyzed by about 10 members of protein arginine methyltransferases (PRMTs). With impacts on a variety of cellular processes, aberrant expression and activity of PRMTs have been shown in many disease conditions. Particularly in oncology, PRMTs are commonly overexpressed in many cancerous tissues and positively correlated with tumor initiation, development and progression. As such, targeting PRMTs is increasingly recognized as an appealing therapeutic strategy for new drug discovery. In the past decade, a great deal of research efforts has been invested in illuminating PRMT functions in diseases and developing chemical probes for the mechanistic study of PRMTs in biological systems. In this review, we provide a brief developmental history of arginine methylation along with some key updates in arginine methylation research, with a particular emphasis on the chemical aspects of arginine methylation. We highlight the research endeavors for the development and application of chemical approaches and chemical tools for the study of functions of PRMTs and arginine methylation in regulating biology and disease.

HBO1 catalyzes lysine benzoylation in mammalian cells.

Lysine benzoylation (Kbz) is a newly discovered protein post-translational modification (PTM). This PTM can be stimulated by benzoate and contributes to gene expression. However, its regulatory enzymes and substrate proteins remain largely unknown, hindering further functional studies. Here we identified and validated the lysine acetyltransferase (KAT) HBO1 as a "writer" of Kbz in mammalian cells. In addition, we report the benzoylome in mammalian cells, identifying 1747 Kbz sites; among them at least 77 are the HBO1-targeted Kbz substrates. Bioinformatics analysis showed that HBO1-targeted Kbz sites were involved in multiple processes, including chromatin remodeling, transcription regulation, immune regulation, and tumor growth. Our results thus identify the regulatory elements of the Kbz pathway and reveal the non-canonical enzymatic activity and functions of HBO1 in cellular physiology.

PRMT1 is an important factor for medulloblastoma cell proliferation and survival.

Aberrant expression of protein arginine methyltransferases (PRMTs) has been implicated in a number of brain tumors, but the role of PRMT1 in medulloblastoma, the most common malignant pediatric brain tumor, remains unexplored. By examining the publicly available databases of pediatric brain tumor collection, we found that PRMT1 was predominantly expressed in medulloblastomas across all the pediatric brain tumors and that the high-level expression of PRMT1 correlated with poor survival of medulloblastoma patients. To determine the role of PRMT1 in medulloblastoma cells, we established an inducible knockdown system and demonstrated that PRMT1 depletion decreased medulloblastoma cell proliferation and induced cell apoptosis. Furthermore, the diamidine compounds, previously shown to exhibit selective PRMT1 inhibition, suppressed medulloblastoma cell viability in a dose-dependent manner. Finally, we observed induction of medulloblastoma cell apoptosis by the potent diamidine compounds at low micromolar concentrations. Together, our results suggest that PRMT1 could be an actionable therapeutic target in medulloblastoma.

Synthesis and Activity of Triazole-Adenosine Analogs as Protein Arginine Methyltransferase 5 Inhibitors.

Protein arginine methyltransferase 5 (PRMT5) is an attractive molecular target in anticancer drug discovery due to its extensive involvement in transcriptional control, RNA processing, and other cellular pathways that are causally related to tumor initiation and progression. In recent years, various compounds have been screened or designed to target either the substrate- or cofactor-binding site of PRMT5. To expand the diversity of chemotypes for inhibitory binding to PRMT5 and other AdoMet-dependent methyltransferases, in this work, we designed a series of triazole-containing adenosine analogs aimed at targeting the cofactor-binding site of PRMT5. Triazole rings have commonly been utilized in drug discovery due to their ease of synthesis and functionalization as bioisosteres of amide bonds. Herein, we utilized the electronic properties of the triazole ring as a novel way to specifically target the cofactor-binding site of PRMT5. A total of about 30 compounds were synthesized using the modular alkyne-azide cycloaddition reaction. Biochemical tests showed that these compounds exhibited inhibitory activity of PRMT5 at varying degrees and several showed single micromolar potency, with clear selectivity for PRMT5 over PRMT1. Docking-based structural analysis showed that the triazole ring plays a key role in binding to the characteristic residue Phe327 in the active pocket of PRMT5, explaining the compounds' selectivity for this type-II enzyme. Overall, this work provides new structure-activity relationship information on the design of AdoMet analogs for selective inhibition of PRMT5. Further structural optimization work will further improve the potency of the top leads.

Methylation of dual-specificity phosphatase 4 controls cell differentiation.

Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity phosphatases (DUSPs), the activities of which are tightly regulated during cell differentiation. Using knockdown screening and single-cell transcriptional analysis, we demonstrate that DUSP4 is the phosphatase that specifically inactivates p38 kinase to promote megakaryocyte (Mk) differentiation. Mechanistically, PRMT1-mediated methylation of DUSP4 triggers its ubiquitinylation by an E3 ligase HUWE1. Interestingly, the mechanistic axis of the DUSP4 degradation and p38 activation is also associated with a transcriptional signature of immune activation in Mk cells. In the context of thrombocytopenia observed in myelodysplastic syndrome (MDS), we demonstrate that high levels of p38 MAPK and PRMT1 are associated with low platelet counts and adverse prognosis, while pharmacological inhibition of p38 MAPK or PRMT1 stimulates megakaryopoiesis. These findings provide mechanistic insights into the role of the PRMT1-DUSP4-p38 axis on Mk differentiation and present a strategy for treatment of thrombocytopenia associated with MDS.

Pharmacophore-based screening of diamidine small molecule inhibitors for protein arginine methyltransferases.

Protein arginine methyltransferases (PRMTs) are essential epigenetic and post-translational regulators in eukaryotic organisms. Dysregulation of PRMTs is intimately related to multiple types of human diseases, particularly cancer. Based on the previously reported PRMT1 inhibitors bearing the diamidine pharmacophore, we performed virtual screening to identify additional amidine-associated structural analogs. Subsequent enzymatic tests and characterization led to the discovery of a top lead K313 (2-(4-((4-carbamimidoylphenyl)amino)phenyl)-1H-indole-6-carboximidamide), which possessed low-micromolar potency with biochemical IC50 of 2.6 µM for human PRMT1. Limited selectivity was observed over some other PRMT isoforms such as CARM1 and PRMT7. Molecular modeling and inhibition pattern studies suggest that K313 is a nonclassic noncompetitive inhibitor to PRMT1. K313 significantly inhibited cell proliferation and reduced the arginine asymmetric dimethylation level in the leukaemia cancer cells.

Metabolic regulation of gene expression by histone lactylation.

The Warburg effect, which originally described increased production of lactate in cancer, is associated with diverse cellular processes such as angiogenesis, hypoxia, polarization of macrophages and activation of T cells. This phenomenon is intimately linked to several diseases including neoplasia, sepsis and autoimmune diseases1,2. Lactate, which is converted from pyruvate in tumour cells, is widely known as an energy source and metabolic by-product. However, its non-metabolic functions in physiology and disease remain unknown. Here we show that lactate-derived lactylation of histone lysine residues serves as an epigenetic modification that directly stimulates gene transcription from chromatin. We identify 28 lactylation sites on core histones in human and mouse cells. Hypoxia and bacterial challenges induce the production of lactate by glycolysis, and this acts as a precursor that stimulates histone lactylation. Using M1 macrophages that have been exposed to bacteria as a model system, we show that histone lactylation has different temporal dynamics from acetylation. In the late phase of M1 macrophage polarization, increased histone lactylation induces homeostatic genes that are involved in wound healing, including Arg1. Collectively, our results suggest that an endogenous 'lactate clock' in bacterially challenged M1 macrophages turns on gene expression to promote homeostasis. Histone lactylation thus represents an opportunity to improve our understanding of the functions of lactate and its role in diverse pathophysiological conditions, including infection and cancer.

Protein arginine methyltransferases: insights into the enzyme structure and mechanism at the atomic level.

Protein arginine methyltransferases (PRMTs) catalyze the methyl transfer to the arginine residues of protein substrates and are classified into three major types based on the final form of the methylated arginine. Recent studies have shown a strong correlation between PRMT expression level and the prognosis of cancer patients. Currently, crystal structures of eight PRMT members have been determined. Kinetic and structural studies have shown that all PRMTs share similar, but unique catalytic and substrate recognition mechanism. In this review, we discuss the structural similarities and differences of different PRMT members, focusing on their overall structure, S-adenosyl-L-methionine-binding pocket, substrate arginine recognition and catalytic mechanisms. Since PRMTs are valuable targets for drug discovery, we also rationally classify the known PRMT inhibitors into five classes and discuss their mechanisms of action at the atomic level.

Downregulation of PRMT1 promotes the senescence and migration of a non-MYCN amplified neuroblastoma SK-N-SH cells.

Protein arginine methyltransferase 1 (PRMT1) catalyzing the formation of asymmetric dimethylarginines has been implicated in cancer development, metastasis, and prognosis. In this study, we investigated the effects of low PRMT1 levels on a non-MYCN amplified neuroblastoma SK-N-SH cell line. Stable PRMT1-knockdown (PRMT1-KD) cells showed reduced growth rates and cell cycle arrest at G2/M. They also exhibited senescent phenotypes and increased p53 expression. p21 and PAI-1, which are two p53 downstream targets critical for senescence, were significantly induced in SK-N-SH cells subjected to either PRMT1-KD or inhibitor treatment. The induction was suppressed by a p53 inhibitor and marginal in a p53-null SK-N-AS cell line, suggesting dependence on p53. In general, the DNA damage and ROS levels of the PRMT1-KD SK-N-SH cells were slightly increased. Their migration activity also increased with the induction of PAI-1. Thus, PRMT1 downregulation released the repression of cellular senescence and migration activity in SK-N-SH cells. These results might partially explain the poor prognostic outcome of low PRMT1 in a non-MYCN-amplified cohort and indicate the multifaceted complexity of PRMT1 as a biological regulator of neuroblastoma.

Defining the epigenetic status of blood cells using a cyanine-based fluorescent probe for PRMT1.

Dynamic regulation of histone modification enzymes such as PRMT1 (protein arginine methyltransferase 1) determines the ordered epigenetic transitions in hematopoiesis. Sorting cells according to the expression levels of histone modification enzymes may further define subpopulations in hematopoietic lineages with unique differentiation potentials that are presently defined by surface markers. We discovered a vital near infrared dye, E84, that fluoresces brightly following binding to PRMT1 and excitation with a red laser. The staining intensity as measured by flow cytometry is correlated with the PRMT1 expression level. Importantly, E84 staining has no apparent negative effect on the proliferation of the labeled cells. Given that long-term hematopoietic stem cells (LT-HSCs) produce low levels of PRMT1, we used E84 to sort LT-HSCs from mouse bone marrow. We found that SLAM (the signalling lymphocyte activation molecule family) marker-positive LT-HSCs were enriched in the E84low cell fraction. We then performed bone marrow transplantations with E84high or E84low Lin-Sca1+Kit+ (LSK) cells and showed that whole blood cell lineages were successfully reconstituted 16 weeks after transplanting 200 E84low LSK cells. Thus, E84 is a useful new tool to probe the role of PRMT1 in hematopoiesis and leukemogenesis. Developing E84 and other small molecules to label histone modification enzymes provides a convenient approach without modifying gene loci to study the interaction between hematopoietic stem/progenitor cell epigenetic status and differentiation state.

Mechanisms and Inhibitors of Histone Arginine Methylation.

Histone methylation plays an important regulatory role in chromatin restructuring and RNA transcription. Arginine methylation that is enzymatically catalyzed by the family of protein arginine methyltransferases (PRMTs) can either activate or repress gene expression depending on cellular contexts. Given the strong correlation of PRMTs with pathophysiology, great interest is seen in understanding molecular mechanisms of PRMTs in diseases and in developing potent PRMT inhibitors. Herein, we reviewed key research advances in the study of biochemical mechanisms of PRMT catalysis and their relevance to cell biology. We highlighted how a random binary, ordered ternary kinetic model for PRMT1 catalysis reconciles the literature reports and endorses a distributive mechanism that the enzyme active site utilizes for multiple turnovers of arginine methylation. We discussed the impacts of histone arginine methylation and its biochemical interplays with other key epigenetic marks. Challenges in developing small-molecule PRMT inhibitors were also discussed.

Lysine benzoylation is a histone mark regulated by SIRT2.

Metabolic regulation of histone marks is associated with diverse biological processes through dynamically modulating chromatin structure and functions. Here we report the identification and characterization of a histone mark, lysine benzoylation (Kbz). Our study identifies 22 Kbz sites on histones from HepG2 and RAW cells. This type of histone mark can be stimulated by sodium benzoate (SB), an FDA-approved drug and a widely used chemical food preservative, via generation of benzoyl CoA. By ChIP-seq and RNA-seq analysis, we demonstrate that histone Kbz marks are associated with gene expression and have physiological relevance distinct from histone acetylation. In addition, we demonstrate that SIRT2, a NAD+-dependent protein deacetylase, removes histone Kbz both in vitro and in vivo. This study therefore reveals a new type of histone marks with potential physiological relevance and identifies possible non-canonical functions of a widely used chemical food preservative.

Detection of PRMT1 inhibitors with stopped flow fluorescence.

Protein arginine methyltransferases (PRMTs) are crucial epigenetic regulators in eukaryotic organisms that serve as histone writers for chromatin remodeling. PRMTs also methylate a variety of non-histone protein substrates to modulate their function and activity. The development of potent PRMT inhibitors has become an emerging and imperative research area in the drug discovery field to provide novel therapeutic agents for treating diseases and as tools to investigate the biological functions of PRMTs. PRMT1 is the major type I enzyme that catalyzes the formation of asymmetric dimethyl arginine, and PRMT1 plays important regulatory roles in signal transduction, transcriptional activation, RNA splicing, and DNA repair. Aberrant expression of PRMT1 is found in many types of cancers, pulmonary diseases, cardiovascular disease, diabetes, and renal diseases. PRMT1 is a highly promising target for therapeutic development. We created a stopped flow fluorescence-based assay for PRMT1 inhibitor detection and characterization that has the advantages of being homogeneous, nonradioactive, and mix-and-measure in nature, allowing for continuous measurement of the methylation reaction and its inhibition. To our knowledge, this is the first continuous assay for PRMT1 reaction detection and inhibitor characterization. The approach is not only capable of quantitatively determining the potency (IC50) of PRMT1 inhibitors but can also distinguish cofactor-competitive inhibitors, substrate-competitive inhibitors, and mixed-type inhibitors.

Chemical Biology Approaches for Investigating the Functions of Lysine Acetyltransferases.

The side-chain acetylation of lysine residues in histones and non-histone proteins catalyzed by lysine acetyltransferases (KATs) represents a widespread posttranslational modification (PTM) in the eukaryotic cells. Lysine acetylation plays regulatory roles in major cellular pathways inside and outside the nucleus. In particular, KAT-mediated histone acetylation has an effect on all DNA-templated epigenetic processes. Aberrant expression and activation of KATs are commonly observed in human diseases, especially cancer. In recent years, the study of KAT functions in biology and disease has greatly benefited from chemical biology tools and strategies. In this Review, we present the past and current accomplishments in the design of chemical biology approaches for the interrogation of KAT activity and function. These methods and probes are classified according to their mechanisms of action and respective applications, with both strengths and limitations discussed.

Blocking Myristoylation of Src Inhibits Its Kinase Activity and Suppresses Prostate Cancer Progression.

Protein N-myristoylation enables localization to membranes and helps maintain protein conformation and function. N-myristoyltransferases (NMT) catalyze co- or posttranslational myristoylation of Src family kinases and other oncogenic proteins, thereby regulating their function. In this study, we provide genetic and pharmacologic evidence that inhibiting the N-myristoyltransferase NMT1 suppresses cell-cycle progression, proliferation, and malignant growth of prostate cancer cells. Loss of myristoylation abolished the tumorigenic potential of Src and its synergy with androgen receptor in mediating tumor invasion. We identified the myristoyl-CoA analogue B13 as a small-molecule inhibitor of NMT1 enzymatic activity. B13 exposure blocked Src myristoylation and Src localization to the cytoplasmic membrane, attenuating Src-mediated oncogenic signaling. B13 exerted its anti-invasive and antitumor effects against prostate cancer cells, with minimal toxic side-effects in vivo Structural optimization based on structure-activity relationships enabled the chemical synthesis of LCL204, with enhanced inhibitory potency against NMT1. Collectively, our results offer a preclinical proof of concept for the use of protein myristoylation inhibitors as a strategy to block prostate cancer progression. Cancer Res; 77(24); 6950-62. ©2017 AACR.

Using Chemistry to Target Neuroblastoma.

Neuroblastoma is a cancer of the neural crest almost exclusively seen in childhood. While children with single, small primary tumors are often cured with surgery alone, the 65% of children with neuroblastoma whose disease has metastasized have less than a 50% chance of surviving five years after diagnosis. Innovative pharmacological strategies are critically needed for these children. Efforts to identify novel targets that afford ablation of neuroblastoma with minimal toxicity to normal tissues are underway. Developing approaches to neuroblastoma include those that target the catecholamine transporter, ubiquitin E3 ligase, the ganglioside GD2, the retinoic acid receptor, the protein kinases ALK and Aurora, and protein arginine N-methyltransferases. Here, as examples of the use of chemistry to combat neuroblastoma, we describe targeting of the protein arginine N-methyltransferases and their role in prolonging the half-life of the neuroblastoma oncoprotein N-Myc, redox signaling in neuroblastoma, and developmentally regulated proteins expressed in primitive neuroblastoma cells but not in mature neural crest elements.

Transient Kinetics Define a Complete Kinetic Model for Protein Arginine Methyltransferase 1.

Protein arginine methyltransferases (PRMTs) are the enzymes responsible for posttranslational methylation of protein arginine residues in eukaryotic cells, particularly within the histone tails. A detailed mechanistic model of PRMT-catalyzed methylation is currently lacking, but it is essential for understanding the functions of PRMTs in various cellular pathways and for efficient design of PRMT inhibitors as potential treatments for a range of human diseases. In this work, we used stopped-flow fluorescence in combination with global kinetic simulation to dissect the transient kinetics of PRMT1, the predominant type I arginine methyltransferase. Several important mechanistic insights were revealed. The cofactor and the peptide substrate bound to PRMT1 in a random manner and then followed a kinetically preferred pathway to generate the catalytic enzyme-cofactor-substrate ternary complex. Product release proceeded in an ordered fashion, with peptide dissociation followed by release of the byproduct S-adenosylhomocysteine. Importantly, the dissociation rate of the monomethylated intermediate from the ternary complex was much faster than the methyl transfer. Such a result provided direct evidence for distributive arginine dimethylation, which means the monomethylated substrate has to be released to solution and rebind with PRMT1 before it undergoes further methylation. In addition, cofactor binding involved a conformational transition, likely an open-to-closed conversion of the active site pocket. Further, the histone H4 peptide bound to the two active sites of the PRMT1 homodimer with differential affinities, suggesting a negative cooperativity mechanism of substrate binding. These findings provide a new mechanistic understanding of how PRMTs interact with their substrates and transfer methyl groups.

Small Molecule Inhibitors of Protein Arginine Methyltransferases.

INTRODUCTION: Arginine methylation is an abundant posttranslational modification occurring in mammalian cells and catalyzed by protein arginine methyltransferases (PRMTs). Misregulation and aberrant expression of PRMTs are associated with various disease states, notably cancer. PRMTs are prominent therapeutic targets in drug discovery. AREAS COVERED: The authors provide an updated review of the research on the development of chemical modulators for PRMTs. Great efforts are seen in screening and designing potent and selective PRMT inhibitors, and a number of micromolar and submicromolar inhibitors have been obtained for key PRMT enzymes such as PRMT1, CARM1, and PRMT5. The authors provide a focus on their chemical structures, mechanism of action, and pharmacological activities. Pros and cons of each type of inhibitors are also discussed. EXPERT OPINION: Several key challenging issues exist in PRMT inhibitor discovery. Structural mechanisms of many PRMT inhibitors remain unclear. There lacks consistency in potency data due to divergence of assay methods and conditions. Physiologically relevant cellular assays are warranted. Substantial engagements are needed to investigate pharmacodynamics and pharmacokinetics of the new PRMT inhibitors in pertinent disease models. Discovery and evaluation of potent, isoform-selective, cell-permeable and in vivo-active PRMT modulators will continue to be an active arena of research in years ahead.

Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing.

RBM15, an RNA binding protein, determines cell-fate specification of many tissues including blood. We demonstrate that RBM15 is methylated by protein arginine methyltransferase 1 (PRMT1) at residue R578, leading to its degradation via ubiquitylation by an E3 ligase (CNOT4). Overexpression of PRMT1 in acute megakaryocytic leukemia cell lines blocks megakaryocyte terminal differentiation by downregulation of RBM15 protein level. Restoring RBM15 protein level rescues megakaryocyte terminal differentiation blocked by PRMT1 overexpression. At the molecular level, RBM15 binds to pre-messenger RNA intronic regions of genes important for megakaryopoiesis such as GATA1, RUNX1, TAL1 and c-MPL. Furthermore, preferential binding of RBM15 to specific intronic regions recruits the splicing factor SF3B1 to the same sites for alternative splicing. Therefore, PRMT1 regulates alternative RNA splicing via reducing RBM15 protein concentration. Targeting PRMT1 may be a curative therapy to restore megakaryocyte differentiation for acute megakaryocytic leukemia.