Methylation Of Arginine Residues Research Articles

OsPRMT6a-mediated arginine methylation of OsJAZ1 regulates jasmonate signaling and spikelet development in rice

Although both protein arginine methylation (PRMT) and jasmonate (JA) signaling are crucial for regulating plant development, the relationship between these processes in the control of spikelet development remains unclear. In this study, we used the CRISPR/Cas9 technology to generate two OsPRMT6a loss-of-function mutants that exhibit various abnormal spikelet structures. Interestingly, we found that OsPRMT6a can methylate arginine residues in JA signal repressors OsJAZ1 and OsJAZ7. We showed that arginine methylation of OsJAZ1 enhances the binding affinity of OsJAZ1 with the JA receptors OsCOI1a and OsCOI1b in the presence of JAs, thereby promoting the ubiquitination of OsJAZ1 by the SCFOsCOI1a/OsCOI1b complex and degradation via the 26S proteasome. This process ultimately releases OsMYC2, a core transcriptional regulator in the JA signaling pathway, to activate or repress JA-responsive genes, thereby maintaining normal plant (spikelet) development. However, in the osprmt6a-1 mutant, reduced arginine methylation of OsJAZ1 impaires the interaction between OsJAZ1 and OsCOI1a/OsCOI1b in the presence of JAs. As a result, OsJAZ1 proteins become more stable, repressing JA responses, thus causing the formation of abnormal spikelet structures. Moreover, we discovered that JA signaling reduces the OsPRMT6a mRNA level in an OsMYC2-dependent manner, thereby establishing a negative feedback loop to balance JA signaling. We further found that OsPRMT6a-mediated arginine methylation of OsJAZ1 likely serves as a switch to tune JA signaling to maintain normal spikelet development under harsh environmental conditions such as high temperatures. Collectively, our study establishes a direct molecular link between arginine methylation and JA signaling in rice.

Protein Arginine Methyltransferases in Pancreatic Ductal Adenocarcinoma: New Molecular Targets for Therapy.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignant disease with a low 5-year overall survival rate. It is the third-leading cause of cancer-related deaths in the United States. The lack of robust therapeutics, absence of effective biomarkers for early detection, and aggressive nature of the tumor contribute to the high mortality rate of PDAC. Notably, the outcomes of recent immunotherapy and targeted therapy against PDAC remain unsatisfactory, indicating the need for novel therapeutic strategies. One of the newly described molecular features of PDAC is the altered expression of protein arginine methyltransferases (PRMTs). PRMTs are a group of enzymes known to methylate arginine residues in both histone and non-histone proteins, thereby mediating cellular homeostasis in biological systems. Some of the PRMT enzymes are known to be overexpressed in PDAC that promotes tumor progression and chemo-resistance via regulating gene transcription, cellular metabolic processes, RNA metabolism, and epithelial mesenchymal transition (EMT). Small-molecule inhibitors of PRMTs are currently under clinical trials and can potentially become a new generation of anti-cancer drugs. This review aims to provide an overview of the current understanding of PRMTs in PDAC, focusing on their pathological roles and their potential as new therapeutic targets.

Arginine Methylation of BRD4 By CARM1 Regulates Its Function in AML

Acute myeloid leukemia (AML) is generally an aggressive and lethal malignancy characterized by the accumulation of immature myeloid cells in the bone marrow and peripheral blood. We have previously shown the dependence of AML, but not normal hematopoiesis, on the presence of coactivator associated arginine methyltransferase 1 (CARM1), which suggests that targeting CARM1 could be useful in the treatment of AML. In preclinical studies, we and others have shown promising effects of CARM1 inhibitors on the growth of hematologic malignancies 1. In order to unearth a potential mechanism for the strong efficacy of CARM1 inhibition or knockdown (KD) in the AML1-ETO (AE+) driven AML mouse model, we employed the BioID system in an AE+ human cell line, SKNO-1, to identify novel substrates and interacting partners of CARM1 that could drive the pathogenesis of the disease. We identified over 500 interactors of CARM1, many of which are novel, including one particularly promising therapeutic target, bromodomain-containing protein 4 (BRD4). When examining the mechanism of action of CARM1 inhibitors, we observed significant overlap between the gene expression changes induced by CARM1 inhibition or KD and BRD4 inhibition or KD. Given evidence that both the histone reader, BRD4, and a histone writer, CARM1, play active roles in AML generation, we examined the relationship between these two proteins in AML. We observed a direct physical interaction between these proteins, using immunoprecipitation and two AE+ AML cell lines. Furthermore, using an overexpression system and tagged fragments of BRD4, we identified the particular interacting regions of BRD4 with CARM1. We also identified BRD4 as a substrate of CARM1 and used mass spectrometry to map the sites of CARM1-dependent BRD4 asymmetric dimethylation, implicating four specific arginine residues in the C-terminus of the protein. This crucial information led us to generate antibodies towards these specific CARM1-dependent methylated arginine residues. Using these novel antibodies, we have shown that small molecule inhibition or KD of CARM1 reduces the methylation of BRD4 while also affecting its intracellular localization. In AE+ leukemia cells, exposure to CARM1 inhibitor results in the reduced chromatin localization of BRD4, while in normal CD34+ HSPCs, under the same conditions, we do not see this effect. These findings suggest a selective effect of CARM1 inhibitors for AML therapy, that may not significantly impair normal hematopoiesis. To identify which BRD4-dependent genes are affected by CARM1 inhibition we utilized chromatin-immunoprecipitation and sequencing (ChIP-seq) coupled with RNA-seq and found the loss of BRD4 from promoters and intragenic regions of several oncogenes, together with loss of RNA expression. Given the importance of CARM1 and BRD4 in the development of AML, we are currently investigating the biological and transcriptional activities present in BRD4 mutants that cannot be methylated by CARM1. While methylation of the N-terminus of BRD4 by PRMTs has been reported 2; we have focused our efforts on the C-terminus of the protein, given the re-localization of BRD4 that occurs when CARM1 is inhibited or knocked down. We continue to define the biological effects of a CARM1-BRD4 signaling axis, to assess the importance of this axis in the pathogenesis of oncogene-driven AML. This study also suggests that inhibiting bromodomain containing proteins like BRD4 can be accomplished by targeting the enzymes that post-translationally modify their function. Further insights will be provided by pre-clinical animal and other studies that are ongoing. 1. Greenblatt SM, Man N, Hamard PJ, et al. CARM1 is essential for myeloid leukemogenesis but dispensable for normal hematopoiesis. Cancer Cell. 2019;35(1):156. doi:10.1016/j.ccell.2018.12.008 2. Liu L, Lin B, Yin S, et al. Arginine methylation of BRD4 by PRMT2/4 governs transcription and DNA repair. Sci Adv. 2022;8(49):eadd8928. doi:10.1126/sciadv.add8928

The potential and challenges of targeting MTAP-negative cancers beyond synthetic lethality.

Approximately 15% of cancers exhibit loss of the chromosomal locus 9p21.3 - the genomic location of the tumour suppressor gene CDKN2A and the methionine salvage gene methylthioadenosine phosphorylase (MTAP). A loss of MTAP increases the pool of its substrate methylthioadenosine (MTA), which binds to and inhibits activity of protein arginine methyltransferase 5 (PRMT5). PRMT5 utilises the universal methyl donor S-adenosylmethionine (SAM) to methylate arginine residues of protein substrates and regulate their activity, notably histones to regulate transcription. Recently, targeting PRMT5, or MAT2A that impacts PRMT5 activity by producing SAM, has shown promise as a therapeutic strategy in oncology, generating synthetic lethality in MTAP-negative cancers. However, clinical development of PRMT5 and MAT2A inhibitors has been challenging and highlights the need for further understanding of the downstream mediators of drug effects. Here, we discuss the rationale and methods for targeting the MAT2A/PRMT5 axis for cancer therapy. We evaluate the current limitations in our understanding of the mechanism of MAT2A/PRMT5 inhibitors and identify the challenges that must be addressed to maximise the potential of these drugs. In addition, we review the current literature defining downstream effectors of PRMT5 activity that could determine sensitivity to MAT2A/PRMT5 inhibition and therefore present a rationale for novel combination therapies that may not rely on synthetic lethality with MTAP loss.

Arginine Methylation Regulates Self-Assembly of Peptides.

Bio-inspired design of peptides represents one facile strategy for development of supramolecular monomers for self-assembly into well-defined nanostructures. Inspired by methylation of arginine during post-translational modification for manipulating protein functions, herein, the controllable self-assembly of peptides via rational incorporation of methylated arginine residues into bola-amphiphilic peptides is reported. A series of bola-amphiphilic peptides are designed and synthesized either containing natural arginine or methylated arginine and investigate the influence of arginine methylation on peptide assembly. This study finds that incorporation of symmetrically di-methylated arginine into oppositely charged hexapeptide hex-SDMAE leads to distinct assembling performance compare to natural peptide hex-RE. The findings demonstrate that the methylation of rationally designed peptide sequences allows for regulation of self-assembly of peptides, thus implying the great potential of arginine methylation in establishing controllable peptide assembling systems and creating in situ formulation of biomedical materials in the future.

The TUDOR domain of SMN is an H3K79me1 histone mark reader.

Spinal muscular atrophy is the leading genetic cause of infant mortality and results from depleted levels of functional survival of motor neuron (SMN) protein by either deletion or mutation of the SMN1 gene. SMN is characterized by a central TUDOR domain, which mediates the association of SMN with arginine methylated (Rme) partners, such as coilin, fibrillarin, and RNA pol II (RNA polymerase II). Herein, we biochemically demonstrate that SMN also associates with histone H3 monomethylated on lysine 79 (H3K79me1), defining SMN as not only the first protein known to associate with the H3K79me1 histone modification but also the first histone mark reader to recognize both methylated arginine and lysine residues. Mutational analyzes provide evidence that SMNTUDOR associates with H3 via an aromatic cage. Importantly, most SMNTUDOR mutants found in spinal muscular atrophy patients fail to associate with H3K79me1.

Structure of the T. brucei kinetoplastid RNA editing substrate-binding complex core component, RESC5.

Kinetoplastid protists such as Trypanosoma brucei undergo an unusual process of mitochondrial uridine (U) insertion and deletion editing termed kinetoplastid RNA editing (kRNA editing). This extensive form of editing, which is mediated by guide RNAs (gRNAs), can involve the insertion of hundreds of Us and deletion of tens of Us to form a functional mitochondrial mRNA transcript. kRNA editing is catalyzed by the 20 S editosome/RECC. However, gRNA directed, processive editing requires the RNA editing substrate binding complex (RESC), which is comprised of 6 core proteins, RESC1-RESC6. To date there are no structures of RESC proteins or complexes and because RESC proteins show no homology to proteins of known structure, their molecular architecture remains unknown. RESC5 is a key core component in forming the foundation of the RESC complex. To gain insight into the RESC5 protein we performed biochemical and structural studies. We show that RESC5 is monomeric and we report the T. brucei RESC5 crystal structure to 1.95 Å. RESC5 harbors a dimethylarginine dimethylaminohydrolase-like (DDAH) fold. DDAH enzymes hydrolyze methylated arginine residues produced during protein degradation. However, RESC5 is missing two key catalytic DDAH residues and does bind DDAH substrate or product. Implications of the fold for RESC5 function are discussed. This structure provides the first structural view of an RESC protein.

Arginine Methylation of the PGC-1α C-Terminus Is Temperature-Dependent.

We set out to determine whether the C-terminus (amino acids 481-798) of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α, UniProt Q9UBK2), a regulatory metabolic protein involved in mitochondrial biogenesis, and respiration, is an arginine methyltransferase substrate. Arginine methylation by protein arginine methyltransferases (PRMTs) alters protein function and thus contributes to various cellular processes. In addition to confirming methylation of the C-terminus by PRMT1 as described in the literature, we have identified methylation by another member of the PRMT family, PRMT7. We performed in vitro methylation reactions using recombinant mammalian PRMT7 and PRMT1 at 37, 30, 21, 18, and 4 °C. Various fragments of PGC-1α corresponding to the C-terminus were used as substrates, and the methylation reactions were analyzed by fluorography and mass spectrometry to determine the extent of methylation throughout the substrates, the location of the methylated PGC-1α arginine residues, and finally, whether temperature affects the deposition of methyl groups. We also employed two prediction programs, PRmePRed and MePred-RF, to search for putative methyltransferase sites. Methylation reactions show that arginine residues R548 and R753 in PGC-1α are methylated at or below 30 °C by PRMT7, while methylation by PRMT1 was detected at these same residues at 30 °C. Computational approaches yielded additional putative methylarginine sites, indicating that since PGC-1α is an intrinsically disordered protein, additional methylated arginine residues have yet to be experimentally verified. We conclude that temperature affects the extent of arginine methylation, with more methylation by PRMT7 occurring below physiological temperature, uncovering an additional control point for PGC-1α.

Akut Pankreas İltihabı Süresince Metillenmiş Arginin Rezidüleri ve İlişkili Amino Asitlerdeki Değişimler

Aim: The extent of the spread of inflammation determines the severity of acute pancreatitis (AP). Methylated arginine residues (MAR), a type of inflammatory mediator, reduce nitric oxide levels and cause vasoconstriction-induced endothelial damage. This study aimed to investigate MAR and related amino acids during acute pancreatic inflammation.
 Material and Method: This prospective, quasi-experimental study was conducted with patients diagnosed with AP and an age-matched control group. The patient samples were taken during the diagnosis and recovery time, whereas during the study for the control group. Mainly, Asymmetric dimethylarginine (ADMA), Arginine (ARG), Citrulline (CIT), and related chemicals were studied via a mass spectrometer.
 Results: A total of 30 patients with AP (mean age=53.3±17.8) and 30 controls (mean age=53.4±18.0) were included in the study. All patients were identified as non-severe (n=8) and severe (n=22). A decrease was detected in the patients' ADMA levels compared to the control group (p=0.01). MAR did not differ concerning disease severity (p > 0.05). However, MAR levels decreased higher in patients with diabetes or chronic kidney disease (CKD). Between the two samplings, the ARG level and ARG to ADMA ratio increased, while the MAR and CIT to ARG ratio decreased.
 Conclusion: Our results showed that MAR levels decreased with AP recovery. The start of a decrease in the high-level blood MAR may indicate the healing of pancreatic inflammation. AP inflammation may be more destructive in patients with diabetes or CKD.

Relationship between protein arginine methyltransferase and cardiovascular disease (Review).

Protein arginine methyltransferases (PRMTs) are widely found in eukaryotes and regulate gene expression and post-translational modifications. PRMT1-PRMT6 have important roles in the pathology of cardiovascular diseases (CVDs), including atherosclerosis, heart failure and myocardial hypertrophy. Although these enzymes are also closely associated with various CVDs, the mechanisms of the involvement of PRMTs in the regulation of CVD have remained largely elusive. PRMTs methylate arginine residues and other factors. The present review describes the roles of PRMT1-PRMT6 in CVD. Furthermore, the biological characteristics of PRMTs and mechanisms by which PRMTs regulate cholesterol metabolism are being introduced. This review aims to provide inspiration for cardiovascular drug research and offer clues for research on the pathogenesis of CVD.

Shotgun proteomic investigation of methyltransferase and methylation profiles in lipopolysaccharide stimulated RAW264.7 murine macrophages.

Arginine methylation is a common post-translational modification which functions as an epigenetic regulator of transcription and plays a key role in various cell signaling pathways. The methylation of arginine residues is catalyzed by protein arginine methyltransferase (PRMT). However, the expression pattern and underlying mechanism of PRMTs and protein methylation profile in lipopolysaccharide (LPS)-induced innate immune responses are poorly understood. Using a shotgun proteomic approach, we found that LPS stimulation increased arginine and proline metabolism and responses to inflammation and bacterial infections. In comparison, cysteine and methionine metabolism, the pentose phosphate pathway, purine metabolism, and protein methylation factors were also decreased in LPS stimulated murine macrophage cell lines. We revealed that LPS stimulation downregulated PRMT1, PRMT5, and protein arginine methylation profiles in RAW264.7 cells using western blot analysis. Additionally, this phenomenon occurred in parallel with nitric oxide accumulation in LPS-induced macrophages. Using inflammation models, we demonstrate for the first time that LPS stimulation decreases PRMTs, leading to the decreasing of arginine methylation in macrophages.

Abstract 3937: PRMT5 inhibition suppress soft tissue sarcomas tumorigenicity through the regulation of glucose metabolism

Abstract Introduction: Soft tissue sarcomas (STS) are a heterogeneous group of rare tumors representing 1% of solid tumors in adults and 15% in children. Despite adequate locoregional treatment, up to 40% of patients will develop metastatic disease. Doxorubicin represents the 1st line standard of care for patients in this setting. However, its activity is limited with a response rate of only 10%, a progression-free survival of less than 6-months. New therapeutic strategies are therefore urgently needed. PRMT5 belongs to a family of enzymes that catalyze the methylation of arginine residues on both histones and non-histone proteins. Recent evidence shows that PRMT5 is upregulated in a wide variety of cancers. Its expression and activity are associated with tumorigenesis and with a decrease in patient survival in several cancers, a rationale for developing highly potent inhibitors. Methods: We analyzed the transcriptome data from 255 translocation-related STS and 389 complex genomics STS with annotated clinical data from the French Sarcoma Group. We then investigated the anti-tumor activity of the first -in class PRMT5 specific inhibitor, GSK3226593 in a panel of 7 cell lines and in two xenograft models established at Institut Bergonié (Bordeaux, France). Results: We found that high PRMT5 gene expression level was significantly associated with worse metastases-free survival in both translocation-related and complex genomics STS (p=0.003 and p=0.009, respectively), support the relevance of PRMT5 as a potential therapeutic target in this indication. We then found that pharmacological inhibition of PRMT5 by using GSK3226593, suppressed the viability and the cell proliferation of 7 STS cell lines in vitro (IC50 range between 0.01 and 1.4 µM) as well as in vivo in two models of xenograft cell lines. This inhibition of cell growth was concomitant with an increase of p53 and p21 expression. However, anti-tumor activity of GSK3226593 was independent of p53 since the compound was still active in shp53 expressing cell lines. Moreover, we did not notice any significant effect of GSK3226593 on cell cycle phases or apoptosis. To decipher the mechanism of action of PRMT5 inhibition in STS, we did a comparative transcriptomic analysis (RNAseq) of STS cells before and after 10 days of treatment with GSK3226593. Gene Ontology and gene set enrichment analysis revealed downregulation of metabolic processes especially glycolysis and mTORC1 pathways. A specific metabolic pathway panel was used to confirm and deepen the underlying molecular mechanism. The downregulation of several genes by GSK3226593 such as PCK2 was confirmed at the protein level by Western blot. Glycolysis inhibition was confirmed by a decrease of glucose uptake. Conclusion: PRMT5 inhibition has anti-tumor activity in vitro and in vivo through the regulation of STS cell metabolism providing a clear rationale for its clinical investigation in this indication. Citation Format: Stephanie Verbeke, Laura Leroy, Aurelien Bourdon, Vanessa Chaire, Elodie Richard, Jean-Philippe Guegan, Alban Bessede, Antoine Italiano. PRMT5 inhibition suppress soft tissue sarcomas tumorigenicity through the regulation of glucose metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3937.

HGG-20. PRMT5 promotes the formation and growth of pediatric high-grade glioma by maintaining tumor stem cell populations

BACKGROUND: Pediatric high-grade gliomas (PHGG) are aggressive, undifferentiated CNS tumors comprising two broad subtypes: diffuse midline glioma with H3K27M mutations (DMG) and cortical high-grade glioma (H3K27-wild-type (wt) PHGG). During normal development, PRMT5 promotes stem cell self-renewal through methylation of arginine residues in histone tails. We hypothesized that PRMT5 controls self-renewal essential to the proliferation of PHGG tumor initiating cells (TICs). METHODS: We identified PRMT5 as potentially oncogenic in PHGG through a screen of 4,139 shRNAs targeting 406 genes with epigenetic activity. To elucidate PRMT5’s activity, we used lentiviral shRNA delivery to knock down (KD) PRMT5 expression in four DMG and one H3K27-wt PHGG cell lines. We performed in vitro growth, cell cycle, apoptosis, limiting dilution and bulk RNA-Seq assays to determine the phenotypic effects of PRMT5 KD. To identify PRMT5’s gene targets, we performed cleavage under targets & release using nuclease (CUT&RUN) followed by qPCR and are currently performing CUT&RUN-Seq. We orthotopically implanted PRMT5 KD PHGG cells into mice and tracked survival, tumor growth and tumor histological characteristics. RESULTS: In vitro, PRMT5 KD reduced cell growth (p<0.001), slowed cell cycle progression and increased apoptosis. PRMT KD also slowed neurosphere formation, demonstrating reduced self-renewal (p<7E-9). Geneset expression analysis showed PRMT5 KD reduced expression of self-renewal genes and increased expression of differentiation genes (FDR<0.0001). In vivo, PRMT5 KD reduced tumor growth, as monitored by bioluminescence and MRI, and aggressiveness, based on Ki-67 staining (p<0.05), leading to increased survival (p<0.001). CUT&RUN-qPCR results showed PRMT5 KD led to decreased expression and H3K4me3 promoter occupancy at PAX3, and decreased expression and increased H3K27me3 occupancy at S100A6. PAX3 and S100A6 are oncogenes that preserve TIC self-renewal. CONCLUSION. In vitro experiments show that PRMT5 KD epigenetically reduces TIC self-renewal. In vitro and in vivo, PRMT5 KD reduced PHGG tumor cell growth and aggressiveness.

TDRD5 Is Required for Spermatogenesis and Oogenesis in Locusta migratoria.

Simple SummaryTudor domain-containing proteins (TDRDs) are a group of evolutionarily conserved and germline-enriched proteins, and most of them function in reproduction and the P-element-induced wimpy testis-interacting RNA (piRNA) pathway. We investigated LmTDRD5, a TDRD5 ortholog in Locusta migratoria. In males, LmTdrd5 knockdown delayed meiosis phase transition and reduced the number of elongated spermatids and sperm count. The expression levels of two haploid germ cell marker genes, LmCREM and LmACT, as well as the sperm tail marker gene LmQrich2 were downregulated. In females, LmTdrd5 knockdown decreased vitellogenin (Vg) and Vg receptor (VgR) transcript levels, thereby affecting ovarian development and oocyte maturation. Therefore, LmTdrd5 may play an important role in locust reproduction, indicative of a conserved primary function of TDRD5.Tudor family proteins exist in all eukaryotic organisms and play a role in many cellular processes by recognizing and binding to proteins with methylated arginine or lysine residues. TDRD5, a member of Tudor domain-containing proteins (TDRDs), has been implicated in the P-element-induced wimpy testis-interacting RNA (piRNA) pathway and germ cell development in some model species, but little is known about its function in other species. Therefore, we identified and characterized LmTDRD5, the TDRD5 ortholog in Locusta migratoria, a hemimetabolous pest. The LmTdrd5 gene has 19 exons that encode a protein possessing a single copy of the Tudor domain and three LOTUS domains at its N-terminus. qRT-PCR analysis revealed a high LmTdrd5 expression level in genital glands. Using RNA interference, LmTdrd5 knockdown in males led to a lag in meiosis phase transition, decreased spermatid elongation and sperm production, and downregulated the expression of the two germ cell-specific transcription factors, LmCREM and LmACT, as well as the sperm tail marker gene LmQrich2. LmTdrd5 knockdown in females reduced the expression levels of vitellogenin (Vg) and Vg receptor (VgR) and impaired ovarian development and oocyte maturation, thus decreasing the hatchability rate. These results demonstrate that LmTdrd5 is essential for germ cell development and fertility in locusts, indicating a conserved function for TDRD5.

SAM-Competitive PRMT5 Inhibitor PF-06939999 Demonstrates Antitumor Activity in Splicing Dysregulated NSCLC with Decreased Liability of Drug Resistance.

Protein arginine methyltransferase 5 (PRMT5) overexpression in hematologic and solid tumors methylates arginine residues on cellular proteins involved in important cancer functions including cell-cycle regulation, mRNA splicing, cell differentiation, cell signaling, and apoptosis. PRMT5 methyltransferase function has been linked with high rates of tumor cell proliferation and decreased overall survival, and PRMT5 inhibitors are currently being explored as an approach for targeting cancer-specific dependencies due to PRMT5 catalytic function. Here, we describe the discovery of potent and selective S-adenosylmethionine (SAM) competitive PRMT5 inhibitors, with in vitro and in vivo characterization of clinical candidate PF-06939999. Acquired resistance mechanisms were explored through the development of drug resistant cell lines. Our data highlight compound-specific resistance mutations in the PRMT5 enzyme that demonstrate structural constraints in the cofactor binding site that prevent emergence of complete resistance to SAM site inhibitors. PRMT5 inhibition by PF-06939999 treatment reduced proliferation of non-small cell lung cancer (NSCLC) cells, with dose-dependent decreases in symmetric dimethyl arginine (SDMA) levels and changes in alternative splicing of numerous pre-mRNAs. Drug sensitivity to PF-06939999 in NSCLC cells associates with cancer pathways including MYC, cell cycle and spliceosome, and with mutations in splicing factors such as RBM10. Translation of efficacy in mouse tumor xenograft models with splicing mutations provides rationale for therapeutic use of PF-06939999 in the treatment of splicing dysregulated NSCLC.

Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System

Protein arginine methyltransferases (PRMTs) methylate arginine residues on a wide variety of proteins that play roles in numerous cellular processes. PRMTs can either mono- or dimethylate arginine guanidino groups symmetrically or asymmetrically. The enzymology of these proteins is a complex and intensely investigated area that requires milligram quantities of high-quality recombinant protein. The baculovirus expression vector system (BEVS) employing Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Spodoptera frugiperda 9 (Sf9) insect cells has been used for expression screening and production of many PRMTs, including PRMT 1, 2, and 4 through 9. To simultaneously screen for the expression of multiple constructs of these proteins, including domains and truncated fragments as well as the full-length proteins, we have applied scalable methods utilizing adjustable and programmable multichannel pipettes, combined with 24- and 96-well plates and blocks. Overall, these method adjustments enabled a large-scale generation of bacmid DNA, recombinant viruses, and protein expression screening. Using culture vessels with a high-fill volume of Sf9 cell suspension helped to overcome space limitations in the production pipeline for single batch large-scale protein production. Here, we describe detailed protocols for the efficient and cost-effective expression of functional PRMTs for biochemical, biophysical, and structural studies.

Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System.

Protein arginine methyltransferases (PRMTs) methylate arginine residues on a wide variety of proteins that play roles in numerous cellular processes. PRMTs can either mono- or dimethylate arginine guanidino groups symmetrically or asymmetrically. The enzymology of these proteins is a complex and intensely investigated area that requires milligram quantities of high-quality recombinant protein. The baculovirus expression vector system (BEVS) employing Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Spodoptera frugiperda 9 (Sf9) insect cells has been used for expression screening and production of many PRMTs, including PRMT 1, 2, and 4 through 9. To simultaneously screen for the expression of multiple constructs of these proteins, including domains and truncated fragments as well as the full-length proteins, we have applied scalable methods utilizing adjustable and programmable multichannel pipettes, combined with 24- and 96-well plates and blocks. Overall, these method adjustments enabled a large-scale generation of bacmid DNA, recombinant viruses, and protein expression screening. Using culture vessels with a high-fill volume of Sf9 cell suspension helped to overcome space limitations in the production pipeline for single batch large-scale protein production. Here, we describe detailed protocols for the efficient and cost-effective expression of functional PRMTs for biochemical, biophysical, and structural studies.

Abstract 1137: PRMT5 inhibition regulates alternative splicing and DNA damage repair pathways in SF3B1 R625G expressing uveal melanoma cells

Abstract PRMT5 (protein arginine methyltransferase 5) is a predominant Type II PRMT that catalyzes symmetric dimethylation of protein arginine residues (sDMA). PRMT5 is overexpressed in many types of cancer and plays roles in multiple essential biological processes to promote cancer growth. Previous studies have shown that PRMT5 is a critical molecule for RNA processing and pre-mRNA splicing. Mechanistically, PRMT5 directly methylates arginine residues of several splicing factors such as Small nuclear ribonucleoprotein (SNRPB and SNRPD3) and Serine and arginine rich splicing factor 1 (SRSF1), which contributes to spliceosome assembly and promotes canonical splicing of many essential genes in cancer cells. In the present study, we examined the effects of PRT543, a potent and selective PRMT5 inhibitor, on alternative splicing in uveal melanoma which frequently express hotspot mutations on Splicing factor 3b subunit 1 (SF3B1). We first confirmed that PRT543-treated MEL202 (SF3B1R625G active mutant) and MEL270 (SF3B1WT) cells show significantly increased global alternative splicing, such as increased retained intron (RI) and skipping exon (SE), determined by delta-PSI (percentage of splice-in) analysis. PRT543 downregulates SF3B1 target genes such as FBXW5, MAP3K7, MBD4 and BRD9 that are associated with increased retention of specific intron sites. Interestingly, downregulation of the SF3B1 target genes are more significant in MEL202 (SF3B1R625G) than MEL270 (SF3B1WT), indicating that PRT543 can regulate the activity of the SF3B1 gain of function mutant. Consistent with previously reported PRMT5 knockout studies in hematological cancer cells, PRT543 also downregulates expression of SRSF1 target genes such as POLD1 and PNKP through increased intron retention in primary and metastatic uveal melanoma cell lines. Furthermore, we uncover that PRT543 strikingly increases retention of a specific intron site of ATM (ex33-34), resulting in a significant reduction of ATM protein levels in PRT543-treated MEL202 and MEL270 cells. Gene set enrichment analysis (GSEA) further reveals that PRT543 significantly and specifically regulates DNA replication and repair pathways in MEL202 cells. Importantly, combining PRT543 with DNA-alkylating agents or PARP inhibitors yields a synergistic reduction in cell viability. In summary, our results suggest that PRMT5 inhibition regulates cancer-associated RNA splicing machinery and the DNA damage response, resulting in synergistic antitumor activity when combined with chemotherapy and/or PARP inhibitors, particularly in cancers with spliceosomal mutations. PRT543 is currently under evaluation in a Phase I clinical trial in patients with advanced solid tumors and hematological malignancies (NCT03886831). Citation Format: Koichi Ito, Venkat Thodima, Jack Carter, Neha Bhagwat, Monisha Sivakumar, Alexander Grego, Joseph Rager, Mizue Terai, Takami Sato, Omar Abdel-Wahab, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. PRMT5 inhibition regulates alternative splicing and DNA damage repair pathways in SF3B1 R625G expressing uveal melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1137.

Front Cover: What Is the Catalytic Mechanism of Enzymatic Histone N‐Methyl Arginine Demethylation and Can It Be Influenced by an External Electric Field? (Chem. Eur. J. 46/2021)

Methylation of arginine residues in histones is an important mechanism of epigenetic regulation, and its dysregulation results in human diseases such as cancer and brain disorders. This study reveals the catalytic mechanism of the demethylation of methylated arginine R3 in H4 histone by a non-heme Fe(II) and 2-Oxoglutarate-Dependent Histone demethylase 4E (KDM4E). Applying QM/MM and MD, we explored both C–H and N–H activations pathways. Importantly, the study revealed that by applying external electric fields (EEFs), we can modulate the C–H and N–H selectivities. More information can be found in the Full Paper by C. Z. Christov et al. (DOI: 10.1002/chem.202101174).

CARM1 Regulates AMPK Signaling in Skeletal Muscle.

SummaryCoactivator-associated arginine methyltransferase 1 (CARM1) is an emerging mediator of skeletal muscle plasticity. We employed genetic, physiologic, and pharmacologic approaches to determine whether CARM1 regulates the master neuromuscular phenotypic modifier AMP-activated protein kinase (AMPK). CARM1 skeletal muscle-specific knockout (mKO) mice displayed reduced muscle mass and dysregulated autophagic and atrophic processes downstream of AMPK. We observed altered interactions between CARM1 and AMPK and its network, including forkhead box protein O1, during muscle disuse. CARM1 methylated AMPK during the early stages of muscle inactivity, whereas CARM1 mKO mitigated progression of denervation-induced atrophy and was accompanied by attenuated phosphorylation of AMPK targets such as unc-51 like autophagy-activating kinase 1Ser555. Lower acetyl-coenzyme A corboxylaseSer79 phosphorylation, as well as reduced peroxisome proliferator-activated receptor-γ coactivator-1α, was also observed in mKO animals following acute administration of the direct AMPK activator MK-8722. Our study suggests that targeting CARM1-AMPK interplay may have broad impacts on neuromuscular health and disease.