Protein arginine methyltransferase 1, a major regulator of biological processes.

Protein arginine methyltransferase 1 (PRMT1) is a major type I arginine methyltransferase that catalyzes the formation of monomethyl and asymmetric dimethylarginine in protein substrates. It was first identified to asymmetrically methylate histone H4 at the third arginine residue forming the H4R3me2a active histone mark. However, several protein substrates are now identified as being methylated by PRMT1. As a result of its association with diverse classes of substrates, PRMT1 regulates several biological processes like chromatin dynamics, transcription, RNA processing, and signal transduction. The review provides an overview of PRMT1 structure, biochemical features, specificity, regulation, and role in cellular functions. We discuss the genomic distribution of PRMT1 and its association with tRNA genes. Further, we explore the different substrates of PRMT1 involved in splicing. In the end, we discuss the proteins that interact with PRMT1 and their downstream effects in diseased states.

Discovery of 2,4-diphenyl-substituted thiazole derivatives as PRMT1 inhibitors and investigation of their anti-cervical cancer effects.

Cervical cancer is one of the most common cancers that affects middle-aged women and the discovery of new drugs to aid clinical management is needed. As an important member of the protein arginine methyltransferases (PRMTs) family, PRMT1 catalyzes the methylation of protein arginine, which can influence multiple biological processes of cancer cells, such as activating epithelial-mesenchymal transformation (EMT) and acquiring resistance to apoptosis. Therefore, PRMT1 can be considered as a potential drug target for cervical cancer. In the current study, a new sub-binding pocket was discovered by molecular modeling, and by introducing a third substitute on the thiazole group to occupy this pocket, a series of compounds were designed and synthesized as potential PRMT1 inhibitors. Of these, two compounds (ZJG51 and ZJG58) exhibited significant inhibitory activities against PRMT1 without significantly inhibiting PRMT5. Both ZJG51 and ZJG58 displayed potent inhibitory effects on the proliferation of four cancer-derived cell lines and ZJG51 exerted relative selectivity against the cervical cancer cell line, HeLa. Further studies showed that ZJG51 inhibited migration and induce the apoptosis of HeLa cells. Mechanistically, ZJG51 significantly regulated PRMT1 related proteins, and indicated that the induction of apoptosis and inhibition of migration by ZJG51 may involve the activation of Caspase 9 and the inhibition of EMT, respectively. Molecular dynamic simulation and free energy calculation showed that ZJG51 can bind to PRMT1 stably and the binding mode was predicted. These data indicated that introducing the third substitute on the five-membered ring could be a future direction for structure-based optimization of PRMT1 inhibitors, and ZJG51 could be an important lead compound to inform the design of more potent inhibitors.

Research Progress on Small-molecule Inhibitors of Protein Arginine Methyltransferase 5 (PRMT5) for Treating Cancer.

BACKGROUND: The protein arginine methyltransferase family includes nine members, with PRMT5 being the major type II arginine methyltransferase. PRMT5 is upregulated in a variety of tumors and promotes tumorigenesis and tumor cell proliferation and metastasis, making it a potential tumor therapy target. Recently, PRMT5 inhibitor research and development have become hotspots in the tumor therapy field. METHODS: We classified and summarized PRMT5 inhibitors according to different binding mechanisms. We mainly analyzed the structure, biological activity, and binding interactions of PRMT5 inhibitors with the PRMT5 enzyme. RESULTS: At present, many PRMT5 inhibitors with various mechanisms of action have been reported, including substrate-competitive inhibitors, SAM-competitive inhibitors, dual substrate-/SAMcompetitive inhibitors, allosteric inhibitors, PRMT5 degraders, MTA-cooperative PRMT5 inhibitors and PPI inhibitors. CONCLUSION: These inhibitors are beneficial to the treatment of tumors. Some drugs are being used in clinical trials. PRMT5 inhibitors have broad application prospects in tumor therapy.

Unraveling the complexity of histone-arginine methyltransferase CARM1 in cancer: From underlying mechanisms to targeted therapeutics.

Coactivator-associated arginine methyltransferase 1 (CARM1), a type I protein arginine methyltransferase (PRMT), has been widely reported to catalyze arginine methylation of histone and non-histone substrates, which is closely associated with the occurrence and progression of cancer. Recently, accumulating studies have demonstrated the oncogenic role of CARM1 in many types of human cancers. More importantly, CARM1 has been emerging as an attractive therapeutic target for discovery of new candidate anti-tumor drugs. Therefore, in this review, we summarize the molecular structure of CARM1 and its key regulatory pathways, as well as further discuss the rapid progress in better understanding of the oncogenic functions of CARM1. Moreover, we further demonstrate several representative targeted CARM1 inhibitors, especially focusing on demonstrating their designing strategies and potential therapeutic applications. Together, these inspiring findings would shed new light on elucidating the underlying mechanisms of CARM1 and provide a clue on discovery of more potent and selective CARM1 inhibitors for the future targeted cancer therapy.

Overexpression of Leishmania major protein arginine methyltransferase 6 reduces parasite infectivity in vivo.

Arginine methylation is catalysed by Protein Arginine Methyltransferases (PRMTs) and can affect how a target protein functions and how it interacts with other macromolecules, which in turn impacts on cell metabolism and gene expression control. Leishmania parasites express five different PRMTs, and although the presence of each individual PRMT is not essential per se, the imbalanced activity of these PRMTs can impact the virulence of Leishmania parasites in vitro and in vivo. Here we created a Leishmania major cell line overexpressing PRMT6 and show that similar to what was observed for the T. brucei homologous enzyme, L. major PRMT6 probably has a narrow substrate range. However, its overexpression notably impairs the infection in mice, with a mild reduction in the number of viable parasites in the lymph nodes. Our results indicate that arginine methylation by LmjPRMT6 plays a significant role in the adaptation of the parasite to the environment found in the mammalian host.

Ribosomal Protein uS5 and Friends: Protein-Protein Interactions Involved in Ribosome Assembly and Beyond.

Ribosomal proteins are fundamental components of the ribosomes in all living cells. The ribosomal protein uS5 (Rps2) is a stable component of the small ribosomal subunit within all three domains of life. In addition to its interactions with proximal ribosomal proteins and rRNA inside the ribosome, uS5 has a surprisingly complex network of evolutionarily conserved non-ribosome-associated proteins. In this review, we focus on a set of four conserved uS5-associated proteins: the protein arginine methyltransferase 3 (PRMT3), the programmed cell death 2 (PDCD2) and its PDCD2-like (PDCD2L) paralog, and the zinc finger protein, ZNF277. We discuss recent work that presents PDCD2 and homologs as a dedicated uS5 chaperone and PDCD2L as a potential adaptor protein for the nuclear export of pre-40S subunits. Although the functional significance of the PRMT3-uS5 and ZNF277-uS5 interactions remain elusive, we reflect on the potential roles of uS5 arginine methylation by PRMT3 and on data indicating that ZNF277 and PRMT3 compete for uS5 binding. Together, these discussions highlight the complex and conserved regulatory network responsible for monitoring the availability and the folding of uS5 for the formation of 40S ribosomal subunits and/or the role of uS5 in potential extra-ribosomal functions.

Effectors and effects of arginine methylation.

Arginine methylation is a ubiquitous and relatively stable post-translational modification (PTM) that occurs in three types: monomethylarginine (MMA), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). Methylarginine marks are catalyzed by members of the protein arginine methyltransferases (PRMTs) family of enzymes. Substrates for arginine methylation are found in most cellular compartments, with RNA-binding proteins forming the majority of PRMT targets. Arginine methylation often occurs in intrinsically disordered regions of proteins, which impacts biological processes like protein-protein interactions and phase separation, to modulate gene transcription, mRNA splicing and signal transduction. With regards to protein-protein interactions, the major 'readers' of methylarginine marks are Tudor domain-containing proteins, although additional domain types and unique protein folds have also recently been identified as methylarginine readers. Here, we will assess the current 'state-of-the-art' in the arginine methylation reader field. We will focus on the biological functions of the Tudor domain-containing methylarginine readers and address other domains and complexes that sense methylarginine marks.

mNeuCode Empowers Targeted Proteome Analysis of Arginine Dimethylation.

Characterization of protein arginine dimethylation presents significant challenges due to its occurrence at the substoichiometric level. To enable a targeted MS/MS analysis of these dimethylation sites, we developed the mNeuCode (methyl-neutron-coding) tag by metabolically labeling methylarginine with stable isotopes during cell culture, which generated a diagnostic peak containing the NeuCode isotopologue signature in a high-resolution MS scan. A software tool, termed NeuCodeFinder, was developed for screening the NeuCode signatures in mass spectra. Therefore, a targeted MS/MS workflow was established for proteome-wide discovery of arginine dimethylation. The efficacy and utility were demonstrated by identifying 176 arginine dimethylation sites residing on 70 proteins in HeLa cells. Among them, 38% of the sites and 29% of the dimethylated proteins are novel, including five novel arginine dimethylation sites on the protein FAM98A, which is a substrate of protein arginine methyltransferase 1 (PRMT1). Our results show that deletion of FAM98A in HeLa cells suppressed cell migration, and importantly, dimethylation-deficient mutation suppressed this process as well. Therefore, the PRMT1-FAM98A pathway mediates cell migration possibly through dimethylation of these newly identified sites of FAM98A. Our study might drive the methodological shift from shotgun-based to targeted proteome analysis for interrogation of the substoichiometric biomolecules by using NeuCode-enabled techniques.

Conjugates of adenosine mimetics and arginine-rich peptides serve as inhibitors and fluorescent probes but not as long-lifetime photoluminescent probes for protein arginine methyltransferases.

The conjugates of an adenosine mimetic and oligo-l-arginine or oligo-d-arginine (ARCs) were initially designed in our research group as inhibitors and photoluminescent probes targeting basophilic protein kinases. Here, we explored a panel of ARCs and their fluorescent derivatives in biochemical assays with members of the protein arginine methyltransferase (PRMT) family, focusing specifically on PRMT1. In the binding/displacement assay with detection of fluorescence anisotropy, we found that ARCs and arginine-rich peptides could serve as high-affinity ligands for PRMT1, whereas the equilibrium dissociation constant values depended dramatically on the number of arginine residues within the compounds. The fluorescently labeled probe ARC-1081 was displaced from its complex with PRMT1 by both S-adenosyl-l-methionine (SAM) and S-adenosyl-l-homocysteine (SAH), indicating binding of the adenosine mimetic of ARCs to the SAM/SAH-binding site within PRMT1. The ARCs that had previously shown microsecond-lifetime photoluminescence in complex with protein kinases did not feature such property in complex with PRMT1, demonstrating the selectivity of the time-resolved readout format. When tested against a panel of PRMT family members in single-dose inhibition experiments, a micromolar concentration of ARC-902 was required for the inhibition of PRMT1 and PRMT7. Overall, our results suggest that the compounds containing multiple arginine residues (including the well-known cell-penetrating peptides) are likely to inhibit PRMT and thus interfere with the epigenetic modification status in complex biological systems, which should be taken into consideration during interpretation of the experimental data.

Identification of PRMT5 inhibitors with novel scaffold structures through virtual screening and biological evaluations.

Protein arginine methyltransferase 5 (PRMT5), an important member in PRMT family, has been validated as a promising anticancer target. In this study, through the combination of virtual screening and biological experiments, we have identified two PRMT5 inhibitors with novel scaffold structures. Among them, compound Y2431 showed moderate activity with IC50 value of 10.09 µM and displayed good selectivity against other methyltransferases. The molecular docking analysis and molecular dynamics (MD) simulations suggested that the compound occupied the substrate-arginine binding site. Furthermore, Y2431 exhibited anti-proliferative activity to leukemia cells by inducing cell cycle arrest. Overall, the hit compound could provide a novel scaffold for further optimization of small-molecule PRMT5 inhibitors.

Design and synthesis of unprecedented 9- and 10-membered cyclonucleosides with PRMT5 inhibitory activity.

Synthesis of medium-sized rings is known to be challenging due to high transannular strain especially for 9- and 10-membered rings. Herein we report design and synthesis of unprecedented 9- and 10-membered purine 8,5'-cyclonucleosides as the first cyclonucleoside PRMT5 inhibitors. The cocrystal structure of PRMT5:MEP50 in complex with the synthesized 9-membered cyclonucleoside 1 revealed its binding mode in the SAM binding pocket of PRMT5.

Functional Study of Leishmania braziliensis Protein Arginine Methyltransferases (PRMTs) Reveals That PRMT1 and PRMT5 Are Required for Macrophage Infection.

In trypanosomatids, regulation of gene expression occurs mainly at the posttranscriptional level, and RNA-binding proteins (RBPs) are key players in determining the fates of transcripts. RBPs are targets of protein arginine methyltransferases (PRMTs), which posttranslationally regulate the RNA-binding capacity and other RBP interactions by transferring methyl groups to arginine residues (R-methylation). Herein, we functionally characterized the five predicted PRMTs in Leishmania braziliensis by gene knockout and endogenous protein HA tagging using CRISPR/Cas9 gene editing. We report that R-methylation profiles vary among Leishmania species and across L. braziliensis lifecycle stages, with the peak PRMT expression occurring in promastigotes. A list of PRMT-interacting proteins was obtained in a single coimmunoprecipitation assay using HA-tagged PRMTs, suggesting a network of putative targets of PRMTs and cooperation between the R-methylation writers. Knockout of each L. braziliensis PRMT led to significant changes in global arginine methylation patterns without affecting cell viability. Deletion of either PRMT1 or PRMT3 disrupted most type I PRMT activity, resulting in a global increase in monomethyl arginine levels. Finally, we demonstrate that L. braziliensis PRMT1 and PRMT5 are required for efficient macrophage infection in vitro, and for axenic amastigote proliferation. The results indicate that R-methylation is modulated across lifecycle stages in L. braziliensis and show possible functional overlap and cooperation among the different PRMTs in targeting proteins. Overall, our data suggest important regulatory roles of these proteins throughout the L. braziliensis life cycle, showing that arginine methylation is important for parasite-host cell interactions.

Identification, Synthesis, and Biological Evaluations of Potent Inhibitors Targeting Type I Protein Arginine Methyltransferases.

CARM1 (coactivator-associated arginine methyltransferase 1), which belongs to type I PRMTs (protein arginine methyltransferases), is a potential therapeutic target for treatment of multiple cancers. In this study, we first identified several hit compounds against CARM1 by structure-based virtual screening (IC50 = 35.51 ± 6.68 to 68.70 ± 8.12 µM) and then carried out chemical structural optimizations, leading to six compounds with significantly improved activities targeting CARM1 (IC50 = 18 ± 2 to 107 ± 6 nM). As a compound with an ethylenediamino motif, the most potent inhibitor, ZL-28-6, also exhibited potent inhibition against other type I PRMTs. Compared to the type I PRMT inhibitor from our previous work (DCPR049_12), ZL-28-6 showed increased potency against CARM1 and decreased activity against other type I PRMTs. Moreover, ZL-28-6 showed better antiproliferation activities toward a series of solid tumor cells than DCPR049_12, indicating its broad spectrum of anticancer activity. In addition, cellular thermal shift and Western blot assays validated that ZL-28-6 could target CARM1 in cells. Taken together, the inhibitor we identified could serve as a potent probe for studying CARM1's biological functions and shed light on the future design of novel CARM1 inhibitors with stronger activities and selectivities.

Fascinating Transformation of SAM-Competitive Protein Methyltransferase Inhibitors from Nucleoside Analogues to Non-Nucleoside Analogues.

The abnormal expression of protein methyltransferase (PMT) has been linked with many diseases such as diabetes, neurological disorders, and cancer. S-Adenyl-l-methionine (SAM) is a universal methyl donor and gets converted to S-adenyl-l-homocysteine (SAH), an endogenous competitive inhibitor of SAM. Initially developed SAM/SAH mimetic nucleoside analogues were pan methyltransferase inhibitors. The gradual understanding achieved through ligand-receptor interaction paved the way for various rational approaches of drug design leading to potent and selective nucleoside inhibitors. The present perspective is based on the systematic evolution of selective SAM-competitive heterocyclic non-nucleoside inhibitors from nucleoside inhibitors. This fascinating transition has resolved several issues inherent to nucleoside analogues such as poor pharmacokinetics leading to poor in vivo efficacy. The perspective has brought together various concepts and strategies of drug design that contributed to this rational transition. We firmly believe that the strategies described herein will serve as a template for the future development of drugs in general.

Cellular pathways influenced by protein arginine methylation: Implications for cancer.

Arginine methylation is an influential post-translational modification occurring on histones, RNA binding proteins, and many other cellular proteins, affecting their function by altering their protein-protein and protein-nucleic acid interactions. Recently, a wealth of information has been gathered, implicating protein arginine methyltransferases (PRMTs), enzymes that deposit arginine methylation, in transcription, pre-mRNA splicing, DNA damage signaling, and immune signaling with major implications for cancer therapy, especially immunotherapy. This review summarizes this recent progress and the current state of PRMT inhibitors, some in clinical trials, as promising drug targets for cancer.

Naturally occurring cancer-associated mutations disrupt oligomerization and activity of protein arginine methyltransferase 1 (PRMT1).

Protein arginine methylation is a posttranslational modification catalyzed by the protein arginine methyltransferase (PRMT) enzyme family. Dysregulated protein arginine methylation is linked to cancer and a variety of other human diseases. PRMT1 is the predominant PRMT isoform in mammalian cells and acts in pathways regulating transcription, DNA repair, apoptosis, and cell proliferation. PRMT1 dimer formation, which is required for methyltransferase activity, is mediated by interactions between a structure called the dimerization arm on one monomer and a surface of the Rossman Fold of the other monomer. Given the link between PRMT1 dysregulation and disease and the link between PRMT1 dimerization and activity, we searched the Catalogue of Somatic Mutations in Cancer (COSMIC) database to identify potential inactivating mutations occurring in the PRMT1 dimerization arm. We identified three mutations that correspond to W215L, Y220N, and M224V substitutions in human PRMT1V2 (isoform 1) (W197L, Y202N, M206V in rat PRMT1V1). Using a combination of site-directed mutagenesis, analytical ultracentrifugation, native PAGE, and activity assays, we found that these conservative substitutions surprisingly disrupt oligomer formation and substantially impair both S-adenosyl-L-methionine (AdoMet) binding and methyltransferase activity. Molecular dynamics simulations suggest that these substitutions introduce novel interactions within the dimerization arm that lock it in a conformation not conducive to dimer formation. These findings provide a clear, if putative, rationale for the contribution of these mutations to impaired arginine methylation in cells and corresponding health consequences.

Cholecalciferol and metformin protect against lipopolysaccharide-induced endothelial dysfunction and senescence by modulating sirtuin-1 and protein arginine methyltransferase-1.

Endothelial cell activation through nuclear factor-kappa-B (NFkB) and mitogen-activated protein kinases leads to increased biosynthesis of pro-inflammatory mediators, cellular injury and vascular inflammation under lipopolysaccharide (LPS) exposure. Recent studies report that LPS up-regulated global methyltransferase activity. In this study, we observed that a combination treatment with metformin (MET) and cholecalciferol (VD) blocked the LPS-induced S-adenosylmethionine (SAM)-dependent methyltransferase (SDM) activity in Eahy926 cells. We found that LPS challenge (i) increased arginine methylation through up-regulated protein arginine methyltransferase-1 (PRMT1) mRNA, intracellular concentrations of asymmetric dimethylarginine (ADMA) and homocysteine (HCY); (ii) up-regulated cell senescence through mitigated sirtuin-1 (SIRT1) mRNA, nicotinamide adenine dinucleotide (NAD+) concentration, telomerase activity and total antioxidant capacity; and (iii) lead to endothelial dysfunction through compromised nitric oxide (NOx) production. However, these LPS-mediated cellular events in Eahy926 cells were restored by the synergistic effect of MET and VD. Taken together, this study identified that the dual compound effect inhibits LPS-induced protein arginine methylation, endothelial senescence and dysfunction through the components of epigenetic machinery, SIRT1 and PRMT1, which is a previously unidentified function of the test compounds. In silico results identified the presence of vitamin D response element (VDRE) sequence on PRMT1 suggesting that VDR could regulate PRMT1 gene expression. Further characterization of the cellular events associated with the dual compound challenge, using gene silencing approach or adenoviral constructs for SIRT1 and/or PRMT1 under inflammatory stress, could identify therapeutic strategies to address the endothelial consequences in vascular inflammation-mediated atherosclerosis.

The macromolecular complexes of histones affect protein arginine methyltransferase activities.

Histone arginine methylation is a key post-translational modification that mediates epigenetic events that activate or repress gene transcription. Protein arginine methyltransferases (PRMTs) are the driving force for the process of arginine methylation, and the core histone proteins have been shown to be substrates for most PRMT family members. However, previous reports of the enzymatic activities of PRMTs on histones in the context of nucleosomes seem contradictory. Moreover, what governs nucleosomal substrate recognition of different PRMT members is not understood. We sought to address this key biological question by examining how different macromolecular contexts where the core histones reside may regulate arginine methylation catalyzed by individual PRMT members (i.e., PRMT1, PRMT3, PRMT4, PRMT5, PRMT6, PRMT7, and PRMT8). Our results demonstrated that the substrate context exhibits a huge impact on the histone arginine methylation activity of PRMTs. Although all the tested PRMTs methylate multiple free histones individually, they show a preference for one particular histone substrate in the context of the histone octamer. We found that PRMT1, PRMT3, PRMT5, PRMT6, PRMT7, and PRMT8 preferentially methylate histone H4, whereas PRMT4/coactivator-associated arginine methyltransferase 1 prefers histone H3. Importantly, neither reconstituted nor cell-extracted mononucleosomes could be methylated by any PRMTs tested. Structural analysis suggested that the electrostatic interaction may play a mechanistic role in priming the substrates for methylation by PRMT enzymes. Taken together, this work expands our knowledge on the molecular mechanisms of PRMT substrate recognition and has important implications for understanding cellular dynamics and kinetics of histone arginine methylation in regulating gene transcription and other chromatin-templated processes.

A Pan-Inhibitor for Protein Arginine Methyltransferase Family Enzymes.

Protein arginine methyltransferases (PRMTs) play important roles in transcription, splicing, DNA damage repair, RNA biology, and cellular metabolism. Thus, PRMTs have been attractive targets for various diseases. In this study, we reported the design and synthesis of a potent pan-inhibitor for PRMTs that tethers a thioadenosine and various substituted guanidino groups through a propyl linker. Compound II757 exhibits a half-maximal inhibition concentration (IC50) value of 5 to 555 nM for eight tested PRMTs, with the highest inhibition for PRMT4 (IC50 = 5 nM). The kinetic study demonstrated that II757 competitively binds at the SAM binding site of PRMT1. Notably, II757 is selective for PRMTs over a panel of other methyltransferases, which can serve as a general probe for PRMTs and a lead for further optimization to increase the selectivity for individual PRMT.

HDAC6 Substrate Discovery Using Proteomics-Based Substrate Trapping: HDAC6 Deacetylates PRMT5 to Influence Methyltransferase Activity.

Histone deacetylase 6 (HDAC6) is upregulated in a variety of tumor cell lines and has been linked to many cellular processes, such as cell signaling, protein degradation, cell survival, and cell motility. HDAC6 is an enzyme that deacetylates the acetyllysine residues of protein substrates, and the discovery of HDAC6 substrates, including tubulin, has revealed many roles of HDAC6 in cell biology. Unfortunately, among the wide variety of acetylated proteins in the cell, only a few are verified as HDAC6 substrates, which limits the full characterization of HDAC6 cellular functions. Substrate trapping mutants were recently established as a tool to discover unanticipated substrates of histone deacetylase 1 (HDAC1). In this study, we applied the trapping approach to identify potential HDAC6 substrates. Among the high confidence protein hits after trapping, protein arginine methyl transferase 5 (PRMT5) was successfully validated as a novel HDAC6 substrate. PRMT5 acetylation enhanced its methyltransferase activity and symmetrical dimethylation of downstream substrates, revealing possible crosstalk between acetylation and methylation. Substrate trapping represents a powerful, systematic, and unbiased approach to discover substrates of HDAC6.