PRMT Family Research Articles

Role of PRMT1 and PRMT5 in Breast Cancer.

Breast cancer is the most common cancer diagnosed in women worldwide. Early-stage breast cancer is curable in ~70-80% of patients, while advanced metastatic breast cancer is considered incurable with current therapies. Breast cancer is a highly heterogeneous disease categorized into three main subtypes based on key markers orientating specific treatment strategies for each subtype. The complexity of breast carcinogenesis is often associated with epigenetic modification regulating different signaling pathways, involved in breast tumor initiation and progression, particularly by the methylation of arginine residues. Protein arginine methyltransferases (PRMT1-9) have emerged, through their ability to methylate histones and non-histone substrates, as essential regulators of cancers. Here, we present an updated overview of the mechanisms by which PRMT1 and PRMT5, two major members of the PRMT family, control important signaling pathways impacting breast tumorigenesis, highlighting them as putative therapeutic targets.

Loss-of-function mutation in PRMT9 causes abnormal synapse development by dysregulation of RNA alternative splicing

Protein arginine methyltransferase 9 (PRMT9) is a recently identified member of the PRMT family, yet its biological function remains largely unknown. Here, by characterizing an intellectual disability associated PRMT9 mutation (G189R) and establishing a Prmt9 conditional knockout (cKO) mouse model, we uncover an important function of PRMT9 in neuronal development. The G189R mutation abolishes PRMT9 methyltransferase activity and reduces its protein stability. Knockout of Prmt9 in hippocampal neurons causes alternative splicing of ~1900 genes, which likely accounts for the aberrant synapse development and impaired learning and memory in the Prmt9 cKO mice. Mechanistically, we discover a methylation-sensitive protein–RNA interaction between the arginine 508 (R508) of the splicing factor 3B subunit 2 (SF3B2), the site that is exclusively methylated by PRMT9, and the pre-mRNA anchoring site, a cis-regulatory element that is critical for RNA splicing. Additionally, using human and mouse cell lines, as well as an SF3B2 arginine methylation-deficient mouse model, we provide strong evidence that SF3B2 is the primary methylation substrate of PRMT9, thus highlighting the conserved function of the PRMT9/SF3B2 axis in regulating pre-mRNA splicing.

Abstract 4596: Targeting arginine methylome in 9p21/MTAP-deleted malignant cancers with a next generation PRMT5-specific inhibitor CTS3497

Abstract It remains largely challenging to treat cancer patients with chromosome 9p21-deletion which occurs in approximately 15% of human cancers. Recently, specific targeting of protein arginine methyltransferase 5 (PRMT5), a key member of the type-II PRMT family and a master epigenetic modulator of arginine methylome essential for cancer progression, has emerged as a promising therapy in various hematological and solid tumors with MTAP deletion serving as a precision biomarker. To optimize the potential of targeting PRMT5, we developed CTS3497, a potent, brain-penetrable, orally bioavailable MTA-cooperative PRMT5 inhibitor, using our EpigenPLUSTM platform. The overall excellent drug-like properties of CTS3497 support fast track IND application, and it is anticipated to enter Phase 1 trials in 2024. CTS3497 demonstrated potent PRMT5 inhibitory activity in cellular pharmacodynamics (PD) assays, evidenced by the suppression of symmetric dimethylarginine (SDMA). Additionally, it exhibited profound cell growth inhibition with a low single-digit nM IC50 and 171-fold high selectivity for MTAPnull over isogenic MTAPwt cell lines. Moreover, CTS3497 displayed strong inhibitory effects on cell growth across diverse panels of MTAPnull cancer cell lines and patient-derived xenograft organoids (PDXOs) spanning various lineages. In vivo, oral administration of CTS3497 resulted in significant tumor regression in multiple MTAP-deleted xenograft models, notably MTAPnull brain tumors. The observed anti-tumor activity correlated with PRMT5 modulation, as indicated by reduced SDMA levels. Impressively, sustained tumor growth inhibition led to the complete tumor regression, with no regrowth even after discontinuation of CTS3497 treatment. The brain penetration property of CTS3497 underscores its therapeutic potential for both primary brain tumors, and brain metastases arising from specific cancers like lung cancer. At the molecular level, PRMT5 inhibition by CTS3497 induced substantial and distinctive RNA splicing defects and differentially expressed gene signatures, impacting key cellular activities downstream of arginine and epigenetic reprogramming in MTAPnull cancer cells. Furthermore, a synergistic anti-tumor effect was observed in multiple MTAP-deleted tumors when combining PRMT5 inhibition with CTS2190, an investigational type-I PRMT inhibitor currently in phase I/II clinical trials. In summary, CTS3497, a PRMT5 inhibitor selectively targeting MTAPnull tumor cells, demonstrated robust and durable anti-tumor responses both in vitro and in vivo across diverse lineages of MTAP-deleted tumors. Its superior ADME properties and favorable safety profiles observed in preclinical studies mark a breakthrough in the next generation epigenetic therapy. CTS3497 emerges as a promising therapeutic option for patients with MTAP-deleted malignancies. Citation Format: Yilin Liu, Long Wang, Hui Shi, Yuanyuan Liu, Qiugeng Ouyang, Xingnian Fu, Meng Wang, Jiannan Guo, Yiqin Wang, Youzhen Wang, Guoliang Xu, Yuan Mi, Haiping Wu. Targeting arginine methylome in 9p21/MTAP-deleted malignant cancers with a next generation PRMT5-specific inhibitor CTS3497 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4596.

Protein arginine methyltransferase 1 is a therapeutic vulnerability in multiple myeloma.

Multiple myeloma (MM) is a devastating plasma cell malignancy characterized by the expansion of aberrant monoclonal plasma cells in the bone marrow, leading to severe clinical manifestations and poor prognosis, particularly in relapsed/refractory cases. Identifying novel therapeutic targets is crucial to improve treatment outcomes in these patients. In this study, we investigated the role of the protein arginine methyltransferase 1 (PRMT1) in MM pathogenesis and explored its potential as a therapeutic target. We observed that PRMT1, responsible for most asymmetric di-methylation in cells, exhibited the highest expression among PRMT family members in MM cell lines and primary MM cells. Importantly, PRMT1 expression was significantly elevated in relapsed/refractory patients compared to newly diagnosed patients. High expression of PRMT1 expression was strongly associated with poor prognosis. We found that genetic or enzymatic inhibition of PRMT1 impaired MM cell growth, induced cell cycle arrest, and triggered cell death. Treatment with MS023, a potent PRMT type I inhibitor, demonstrated a robust inhibitory effect on the viability of primary cells isolated from newly diagnosed and proteasome inhibitor-relapsed/refractory patients in a dose-dependent manner. Suppression of PRMT1 downregulated genes related to cell division and upregulated genes associated with apoptosis pathway. We also found that genes related to immune response and lymphocyte activation were significantly upregulated in PRMT1-suppressed cells. Notably, the activation status of T cells was strikingly enhanced upon co-culturing with PRMT1-KO MM cells. In vivo studies using a xenograft model revealed that targeting PRMT1 by either CRISPR/Cas9-mediated knockout or MS023 treatment significantly attenuated MM tumor growth and prolonged the survival of tumor-bearing mice. Histological analysis further confirmed increased apoptotic cell death in MS023-treated tumors. Collectively, our findings establish PRMT1 as an indispensable and novel therapeutic vulnerability in MM. The elevated expression of PRMT1 in relapsed/refractory patients underscores its potential as a target for overcoming treatment resistance. Moreover, our results highlight the efficacy of MS023 as a promising therapeutic agent against MM, offering new avenues for therapeutic approaches in relapsed/refractory MM.

Elucidating the role of PRMTs in prostate cancer using open access databases and a patient cohort dataset.

Protein arginine methylation is an understudied epigenetic mechanism catalyzed by enzymes known as Protein Methyltransferases of Arginine (PRMTs), while the opposite reaction is performed by Jumonji domain- containing protein 6 (JMJD6). There is increasing evidence that PRMTs are deregulated in prostate cancer (PCa). In this study, the expression of two PRMT members, PRMT2 and PRMT7 as well as JMJD6, a demethylase, was analyzed in PCa. Initially, we retrieved data from The Cancer Genome Atlas (TCGA) project and the Gene Expression Omnibus (GEO) database to explore the differential expression of various PRMT family members in patients with PCa and then applied immunohistochemistry in a patient cohort across the spectrum of PCa, including non-neoplastic prostate tissue and lymph node metastatic foci. The results from the TCGA analysis revealed that PRMT7, PRMT6 and PRMT3 expression increased while PRMT2, PRMT9 and JMJD6 levels decreased in the tumor compared to non-neoplastic prostate. Results from the GEO datasets were similar, albeit not identical with the TCGA results, with PRMT7 and PRMT3 being upregulated and PRMT2 and JMJD6 being downregulated in the tumor compared to non-neoplastic tissue in some of them. In addition, PRMT7 levels decreased with stage and grade progression in the TCGA analysis. In the patient cohort, both PRMTs and JMJD6 were overexpressed in PCa compared to non-neoplastic tissue, and nuclear PRMT2 and JMJD6 were upregulated in lymph node metastasis, too. PRMT7 and JMJD6 expression were upregulated with the progression of stage and JMJD6 was also increased with the elevation of grade. After androgen ablation therapy, nuclear expression of PRMT7 and JMJD6 were elevated compared to untreated tumors. PRMT2, PRMT7 and JMD6 were also correlated with markers of EMT and cell cycle regulators. Finally, our findings indicate that PRMTs and JMJD6 are involved in prostate cancer progression and revealed a potential interplay of PRMTs with EMT mediators, underscoring the need for therapeutic targeting of arginine methylation in prostate cancer.

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 10.09μM 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.

Protein Arginine Methyltransferase 1 and its Dynamic Regulation Associated with Cellular Processes and Diseases

Post-translational modifications (PTMs) of proteins influence protein degradation, protein-protein interactions, expression of genes, and intracellular signal transduction, and thereby regulate major life processes. Among the PTMs occurring within the cytoplasm and nucleus, the most commonly studied one is the arginine methylation of proteins catalyzed by PRMTs. PRMT1 is the most excellent and extensively studied member of the PRMT family. PRMT1 occurs in various isoforms, and the unique sequence splicing of each of these isoforms encodes differential proteins that exhibit different cellular localization, substrate specificity, and enzyme activity. In addition to methylating histones, PRMT1 also methylates a large number of non-histone substrates that regulate a broad range of cellular processes. In recent years, research has been revealing an increasing number of pathological diseases caused by the misregulation and aberrant expression of PRMT1, demonstrating the potential of PRMT1 as an effective biomarker for drug targets. In this context, the present study discusses the structural characteristics and the biological functions of PRMT1.

Systematic investigation of PRMT6 substrate recognition reveals broad specificity with a preference for an RG motif or basic and bulky residues

Protein arginine methyltransferase 6 (PRMT6) catalyses the asymmetric dimethylation of arginines on numerous substrate proteins within the human cell. In particular, PRMT6 methylates histone H3 arginine 2 (H3R2) which affects both gene repression and activation. However, the substrate specificity of PRMT6 has not been comprehensively analysed. Here, we systematically characterise the substrate recognition motif of PRMT6, finding that it has broad specificity and recognises the RG motif. Working with a H3 tail peptide as a template, on which we made 204 amino acid substitutions, we use targeted mass spectrometry to measure their effect on PRMT6 invitro activity. We first show that PRMT6 methylates R2 and R8 in the H3 peptide, although H3R8 is methylated with lower efficiency and is not an invivo PRMT6 substrate. We then quantify the effect of 194 of these amino acid substitutions on methylation at both H3R2 and H3R8. In both cases, we find that PRMT6 tolerates essentially any amino acid substitution in the H3 peptide, but that positively charged and bulky residues are preferred near the target arginine. We show that PRMT6 also has preference for glycine, but only in the position immediately following the target arginine. This indicates that PRMT6 recognises the RG motif rather than the RGG motif. We further confirm this preference for the RG motif on another PRMT6 substrate, histone H4R3. This broad specificity and recognition of RG rather than RGG are distinctive among the PRMT family and has implications for the development of drugs to selectively target PRMT6. DATABASES: Panorama Public (https://panoramaweb.org/PRMT6motif.url); ProteomeXchange (PXD016711).

PRMT4 drives post-ischemic angiogenesis via YB1/VEGF signaling.

Angiogenesis is an integral process in many ischemic disorders, and vascular endothelial growth factor (VEGF) plays an important role in it. Protein arginine methyltransferase 4 (PRMT4), a member of the type I PRMT family, is involved in various biological activities, but its role in endothelial cell (EC) remains elusive. Here, we aimed to investigate the role of PRMT4 in ischemic angiogenesis and explore the possible underlying mechanism. We found that PRMT4 was upregulated in ischemic muscles, and VEGF treatment potentiated its expression in ECs. In vitro, adenovirus-mediated PRMT4 overexpression promoted, while its gene disruption inhibited, EC proliferation, migration, and tube formation. In an in vivo hindlimb ischemia model, forced expression of PRMT4 in ECs showed accelerated blood flow recovery and increased capillary density, whereas its knockdown exhibited the opposite effect. Mechanistically, PRMT4 activated the transcription of VEGF via the interaction with Y-box binding protein-1 (YB1), leading to accelerated angiogenesis. Interestingly, the loss of YB1 partially abolished PRMT4-mediated angiogenesis in vitro. Collectively, our data revealed that PRMT4 promoted angiogenesis through interacting with YB1 and the consequential VEGF upregulation, suggesting that PRMT4 may present as a potential therapeutic target in ischemic angiogenesis. KEY MESSAGES: •PRMT4 is induced by VEGF and upregulated in a hindlimb ischemia model. •PRMT4 promotes angiogenesis both in vitro and in vivo. •PRMT4 regulates VEGF expression through interacting with YB1. •YB1 knockdown retards PRMT4-mediated angiogenic effects in vitro.

Rational Design and Synthesis of Selective PRMT4 Inhibitors: A New Chemotype for Development of Cancer Therapeutics**

Protein arginine N-methyl transferase 4 (PRMT4) asymmetrically dimethylates the arginine residues of histone H3 and nonhistone proteins. The overexpression of PRMT4 in several cancers has stimulated interest in the discovery of inhibitors as biological tools and, potentially, therapeutics. Although several PRMT4 inhibitors have been reported, most display poor selectivity against other members of the PRMT family of methyl transferases. Herein, we report the structure-based design of a new class of alanine-containing 3-arylindoles as potent and selective PRMT4 inhibitors, and describe key structure-activity relationships for this class of compounds.

Dysregulacja poziomu metylotransferaz argininy w patogenezie chorób nowotworowych

Streszczenie Metylacja argininy uznawana jest za jedną z najtrwalszych i najczęściej występujących modyfikacji potranslacyjnych. Reakcja przeniesienia grupy metylowej z S-adenyzolometioniny na aminową resztę argininy katalizowana jest przez metylotransferazy argininy (PRMT). W organizmie człowieka znanych jest dziewięć enzymów z rodziny PRMT, nazwanych zgodnie z kolejnością odkrycia PRMT1-PRMT9. Ze względu na produkt katalizowanej reakcji metylotransferazy argininy podzielono na trzy klasy: I, II, III. Produktami ich aktywności są odpowiednio: asymetryczna dimetyloarginina (ADMA), symetryczna dimetyloarginina (SDMA) oraz monometyloarginina (MMA). Powstałe modyfikacje w istotny sposób wpływają na strukturę chromatyny, dzięki czemu mogą pełnić funkcję koaktywatorów i supresorów transkrypcji. Metylacja argininy pełni wiele krytycznych funkcji, niezbędnych do prawidłowego funkcjonowania organizmu. Uczestniczy m.in. w kontroli transdukcji sygnału, splicingu mRNA oraz reguluje podstawowe procesy komórkowe, takie jak: proliferacja, różnicowanie, migracja i apoptoza. Coraz więcej dowodów wskazuje, że dysregulacja poziomu PRMT może powadzić do transformacji nowotworowej. Związek między podwyższonym poziomem PRMT a chorobą nowotworową udowodniono m.in. w raku: piersi, jajnika, płuc i jelita grubego. Aktywność metylotransferaz argininy może być regulowana za pomocą małocząsteczkowych inhibitorów PRMT. Obecnie trzy substancje hamujące aktywność PRMT znajdują się w fazie badań klinicznych i wykazują działanie przeciwnowotworowe wobec nowotworów hematologicznych. Przypuszcza się, że zastosowanie swoistych inhibitorów PRMT może się okazać nowym, skutecznym i bezpiecznym sposobem zwalczania chorób onkologicznych.

PRMT5 function and targeting in cancer.

Protein methyl transferases play critical roles in numerous regulatory pathways that underlie cancer development, progression and therapy-response. Here we discuss the function of PRMT5, a member of the nine-member PRMT family, in controlling oncogenic processes including tumor intrinsic, as well as extrinsic microenvironmental signaling pathways. We discuss PRMT5 effect on histone methylation and methylation of regulatory proteins including those involved in RNA splicing, cell cycle, cell death and metabolic signaling. In all, we highlight the importance of PRMT5 regulation and function in cancer, which provide the foundation for therapeutic modalities targeting PRMT5.

PRMT5, regulated by lncRNA ZFAS1/miR-150-5p, promoted androgen-independent prostate cancer migration and invasion

IntroductionProstate cancer (PCa) is the most common male genitourinary malignancy in the world. The protein arginine methyltransferase 5 (PRMT5) is one of the main members of the type II PRMT family, which was reported to regulate androgen-dependent PCa cell proliferation. However, the upstream regulators of PRMT5 and its effects on androgen-independent PCa metastasis remained unclear. In the present study, we investigated whether PRMT5 could be a novel diagnostic marker and be used as a therapeutic target in PCa, to explore the possible molecular mechanism, and to understand the clinical importance of PRMT5 in PCa.Material and methodsThe present study evaluated PRMT5 expression levels in PCa and normal prostate samples using public datasets, including TCGA, GEPIA and GSE21032. Furthermore, CCK-8 assay, flow cytometer assay, and transwell assay were conducted to detect the roles of PRMT5. Luciferase reporter assay was used to determine the relationship among ZFAS1/miR-150-5p/PRMT5.ResultsOur results showed that PRMT5 was overexpressed in PCa samples. PRMT5 significantly promoted androgen-dependent PCa proliferation and cell cycle progression and suppressed cell apoptosis. However, PRMT5 did not affect androgen-independent PCa proliferation but it could significantly induce androgen-independent PCa metastasis. Knockdown of PRMT5 suppressed, whereas overexpression of PRMT5 induced, cell migration and invasion in androgen-independent DU145 and PC-3 cells. Moreover, our results showed that the ZFAS1/miR-150-5p axis regulated PRMT5 expression in PCa cells. Furthermore, the study showed that ZFAS1 and PRMT5 were overexpressed and miR-150-5p was down-regulated in PCa samples. Higher expression levels of ZFAS1 and PRMT5 were correlated with shorter disease-free survival time in PCa patients.ConclusionsThese results showed that PRMT5 may be a therapeutic target for PCa.

Protein Arginine Methyltransferase 7 (PRMT7): A Human Enzyme Often Overexpressed in Cancer is Most Active Under Non‐physiological Conditions

Arginine methylation is a ubiquitous eukaryotic posttranslational modification that can modulate transcriptional activation and suppression. Protein arginine methyltransferase 7 (PRMT7), one of the nine mammalian PRMT family members, catalyzes only monomethylation of a terminal guanidino nitrogen atom on its substrates. In an effort to understand the role of PRMT7 in the cell, we characterized a recombinant human PRMT7 expressed as a GST‐fusion protein in bacterial cells. Using S‐adenosyl‐L‐[methyl‐3H]methionine (3H‐AdoMet) as a substrate, we measured the rate of methylation of a synthetic peptide containing residues 23–37 of human histone H2B. Although the physiological methyl‐accepting substrates of PRMT7 are not known, this peptide contains major in vitro monomethylation sites at arginine residues 29, 31, and 33. A P81 phosphocellulose binding assay was performed that separates the 3H‐methylated product from unused 3H‐AdoMet. We confirmed the unusual temperature dependence of PRMT7, where optimal activity was found at about 20 °C with only about 30% of this activity at 37 °C. We found that the Km of PRMT7 for the H2B‐derived peptide was about 10 μM at 20 °C and about 20 μM at 37 °C. The Km for AdoMet was about 0.5 μM at both temperatures. Additionally we showed that physiological salt conditions can significantly decrease the activity. At 150 mM concentrations, we found only about 10% activity with NaCl or KCl, but about 80–90% activity with potassium acetate or ammonium acetate. We also detected a loss of activity with high ionic strength. These results suggest that there may be a specific inhibition by chloride ion as well as by ionic strength. Finally we show that PRMT7 activity is optimal in alkaline environments. The activity of PRMT7 at the near‐physiological pH of 7.5 is only about 20% that at pH 8.5. Taken together, these results open the possibility that PRMT7 can respond to cells undergoing temperature, salt, or pH stress. These findings demonstrate a potential mechanism for the in vivo regulation of arginine methylation by PRMT7 and provide clues to why PRMT7 has pleiotropic effects involved with transcriptional regulation and translation in the cell.Support or Funding InformationThis work was supported by National Science Foundation Grant MCB‐1714569 (to S.G.C), by a predoctoral Ruth L. Kirschstein National Service Award GM007185 (to T.L.L), and by an Amgen Scholars Award (to A.V.).

PRMT1 promotes pancreatic cancer growth and predicts poor prognosis.

Protein arginine methyltransferase 1 (PRMT1) is the founding member of the PRMT family of proteins, whose members catalyze methylation of arginine residues in various proteins. Although several studies have reported upregulation of PRMT1 in various cancer types, the expression pattern and the underlying mechanism of PRMT1 action in pancreatic ductal adenocarcinoma (PDAC) are still unclear. Immunohistochemistry staining as well as RT-PCR was used to determine the expression pattern of PRMT1 in clinical PDAC samples. Lentivirus packaging and transfection were employed to construct cell lines with PRMT1 overexpression or knockdown. MTT and crystal violet assays were used to determine the proliferation rates of PDAC cells. β-catenin transcription activity was measured using a TOPFlash assay. PRMT1 binding to the promoter region of CTNNB1 was determined by ChIP-qPCR assay. Elevated PRMT1 expression was found in PDAC tissue samples compared to noncancerous normal tissues in 41 patients using a real-time PCR assay and in 90 patients using a tissue microarray (TMA) in conjunction with immunohistochemistry. Analysis of the PRMT1 expression data and PDAC clinical features revealed that PRMT1 expression was significantly correlated with PDAC tumor size and prognosis in postoperative patients. Additional functional experiments revealed that PRMT1 expression promoted the growth of pancreatic cancer-derived cells, both in vitro and in vivo. Mechanistically, we found that PRMT1 increased the cellular β-catenin level. We also found that PRMT1 and β-catenin were co-expressed in TCGA and GTEx datasets containing 370 samples. Collectively, our study provides novel insight into the expression and function of PRMT1 in PDAC and indicates that PRMT1 may serve as a therapeutic target for treating patients with pancreatic ductal adenocarcinoma.

The Development of Tetrazole Derivatives as Protein Arginine Methyltransferase I (PRMT I) Inhibitors.

Protein arginine methyltransferase 1 (PRMT1) can catalyze protein arginine methylation by transferring the methyl group from S-adenosyl-L-methionine (SAM) to the guanidyl nitrogen atom of protein arginine, which influences a variety of biological processes. The dysregulation of PRMT1 is involved in a diverse range of diseases, including cancer. Therefore, there is an urgent need to develop novel and potent PRMT1 inhibitors. In the current manuscript, a series of 1-substituted 1H-tetrazole derivatives were designed and synthesized by targeting at the substrate arginine-binding site on PRMT1, and five compounds demonstrated significant inhibitory effects against PRMT1. The most potent PRMT1 inhibitor, compound 9a, displayed non-competitive pattern with respect to either SAM or substrate arginine, and showed the strong selectivity to PRMT1 compared to PRMT5, which belongs to the type II PRMT family. It was observed that the compound 9a inhibited the functions of PRMT1 and relative factors within this pathway, and down-regulated the canonical Wnt/β-catenin signaling pathway. The binding of compound 9a to PRMT1 was carefully analyzed by using molecular dynamic simulations and binding free energy calculations. These studies demonstrate that 9a was a potent PRMT1 inhibitor, which could be used as lead compound for further drug discovery.

The arginine methyltransferase PRMT5 and PRMT1 distinctly regulate the degradation of anti-apoptotic protein CFLARL in human lung cancer cells

BackgroundCFLARL, also known as c-FLIPL, is a critical anti-apoptotic protein that inhibits activation of caspase 8 in mammalian cells. Previous studies have shown that arginine 122 of CFLARL can be mono-methylated. However, the precise role of arginine methyltransferase of CFLARL remains unknown. PRMT5 and PRMT1, which are important members of the PRMT family, catalyze the transfer of methyl groups to the arginine of substrate proteins. PRMT5 can monomethylate or symmetrically dimethylate arginine residues, while PRMT1 can monomethylate or asymmetrically dimethylate arginine residues.MethodsLung cancer cells were cultured following the standard protocol and the cell lysates were prepared to detect the given proteins by Western Blot analysis, and the protein interaction was assayed by co-immunoprecipitation (Co-IP) or GST pull-down assay. CFLARL ubiquitination level was evaluated by proteasomal inhibitor treatment combined with HA-Ub transfection and WB assay. PRMT1 and PRMT5 genes were knocked down by siRNA technique.ResultsWe show that PRMT5 up-regulated the protein levels of CFLARL by decreasing the ubiquitination and increasing its protein level. Additionally, PRMT1 down-regulated the protein level of CFLARL by increasing the ubiquitination and degradation. The overexpression of PRMT5 can inhibit the interaction between CFLARL and ITCH, which has been identified as an E3 ubiquitin ligase of CFLARL, while overexpressed PRMT1 enhances the interaction between CFLARL and ITCH. Furthermore, we verified that dead mutations of PRMT5 or PRMT1 have the same effects on CFLARL as the wild-type ones have, suggesting it is the physical interaction between CFLAR and PRMT1/5 that regulates CFLARL degradation other than its enzymatic activity. Finally, we showed that PRMT5 and PRMT1 could suppress or facilitate apoptosis induced by doxorubicin or pemetrexed by affecting CFLARL in NSCLC cells.ConclusionsPRMT5 and PRMT1 mediate the distinct effects on CFLARL degradation by regulating the binding of E3 ligase ITCH in NSCLC cells. This study identifies a cell death mechanism that is fine-tuned by PRMT1/5 that modulate CFLARL degradation in human NSCLC cells.

PGC‐1α, a Novel Substrate for Protein Arginine Methyltransferase 7?

Peroxisome proliferator‐activated receptor γ coactivator 1α (PGC‐1α) is a protein that plays an important role in the regulation of energy metabolism. It participates in the regulation of both carbohydrate and lipid metabolism and is activated by insulin. For these reasons, it has been implicated in obesity, aging, diabetes and cardiomyopathy. Despite many studies on the role of this master regulator, the precise way it becomes regulated remains unknown. A recent study found that PGC‐1α becomes phosphorylated at Ser570 by kinase B (Akt/PKB) and this stops it from promoting gluconeogenesis and fatty acid oxidation. PGC‐1α is also modified by protein arginine methyltransferase 1 (PRMT1) at several arginine residues, which enhance its regulation. In this study, we set out to determine whether PGC‐1α is modified by PRMT7, another member of the PRMT family. We hypothesize that PRMT7 methylates PGC‐1α because it contains many RXR motifs, a known PRMT7 motif. In addition, they both are active at similar temperatures. To test this hypothesis, truncated constructs corresponding to PGC‐1α and full‐length PRMT7 were expressed and purified in bacterial cells. In vitro methylation reactions using tritiated AdoMet were carried out. Through fluorography of the methylation reactions, it was determined that PRMT7 methylates PGC‐1α within amino acid residues 532–640. Upon confirmation of the methylated arginine residue(s) through immunoblotting and mass spectrometry, further studies will be carried out to investigate the function of arginine methylation by PRMT7. These results will enhance our understanding of PGC‐1α with the broader goal of understanding its regulation in the context of obesity and diabetes.Support or Funding InformationCSULA Minority Access to Research Careers‐Undergraduate Student Training for Academic Research (MARC‐U*STAR), Grant: T34 GM008228 and Louis Stokes Alliance for Minority Participation (LSAMP), Grant: HRD‐1302873This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

What can protein methylation tell us about histones, ribosomes, translation factors and the biology of cancer and aging?

The chemical diversity of proteins is enhanced by posttranslational methylation of most of the 20 major ribosomally‐incorporated amino acids. The most abundant of these modifications occur at the side chains of lysine and arginine residues, although methylation is also known for histidine, glutamine, asparagine, glutamate, cysteine, and methionine residues, as well as for the alpha‐amino groups of several N‐terminal residues and the alpha‐carboxyl groups of leucine and isoprenylcysteine C‐terminal residues. Methylation reactions also can tag age‐damaged D‐aspartyl and L‐isoaspartyl residues for repair reactions.Protein arginine methylation in mammalian cells is largely catalyzed by the nine members of the PRMT family. We have focused our work on the PRMT5 and PRMT9 species that catalyze symmetric dimethylarginine formation, as well as the PRMT7 species that only catalyzes monomethylarginine formation. PRMT9 appears to be specific for the modification of the SF3B2 splicing factor, whereas PRMT5 and PRMT7 appear to have many physiological substrates, including histones. The latter methylation reactions represent a crucial part of the histone code, and recognition of these methylated residues by protein interaction domains modulates transcription. We have recently explored crosstalk between PRMT5 and PRMT7 in the modification of histone H4. In in vitro experiments using a peptide corresponding to the N‐terminal 21 amino acid residues of histone H4, we showed that the activity of the PRMT5 enzyme at the arginine‐3 residue is greatly enhanced by PRMT7‐dependent methylation of arginine‐17 and/or arginine‐19. These experiments provide evidence that PRMTs can cooperate with each other to affect the final degree of protein modification and epigenetic control. Surprisingly, PRMT7 has very little activity in vitro at 37 °C; it is most active between 10 and 20 °C. This result suggests that PRMT7 may respond to cold stress in the organism.Methylation reactions, including modification at arginine residues, can occur predominately on proteins of the translational apparatus. In the yeast Saccharomyces cerevisiae, some 51 of the approximately 70 methyltransferases modify rRNA (13 enzymes), mRNA (2 enzymes), tRNA (16 enzymes), translation factors (10 enzymes), and ribosomal proteins (10 enzymes). Of particular interest, we have shown that the Sfm1, Hpm1, Rkm3, and Rkm4 enzymes modify arginine, histidine, and lysine residues buried deep within the ribosome; these modified residues may function in protein–RNA interactions rather than protein–protein interactions.Support or Funding InformationThis work is funded by the National Science Foundation Grant MCB‐1714569.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phe71 in Type III Trypanosomal Protein Arginine Methyltransferase 7 (TbPRMT7) Restricts the Enzyme to Monomethylation.

Protein arginine methyltransferase 7 (PRMT7) is unique within the PRMT family as it is the only isoform known to exclusively make monomethylarginine (MMA). Given its role in epigenetics, the mechanistic basis for the strict monomethylation activity is under investigation. It is thought that PRMT7 enzymes are unable to add a second methyl group because of steric hindrance in the active site that restricts them to monomethylation. To test this, we probed the active site of trypanosomal PRMT7 (TbPRMT7) using accelerated molecular dynamics, site-directed mutagenesis, kinetic, binding, and product analyses. Both the dynamics simulations and experimental results show that the mutation of Phe71 to Ile converts the enzyme from a type III methyltransferase into a mixed type I/II, that is, an enzyme that can now perform dimethylation. In contrast, the serine and alanine mutants of Phe71 preserve the type III behavior of the native enzyme. These results are inconsistent with a sterics-only model to explain product specificity. Instead, molecular dynamics simulations of these variants bound to peptides show hydrogen bonding between would-be substrates and Glu172 of TbPRMT7. Only in the case of the Phe71 to Ile mutation is this interaction between MMA and the enzyme maintained, and the geometry for optimal SN2 methyl transfer is obtained. The results of these studies highlight the benefit of combined computational and experimental methods in providing a better understanding for how product specificity is dictated by PRMTs.