RESUMEN
Proper protein arginine methylation by protein arginine methyltransferase 1 (PRMT1) is critical for maintaining cellular health, while dysregulation is often associated with disease. How the activity of PRMT1 is regulated is therefore paramount, but is not clearly understood. Several studies have observed higher order oligomeric species of PRMT1, but it is unclear if these exist at physiological concentrations and there is confusion in the literature about how oligomerization affects activity. We therefore sought to determine which oligomeric species of PRMT1 are physiologically relevant, and quantitatively correlate activity with specific oligomer forms. Through quantitative western blotting, we determined that concentrations of PRMT1 available in a variety of human cell lines are in the sub-micromolar to low micromolar range. Isothermal spectral shift binding data were modeled to a monomer/dimer/tetramer equilibrium with an EC50 for tetramer dissociation of ~20 nM. A combination of sedimentation velocity and Native polyacrylamide gel electrophoresis experiments directly confirmed that the major oligomeric species of PRMT1 at physiological concentrations would be dimers and tetramers. Surprisingly, the methyltransferase activity of a dimeric PRMT1 variant is similar to wild type, tetrameric PRMT1 with some purified substrates, but dimer and tetramer forms of PRMT1 show differences in catalytic efficiencies and substrate specificity for other substrates. Our results define an oligomerization paradigm for PRMT1, show that the biophysical characteristics of PRMT1 are poised to support a monomer/dimer/tetramer equilibrium in vivo, and suggest that the oligomeric state of PRMT1 could be used to regulate substrate specificity.
Asunto(s)
Multimerización de Proteína , Proteína-Arginina N-Metiltransferasas , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Humanos , Especificidad por Sustrato , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , MetilaciónRESUMEN
Glycine- and arginine-rich (GAR) motifs, commonly found in RNA-binding and -processing proteins, can be symmetrically (SDMA) or asymmetrically (ADMA) dimethylated at the arginine residue by protein arginine methyltransferases. Arginine-methylated protein motifs are usually read by Tudor domain-containing proteins. Here, using a GFP-Trap, we identify a non-Tudor domain protein, squamous cell carcinoma antigen recognized by T cells 3 (SART3), as a reader for SDMA-marked GAR motifs. Structural analysis and mutagenesis of SART3 show that aromatic residues lining a groove between two adjacent aromatic-rich half-a-tetratricopeptide (HAT) repeat domains are essential for SART3 to recognize and bind to SDMA-marked GAR motif peptides, as well as for the interaction between SART3 and the GAR-motif-containing proteins fibrillarin and coilin. Further, we show that the loss of this reader ability affects RNA splicing. Overall, our findings broaden the range of potential SDMA readers to include HAT domains.
Asunto(s)
Secuencias de Aminoácidos , Arginina , Glicina , Arginina/metabolismo , Arginina/química , Humanos , Glicina/metabolismo , Glicina/química , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Unión Proteica , Empalme del ARN , Células HEK293 , Metilación , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/química , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteína-Arginina N-Metiltransferasas/químicaRESUMEN
ß-Thalassemia is an inherited genetic disorder associated with ß-globin chain synthesis, which ultimately becomes anemia. Adenosine-2,3-dialdehyde, by inhibiting arginine methyl transferase 5 (PRMT5), can induce fetal hemoglobin (HbF) levels. Hence, the materialization of PRMT5 inhibitors is considered a promising therapy in the management of ß-thalassemia. This study conducted a virtual screening of certain compounds similar to 5'-deoxy-5'methyladenosine (3XV) using the PubChem database. The top 10 compounds were chosen based on the best docking scores, while their interactions with the PRMT5 active site were analyzed. Further, the top two compounds demonstrating the lowest binding energy were subjected to drug-likeness analysis and pharmacokinetic property predictions, followed by molecular dynamics simulation studies. Based on the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) score and molecular interactions, (3R,4S)-2-(6-aminopurin-9-yl)-5-[(4-ethylcyclohexyl)sulfanylmethyl]oxolane-3,4-diol (TOP1) and 2-(6-Aminopurin-9-yl)-5-[(6-aminopurin-9-yl)methylsulfanylmethyl]oxolane-3,4-diol (TOP2) were identified as potential hit compounds, while TOP1 exhibited higher binding affinity and stabler binding capabilities than TOP2 during molecular dynamics simulation (MDS) analysis. Taken together, the outcomes of our study could aid researchers in identifying promising PRMT5 inhibitors. Moreover, further investigations through in vivo and in vitro experiments would unquestionably confirm that this compound could be employed as a therapeutic drug in the management of ß-thalassemia.
Asunto(s)
Inhibidores Enzimáticos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteína-Arginina N-Metiltransferasas , Talasemia beta , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/metabolismo , Talasemia beta/tratamiento farmacológico , Humanos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Descubrimiento de Drogas , Unión Proteica , Dominio Catalítico , Adenosina/análogos & derivados , Adenosina/química , Adenosina/farmacologíaRESUMEN
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.
Asunto(s)
Histonas , Proteína-Arginina N-Metiltransferasas , Cromatina , Histonas/genética , Histonas/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/metabolismoRESUMEN
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.
Asunto(s)
Inhibidores Enzimáticos , Neoplasias del Cuello Uterino , Femenino , Humanos , Persona de Mediana Edad , Células HeLa , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Compuestos de Bifenilo , Neoplasias del Cuello Uterino/tratamiento farmacológico , Arginina , Proteína-Arginina N-Metiltransferasas/química , Proteínas Represoras/metabolismoRESUMEN
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.
Asunto(s)
Neoplasias , Proteína-Arginina N-Metiltransferasas , Humanos , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/metabolismo , Neoplasias/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Línea Celular Tumoral , Arginina/metabolismoRESUMEN
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.
Asunto(s)
Amigos , Proteínas Ribosómicas , Humanos , Proteínas Ribosómicas/metabolismo , Proteína-Arginina N-Metiltransferasas/química , Ribosomas/metabolismo , ARN Ribosómico/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismoRESUMEN
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.
Asunto(s)
Neoplasias , Proteína-Arginina N-Metiltransferasas , Humanos , Histonas/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/metabolismoRESUMEN
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.
Asunto(s)
Leishmania major , Parásitos , Ratones , Animales , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Parásitos/metabolismo , Metilación , Arginina/metabolismo , MamíferosRESUMEN
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.
Asunto(s)
Procesamiento Proteico-Postraduccional , Proteínas de Unión al ARN , Arginina/química , Arginina/genética , Arginina/metabolismo , Metilación , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
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.
Asunto(s)
Arginina , Proteoma , Humanos , Proteoma/análisis , Arginina/química , Espectrometría de Masas en Tándem , Células HeLa , Programas Informáticos , Proteína-Arginina N-Metiltransferasas/química , Proteínas Represoras/metabolismo , ProteínasRESUMEN
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.
Asunto(s)
Adenosina , Proteína-Arginina N-Metiltransferasas , Adenosina/química , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/metabolismo , Colorantes Fluorescentes , Arginina/química , Arginina/metabolismo , Péptidos/química , Proteínas QuinasasRESUMEN
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.
Asunto(s)
Inhibidores Enzimáticos , Proteína-Arginina N-Metiltransferasas , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Simulación del Acoplamiento Molecular , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/metabolismo , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteína-Arginina N-Metiltransferasas , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína-Arginina N-Metiltransferasas/químicaRESUMEN
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.
Asunto(s)
Leishmania braziliensis , Proteína-Arginina N-Metiltransferasas , Arginina/metabolismo , Leishmania braziliensis/genética , Macrófagos/metabolismo , Metilación , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismoRESUMEN
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.
Asunto(s)
Inhibidores Enzimáticos/uso terapéutico , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Animales , Sitios de Unión , Línea Celular Tumoral , Ensayos Clínicos como Asunto , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Unión Proteica , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/metabolismo , S-Adenosilmetionina/químicaRESUMEN
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.
Asunto(s)
Inhibidores Enzimáticos , Proteína-Arginina N-Metiltransferasas , Arginina , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Unión Proteica , Proteína-Arginina N-Metiltransferasas/químicaRESUMEN
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.
Asunto(s)
Arginina/química , Metilación , Neoplasias/metabolismo , Procesamiento Proteico-Postraduccional , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Empalme Alternativo , Animales , Antígeno B7-H1/metabolismo , Sistemas CRISPR-Cas , Comunicación Celular , Línea Celular Tumoral , Daño del ADN , Reparación del ADN , Inhibidores Enzimáticos/farmacología , Epigénesis Genética , Histonas , Humanos , Sistema Inmunológico , Inmunoterapia/métodos , Ratones , Ratones Noqueados , Proteína-Arginina N-Metiltransferasas/química , Empalme del ARN , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
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.
Asunto(s)
Antioxidantes/farmacología , Colecalciferol/farmacología , Metformina/farmacología , Sustancias Protectoras/farmacología , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Represoras/metabolismo , Sirtuina 1/metabolismo , Arginina/análogos & derivados , Arginina/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular , Senescencia Celular/efectos de los fármacos , Endotelio/efectos de los fármacos , Homocisteína/metabolismo , Humanos , Lipopolisacáridos/toxicidad , Metilación/efectos de los fármacos , NAD/metabolismo , Óxido Nítrico/metabolismo , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/química , Proteínas Represoras/genética , S-Adenosilmetionina/metabolismo , Sirtuina 1/genética , Telomerasa/metabolismo , Elemento de Respuesta a la Vitamina DRESUMEN
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.