Methylation of elongation factor 1A by yeast Efm4 or human eEF1A-KMT2 involves a beta-hairpin recognition motif and crosstalks with phosphorylation.

Translation elongation factor 1A (eEF1A) is an essential and highly conserved protein required for protein synthesis in eukaryotes. In both Saccharomyces cerevisiae and human, five different methyltransferases methylate specific residues on eEF1A, making eEF1A the eukaryotic protein targeted by the highest number of dedicated methyltransferases after histone H3. eEF1A methyltransferases are highly selective enzymes, only targeting eEF1A and each targeting just one or two specific residues in eEF1A. However, the mechanism of this selectivity remains poorly understood. To reveal how S. cerevisiae elongation factor methyltransferase 4 (Efm4) specifically methylates eEF1A at K316, we have used AlphaFold-Multimer modeling in combination with crosslinking mass spectrometry (XL-MS) and enzyme mutagenesis. We find that a unique beta-hairpin motif, which extends out from the core methyltransferase fold, is important for the methylation of eEF1A K316 in vitro. An alanine mutation of a single residue on this beta-hairpin, F212, significantly reduces Efm4 activity in vitro and in yeast cells. We show that the equivalent residue in human eEF1A-KMT2 (METTL10), F220, is also important for its activity towards eEF1A in vitro. We further show that the eEF1A guanine nucleotide exchange factor, eEF1Bα, inhibits Efm4 methylation of eEF1A in vitro, likely due to competitive binding. Lastly, we find that phosphorylation of eEF1A at S314 negatively crosstalks with Efm4-mediated methylation of K316. Our findings demonstrate how protein methyltransferases can be highly selective towards a single residue on a single protein in the cell.

Genetic Alterations of SMYD4 in Solid Tumors Using Integrative Multi-Platform Analysis.

SMYD4 is a member of the SMYD family that has lysine methyltransferase function. Little is known about the roles of SMYD4 in cancer. The aim of this study is to investigate genetic alterations in the SMYD4 gene across the most prevalent solid tumors and determine its potential as a biomarker. We performed an integrative multi-platform analysis of the most common mutations, copy number alterations (CNAs), and mRNA expression levels of the SMYD family genes using cohorts available at the Cancer Genome Atlas (TCGA), cBioPortal, and the Catalogue of Somatic Mutations in Cancer (COSMIC). SMYD genes displayed a lower frequency of mutations across the studied tumors, with none of the SMYD4 mutations detected demonstrating sufficient discriminatory power to serve as a biomarker. In terms of CNAs, SMYD4 consistently exhibited heterozygous loss and downregulation across all tumors evaluated. Moreover, SMYD4 showed low expression in tumor samples compared to normal samples, except for stomach adenocarcinoma. SMYD4 demonstrated a frequent negative correlation with other members of the SMYD family and a positive correlation between CNAs and mRNA expression. Additionally, patients with low SMYD4 expression in STAD and LUAD tumors exhibited significantly poorer overall survival. SMYD4 demonstrated its role as a tumor suppressor in the majority of tumors evaluated. The consistent downregulation of SMYD4, coupled with its association with cancer progression, underscores its potential usefulness as a biomarker.

Inhibiting PRMT5 induces DNA damage and increases anti-proliferative activity of Niraparib, a PARP inhibitor, in models of breast and ovarian cancer.

BACKGROUND: Inhibitors of Poly (ADP-Ribose) Polymerases (PARP) provide clinical benefit to patients with breast and ovarian cancers, by compromising the DNA repair activity of cancer cells. Although these agents extend progression-free survival in many patients, responses can be short lived with many patients ultimately progressing. Identification of combination partners that increase dependence of cancer cells to the DNA repair activity of PARPs may represent a strategy to increase the utility of PARP inhibitors. Protein arginine methyltransferase 5 (PRMT5) regulates DNA damage response pathways through splicing and protein modification, and inhibitors of PRMT5 have recently entered clinical trials. METHODS: The effect of PRMT5 inhibition on the levels of DNA damage and repair markers including γH2AX, RAD51, and 53BP1 was determined using high content immunofluorescent imaging. The anti-proliferative activity of the combination of PRMT5 and PARP inhibitors was evaluated using in vitro models of breast and ovarian cancers using both cell lines and ex vivo patient derived xenografts. Finally, the combinations of PRMT5 and PARP inhibitors were evaluated in cell line xenograft models in vivo. RESULTS: Inhibition of PRMT5 by GSK3326595 led to increased levels of markers of DNA damage. The addition of GSK3326595 to the PARP inhibitor, niraparib, resulted in increased growth inhibition of breast and ovarian cancer cell lines and patient derived spheroids. In vivo, the combination improved the partial effects on tumor growth inhibition achieved by either single agent, producing complete tumor stasis and regression. CONCLUSION: These data demonstrate that inhibition of PRMT5 induced signatures of DNA damage in models of breast and ovarian cancer. Furthermore, combination with the PARP inhibitor, Niraparib, resulted in increased anti-tumor activity in vitro and in vivo. Overall, these data suggest inhibition of PRMT5 as a mechanism to broaden and enhance the clinical application of PARP inhibitors.

Rapid and direct measurement of methyltransferase activity in about 30 min.

Protein arginine methylation is a widespread eukaryotic posttranslational modification that occurs to both histone and non-histone proteins. The S-adenosyl-L-methionine (AdoMet or SAM)-dependent modification is catalyzed by the protein arginine methyltransferase (PRMT) family of enzymes. In the last several years a series of both direct and indirect assay formats have been described that allow the rate of methylation to be measured. Here we provide a detailed protocol to directly measure PRMT activity using radiolabeled AdoMet, reversed-phase resin-filled pipette tips (ZipTips®) and a liquid scintillation counter. Because the ZipTips® based quantitation relies only on the straightforward separation of unreacted AdoMet from a methylated substrate, this protocol should be readily adaptable to other methyltransferases. The method is fast, simple to employ with both peptide and protein substrates, and produces very little radioactive waste.

Development of fluorescence polarization-based competition assay for nicotinamide N-methyltransferase.

Methylation-mediated pathways play important roles in the progression of various diseases. Thus, targeting methyltransferases has proven to be a promising strategy for developing novel therapies. Nicotinamide N-methyltransferase (NNMT) is a major metabolic enzyme involved in epigenetic regulation through catalysis of methyl transfer from the cofactor S-adenosyl-l-methionine onto nicotinamide and other pyridines. Accumulating evidence infers that NNMT is a novel therapeutic target for a variety of diseases such as cancer, diabetes, obesity, cardiovascular and neurodegenerative diseases. Therefore, there is an urgent need to discover potent and specific inhibitors for NNMT to assess its therapeutical potential. Herein, we reported the design and synthesis of a fluorescent probe II138, exhibiting a Kd value of 369 ± 14 nM for NNMT. We also established a fluorescence polarization (FP)-based competition assay for evaluation of NNMT inhibitors. Importantly, the unique feature of this FP competition assay is its capability to identify inhibitors that interfere with the interaction of the NNMT active site directly or allosterically. In addition, this assay performance is robust with a Z'factor of 0.76, indicating its applicability in high-throughput screening for NNMT inhibitors.

Targeting protein methylation: from chemical tools to precision medicines.

The methylation of proteins is integral to the execution of many important biological functions, including cell signalling and transcriptional regulation. Protein methyltransferases (PMTs) are a large class of enzymes that carry out the addition of methyl marks to a broad range of substrates. PMTs are critical for normal cellular physiology and their dysregulation is frequently observed in human disease. As such, PMTs have emerged as promising therapeutic targets with several inhibitors now in clinical trials for oncology indications. The discovery of chemical inhibitors and antagonists of protein methylation signalling has also profoundly impacted our general understanding of PMT biology and pharmacology. In this review, we present general principles for drugging protein methyltransferases or their downstream effectors containing methyl-binding modules, as well as best-in-class examples of the compounds discovered and their impact both at the bench and in the clinic.

Mechanistic Insights into the Allosteric Regulation of the Clr4 Protein Lysine Methyltransferase by Autoinhibition and Automethylation.

Clr4 is a histone H3 lysine 9 methyltransferase in Schizosaccharomyces pombe that is essential for heterochromatin formation. Previous biochemical and structural studies have shown that Clr4 is in an autoinhibited state in which an autoregulatory loop (ARL) blocks the active site. Automethylation of lysine residues in the ARL relieves autoinhibition. To investigate the mechanism of Clr4 regulation by autoinhibition and automethylation, we exchanged residues in the ARL by site-directed mutagenesis leading to stimulation or inhibition of automethylation and corresponding changes in Clr4 catalytic activity. Furthermore, we demonstrate that Clr4 prefers monomethylated (H3K9me1) over unmodified (H3K9me0) histone peptide substrates, similar to related human enzymes and, accordingly, H3K9me1 is more efficient in overcoming autoinhibition. Due to enzyme activation by automethylation, we observed a sigmoidal dependence of Clr4 activity on the AdoMet concentration, with stimulation at high AdoMet levels. In contrast, an automethylation-deficient mutant showed a hyperbolic Michaelis-Menten type relationship. These data suggest that automethylation of the ARL could act as a sensor for AdoMet levels in cells and regulate the generation and maintenance of heterochromatin accordingly. This process could connect epigenome modifications with the metabolic state of cells. As other human protein lysine methyltransferases (for example, PRC2) also use automethylation/autoinhibition mechanisms, our results may provide a model to describe their regulation as well.

The ribosome: A hot spot for the identification of new types of protein methyltransferases.

Cellular physiology depends on the alteration of protein structures by covalent modification reactions. Using a combination of bioinformatic, genetic, biochemical, and mass spectrometric approaches, it has been possible to probe ribosomal proteins from the yeast Saccharomyces cerevisiae for post-translationally methylated amino acid residues and for the enzymes that catalyze these modifications. These efforts have resulted in the identification and characterization of the first protein histidine methyltransferase, the first N-terminal protein methyltransferase, two unusual types of protein arginine methyltransferases, and a new type of cysteine methylation. Two of these enzymes may modify their substrates during ribosomal assembly because the final methylated histidine and arginine residues are buried deep within the ribosome with contacts only with RNA. Two of these modifications occur broadly in eukaryotes, including humans, whereas the others demonstrate a more limited phylogenetic range. Analysis of strains where the methyltransferase genes are deleted has given insight into the physiological roles of these modifications. These reactions described here add diversity to the modifications that generate the typical methylated lysine and arginine residues previously described in histones and other proteins.

Unique Features of Human Protein Arginine Methyltransferase 9 (PRMT9) and Its Substrate RNA Splicing Factor SF3B2.

Human protein arginine methyltransferase (PRMT) 9 symmetrically dimethylates arginine residues on splicing factor SF3B2 (SAP145) and has been functionally linked to the regulation of alternative splicing of pre-mRNA. Site-directed mutagenesis studies on this enzyme and its substrate had revealed essential unique residues in the double E loop and the importance of the C-terminal duplicated methyltransferase domain. In contrast to what had been observed with other PRMTs and their physiological substrates, a peptide containing the methylatable Arg-508 of SF3B2 was not recognized by PRMT9 in vitro. Although amino acid substitutions of residues surrounding Arg-508 had no great effect on PRMT9 recognition of SF3B2, moving the arginine residue within this sequence abolished methylation. PRMT9 and PRMT5 are the only known mammalian enzymes capable of forming symmetric dimethylarginine (SDMA) residues as type II PRMTs. We demonstrate here that the specificity of these enzymes for their substrates is distinct and not redundant. The loss of PRMT5 activity in mouse embryo fibroblasts results in almost complete loss of SDMA, suggesting that PRMT5 is the primary SDMA-forming enzyme in these cells. PRMT9, with its duplicated methyltransferase domain and conserved sequence in the double E loop, appears to have a unique structure and specificity among PRMTs for methylating SF3B2 and potentially other polypeptides.

New small molecule inhibitors of histone methyl transferase DOT1L with a nitrile as a non-traditional replacement for heavy halogen atoms.

A number of new nucleoside derivatives are disclosed as inhibitors of DOT1L activity. SARs established that DOT1L inhibition could be achieved through incorporation of polar groups and small heterocycles at the 5-position (5, 6, 12) or by the application of alternative nitrogenous bases (18). Based on these results, CN-SAH (19) was identified as a potent and selective inhibitor of DOT1L activity where the polar 5-nitrile group was shown by crystallography to bind in the hydrophobic pocket of DOT1L. In addition, we show that a polar nitrile group can be used as a non-traditional replacement for heavy halogen atoms.

A direct, ratiometric, and quantitative MALDI-MS assay for protein methyltransferases and acetyltransferases.

Protein methylation and acetylation play important roles in biological processes, and misregulation of these modifications is involved in various diseases. Therefore, it is critical to understand the activities of the enzymes responsible for these modifications. Herein we describe a sensitive method for ratiometric quantification of methylated and acetylated peptides via MALDI-MS by direct spotting of enzymatic methylation and acetylation reaction mixtures without tedious purification procedures. The quantifiable detection limit for peptides with our method is approximately 10 fmol. This is achieved by increasing the signal-to-noise ratio through the addition of NH4H2PO4 to the matrix solution and reduction of the matrix α-cyanohydroxycinnamic acid concentration to 2 mg/ml. We have demonstrated the application of this method in enzyme kinetic analysis and inhibition studies. The unique feature of this method is the simultaneous quantification of multiple peptide species for investigation of processivity mechanisms. Its wide buffer compatibility makes it possible to be adapted to investigate the activity of any protein methyltransferase or acetyltransferase.

The interplay mechanism between IDH mutation, MGMT-promoter methylation, and PRMT5 activity in the progression of grade 4 astrocytoma: unraveling the complex triad theory.

Background: The dysregulation of Isocitrate dehydrogenase (IDH) and the subsequent production of 2-Hydroxyglutrate (2HG) may alter the expression of epigenetic proteins in Grade 4 astrocytoma. The interplay mechanism between IDH, O-6-methylguanine-DNA methyltransferase (MGMT)-promoter methylation, and protein methyltransferase proteins-5 (PRMT5) activity, with tumor progression has never been described. Methods: A retrospective cohort of 34 patients with G4 astrocytoma is classified into IDH-mutant and IDH-wildtype tumors. Both groups were tested for MGMT-promoter methylation and PRMT5 through methylation-specific and gene expression PCR analysis. Inter-cohort statistical significance was evaluated. Results: Both IDH-mutant WHO grade 4 astrocytomas (n = 22, 64.7%) and IDH-wildtype glioblastomas (n = 12, 35.3%) had upregulated PRMT5 gene expression except in one case. Out of the 22 IDH-mutant tumors, 10 (45.5%) tumors showed MGMT-promoter methylation and 12 (54.5%) tumors had unmethylated MGMT. All IDH-wildtype tumors had unmethylated MGMT. There was a statistically significant relationship between MGMT-promoter methylation and IDH in G4 astrocytoma (p-value = 0.006). Statistically significant differences in progression-free survival (PFS) were also observed among all G4 astrocytomas that expressed PRMT5 and received either temozolomide (TMZ) or TMZ plus other chemotherapies, regardless of their IDH or MGMT-methylation status (p-value=0.0014). Specifically, IDH-mutant tumors that had upregulated PRMT5 activity and MGMT-promoter methylation, who received only TMZ, have exhibited longer PFS. Conclusions: The relationship between PRMT5, MGMT-promoter, and IDH is not tri-directional. However, accumulation of D2-hydroxyglutarate (2-HG), which partially activates 2-OG-dependent deoxygenase, may not affect their activities. In IDH-wildtype glioblastomas, the 2HG-2OG pathway is typically inactive, leading to PRMT5 upregulation. TMZ alone, compared to TMZ-plus, can increase PFS in upregulated PRMT5 tumors. Thus, using a PRMT5 inhibitor in G4 astrocytomas may help in tumor regression.

Defining the elusive oncogenic role of the methyltransferase TMT1B.

Methyltransferases are enzymes fundamental to a wide range of normal biological activities that can become dysregulated during oncogenesis. For instance, the recent description of the methyltransferase-like (METTL) family of enzymes, has demonstrated the importance of the N6-adenosine-methyltransferase (m6A) modification in transcripts in the context of malignant transformation. Because of their importance, numerous METTL family members have been biochemically characterized to identify their cellular substrates, however some members such as METTL7B, recently renamed TMT1B and which is the subject of this review, remain enigmatic. First identified in the stacked Golgi, TMT1B is also localized to the endoplasmic reticulum as well as lipid droplets and has been reported as being upregulated in a wide range of cancer types including lung cancer, gliomas, and leukemia. Interestingly, despite evidence that TMT1B might act on protein substrates, it has also been shown to act on small molecule alkyl thiol substrates such as hydrogen sulfide, and its loss has been found to affect cellular proliferation and migration. Here we review the current evidence for TMT1B's activity, localization, and potential biological role in the context of both normal and cancerous cell types.

Specificity Analysis of Protein Methyltransferases and Discovery of Novel Substrates Using SPOT Peptide Arrays.

Posttranslational methylation of amino acid side chains in proteins mainly occurs on lysine, arginine, glutamine, and histidine residues. It is introduced by different protein methyltransferases (PMTs) and regulates many aspects of protein function including stability, activity, localization, and protein/protein interactions. Although the biological effects of PMTs are mediated by their methylation substrates, the full substrate spectrum of most PMTs is not known. For many PMTs, their activity on a particular potential substrate depends, among other factors, on the peptide sequence containing the target residue for methylation. In this protocol, we describe the application of SPOT peptide arrays to investigate the substrate specificity of PMTs and identify novel substrates. Methylation of SPOT peptide arrays makes it possible to study the methylation of many different peptides in one experiment at reasonable costs and thereby provides detailed information about the specificity of the PMT under investigation. In these experiments, a known substrate sequence is used as template to design a SPOT peptide array containing peptides with single amino acid exchanges at all positions of the sequence. Methylation of the array with the PMT provides detailed preferences for each amino acid at each position in the substrate sequence, yielding a substrate sequence specificity profile. This information can then be used to identify novel potential PMT substrates by in silico data base searches. Methylation of novel substrate candidates can be validated in SPOT arrays at peptide level, followed by validation at protein level in vitro and in cells.

PRMT5: An Emerging Target for Pancreatic Adenocarcinoma.

The overall survival of pancreatic ductal adenocarcinoma (PDAC) remains poor and its incidence is rising. Targetable mutations in PDAC are rare, thus novel therapeutic approaches are needed. Protein arginine methyltransferase 5 (PRMT5) overexpression is associated with worse survival and inhibition of PRMT5 results in decreased cancer growth across multiple cancers, including PDAC. Emerging evidence also suggests that altered RNA processing is a driver in PDAC tumorigenesis and creates a partial dependency on this process. PRMT5 inhibition induces altered splicing and this vulnerability can be exploited as a novel therapeutic approach. Three possible biological pathways underpinning the action of PRMT5 inhibitors are discussed; c-Myc regulation appears central to its action in the PDAC setting. Whilst homozygous MTAP deletion and symmetrical dimethylation levels are associated with increased sensitivity to PRMT5 inhibition, neither measure robustly predicts its growth inhibitory response. The immunomodulatory effect of PRMT5 inhibitors on the tumour microenvironment will also be discussed, based on emerging evidence that PDAC stroma has a significant bearing on disease behaviour and response to therapy. Lastly, with the above caveats in mind, current knowledge gaps and the implications and rationales for PRMT5 inhibitor development in PDAC will be explored.

The role of protein methyltransferases as potential novel therapeutic targets in squamous cell carcinoma of the head and neck.

Squamous cell carcinoma of the head and neck is a lethal disease with suboptimal survival outcomes and standard therapies with significant comorbidities. Whole exome sequencing data recently revealed an abundance of genetic and expression alterations in a family of enzymes known as protein methyltransferases in a variety of cancer types, including squamous cell carcinoma of the head and neck. These enzymes are mostly known for their chromatin-modifying functions through methylation of various histone substrates, though evidence supports their function also through methylation of non-histone substrates. This review summarizes the current knowledge on the function of protein methyltransferases in squamous cell carcinoma of the head and neck and highlights their promising potential as the next generation of therapeutic targets in this disease.

Molecular Evolution of the Substrate Specificity of Chloroplastic Aldolases/Rubisco Lysine Methyltransferases in Plants.

Rubisco and fructose-1,6-bisphosphate aldolases (FBAs) are involved in CO2 fixation in chloroplasts. Both enzymes are trimethylated at a specific lysine residue by the chloroplastic protein methyltransferase LSMT. Genes coding LSMT are present in all plant genomes but the methylation status of the substrates varies in a species-specific manner. For example, chloroplastic FBAs are naturally trimethylated in both Pisum sativum and Arabidopsis thaliana, whereas the Rubisco large subunit is trimethylated only in the former species. The in vivo methylation status of aldolases and Rubisco matches the catalytic properties of AtLSMT and PsLSMT, which are able to trimethylate FBAs or FBAs and Rubisco, respectively. Here, we created chimera and site-directed mutants of monofunctional AtLSMT and bifunctional PsLSMT to identify the molecular determinants responsible for substrate specificity. Our results indicate that the His-Ala/Pro-Trp triad located in the central part of LSMT enzymes is the key motif to confer the capacity to trimethylate Rubisco. Two of the critical residues are located on a surface loop outside the methyltransferase catalytic site. We observed a strict correlation between the presence of the triad motif and the in vivo methylation status of Rubisco. The distribution of the motif into a phylogenetic tree further suggests that the ancestral function of LSMT was FBA trimethylation. In a recent event during higher plant evolution, this function evolved in ancestors of Fabaceae, Cucurbitaceae, and Rosaceae to include Rubisco as an additional substrate to the archetypal enzyme. Our study provides insight into mechanisms by which SET-domain protein methyltransferases evolve new substrate specificity.

Aryl Pyrazoles as Potent Inhibitors of Arginine Methyltransferases: Identification of the First PRMT6 Tool Compound.

A novel aryl pyrazole series of arginine methyltransferase inhibitors has been identified. Synthesis of analogues within this series yielded the first potent, selective, small molecule PRMT6 inhibitor tool compound, EPZ020411. PRMT6 overexpression has been reported in several cancer types suggesting that inhibition of PRMT6 activity may have therapeutic utility. Identification of EPZ020411 provides the field with the first small molecule tool compound for target validation studies. EPZ020411 shows good bioavailability following subcutaneous dosing in rats making it a suitable tool for in vivo studies.