Protein Methyltransferases Research Articles

Chemical probes of histone lysine methyltransferases.

Growing evidence suggests that histone methyltransferases (HMTs, also known as protein methyltransferases (PMTs)) play an important role in diverse biological processes and human diseases by regulating gene expression and the chromatin state. Therefore, HMTs have been increasingly recognized by the biomedical community as a class of potential therapeutic targets. High quality chemical probes of HMTs, as tools for deciphering their physiological functions and roles in human diseases and testing therapeutic hypotheses, are critical for advancing this promising field. In this review, we focus on the discovery, characterization, and biological applications of chemical probes for HMTs.

Identifying novel selective non-nucleoside DNA methyltransferase 1 inhibitors through docking-based virtual screening.

The DNA methyltransferases (DNMTs) found in mammals include DNMT1, DNMT3A, and DNMT3B and are attractive targets in cancer chemotherapy. DNMT1 was the first among the DNMTs to be characterized, and it is responsible for maintaining DNA methylation patterns. A number of DNMT inhibitors have been reported, but most of them are nucleoside analogs that can lead to toxic side effects and lack specificity. By combining docking-based virtual screening with biochemical analyses, we identified a novel compound, DC_05. DC_05 is a non-nucleoside DNMT1 inhibitor with low micromolar IC50 values and significant selectivity toward other AdoMet-dependent protein methyltransferases. Through a process of similarity-based analog searching, compounds DC_501 and DC_517 were found to be more potent than DC_05. These three potent compounds significantly inhibited cancer cell proliferation. The structure-activity relationship (SAR) and binding modes of these inhibitors were also analyzed to assist in the future development of more potent and more specific DNMT1 inhibitors.

Neural crest specification and migration independently require NSD3-related lysine methyltransferase activity.

Neural crest precursors express genes that cause them to become migratory, multipotent cells, distinguishing them from adjacent stationary neural progenitors in the neurepithelium. Histone methylation spatiotemporally regulates neural crest gene expression; however, the protein methyltransferases active in neural crest precursors are unknown. Moreover, the regulation of methylation during the dynamic process of neural crest migration is unclear. Here we show that the lysine methyltransferase NSD3 is abundantly and specifically expressed in premigratory and migratory neural crest cells. NSD3 expression commences before up-regulation of neural crest genes, and NSD3 is necessary for expression of the neural plate border gene Msx1, as well as the key neural crest transcription factors Sox10, Snail2, Sox9, and FoxD3, but not gene expression generally. Nevertheless, only Sox10 histone H3 lysine 36 dimethylation requires NSD3, revealing unexpected complexity in NSD3-dependent neural crest gene regulation. In addition, by temporally limiting expression of a dominant negative to migratory stages, we identify a novel, direct requirement for NSD3-related methyltransferase activity in neural crest migration. These results identify NSD3 as the first protein methyltransferase essential for neural crest gene expression during specification and show that NSD3-related methyltransferase activity independently regulates migration.

Abstract 379: TARBP1, a member of the SPOUT methyltransferase family, is involved in human carcinogenesis

Abstract It has recently been recognized that protein methylation plays a critical role in many biological processes, and its deregulation has been observed in various kinds of diseases, including cancer. To date, a number of protein methyltransferases have already been reported to be associated with human carcinogenesis. Although many protein methyltransferases belong to the SET-domain containing protein family or the protein arginine methyltransferase (PRMT) family, a series of other methyltransferase families have also been discovered by bioinformatics-based approaches. On the contrary, physiological functions of these methyltransferases, especially, the significance in human disease like cancer, still remains unknown. In this study, we demonstrated that expression levels of TARBP1 (TAR RNA-Binding Protein 1), a member of the SPOUT methyltransferase family, are significantly up-regulated in many types of cancer tissues compared to corresponding non-tumor tissues. In addition, knockdown of TARBP1 resulted in the suppression of cancer cell growth, indicating that TARBP1 seems to regulate the proliferation of cancer cells. Since expression levels of TARBP1 in normal tissues are significantly low, TARBP1 may be a good new target for anti-cancer therapy. Citation Format: Makoto Nakakido, Yusuke Nakamura, Ryuji Hamamoto. TARBP1, a member of the SPOUT methyltransferase family, is involved in human carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 379. doi:10.1158/1538-7445.AM2014-379

Reaction coupling between wild-type and disease-associated mutant EZH2.

EZH2 and EZH1 are protein methyltransferases (PMTs) responsible for histone H3, lysine 27 (H3K27) methylation. Trimethylation of H3K27 (H3K27me3) is a hallmark of many cancers, including non-Hodgkin lymphoma (NHL). Heterozygous EZH2 point mutations at Tyr641, Ala677, and Ala687 have been observed in NHL. The Tyr641 mutations enhance activity on H3K27me2 but have weak or no activity on unmethylated H3K27, whereas the Ala677 and Ala687 mutations use substrates of all methylation states effectively. It has been proposed that enzymatic coupling of the wild-type and mutant enzymes leads to the oncogenic H3K27me3 mark in mutant-bearing NHL. We show that coupling with the wild-type enzyme is needed to achieve H3K27me3 for several mutants, but that others are capable of achieving H3K27me3 on their own. All forms of PRC2 (wild-type and mutants) display kinetic signatures that are consistent with a distributive mechanism of catalysis.

(R)-PFI-2 is a potent and selective inhibitor of SETD7 methyltransferase activity in cells.

SET domain containing (lysine methyltransferase) 7 (SETD7) is implicated in multiple signaling and disease related pathways with a broad diversity of reported substrates. Here, we report the discovery of (R)-PFI-2-a first-in-class, potent (Ki (app) = 0.33 nM), selective, and cell-active inhibitor of the methyltransferase activity of human SETD7-and its 500-fold less active enantiomer, (S)-PFI-2. (R)-PFI-2 exhibits an unusual cofactor-dependent and substrate-competitive inhibitory mechanism by occupying the substrate peptide binding groove of SETD7, including the catalytic lysine-binding channel, and by making direct contact with the donor methyl group of the cofactor, S-adenosylmethionine. Chemoproteomics experiments using a biotinylated derivative of (R)-PFI-2 demonstrated dose-dependent competition for binding to endogenous SETD7 in MCF7 cells pretreated with (R)-PFI-2. In murine embryonic fibroblasts, (R)-PFI-2 treatment phenocopied the effects of Setd7 deficiency on Hippo pathway signaling, via modulation of the transcriptional coactivator Yes-associated protein (YAP) and regulation of YAP target genes. In confluent MCF7 cells, (R)-PFI-2 rapidly altered YAP localization, suggesting continuous and dynamic regulation of YAP by the methyltransferase activity of SETD7. These data establish (R)-PFI-2 and related compounds as a valuable tool-kit for the study of the diverse roles of SETD7 in cells and further validate protein methyltransferases as a druggable target class.

Large-scale, protection-free synthesis of Se-adenosyl-L-selenomethionine analogues and their application as cofactor surrogates of methyltransferases.

S-Adenosyl-l-methionine (SAM) analogues have previously demonstrated their utility as chemical reporters of methyltransferases. Here we describe the facile, large-scale synthesis of Se-alkyl Se-adenosyl-l-selenomethionine (SeAM) analogues and their precursor, Se-adenosyl-l-selenohomocysteine (SeAH). Comparison of SeAM analogues with their equivalent SAM analogues suggests that sulfonium-to-selenonium substitution can enhance their compatibility with certain protein methyltransferases, favoring otherwise less reactive SAM analogues. Ready access to SeAH therefore enables further application of SeAM analogues as chemical reporters of diverse methyltransferases.

Uncovering the protein lysine and arginine methylation network in Arabidopsis chloroplasts.

Post-translational modification of proteins by the addition of methyl groups to the side chains of Lys and Arg residues is proposed to play important roles in many cellular processes. In plants, identification of non-histone methylproteins at a cellular or subcellular scale is still missing. To gain insights into the extent of this modification in chloroplasts we used a bioinformatics approach to identify protein methyltransferases targeted to plastids and set up a workflow to specifically identify Lys and Arg methylated proteins from proteomic data used to produce the Arabidopsis chloroplast proteome. With this approach we could identify 31 high-confidence Lys and Arg methylation sites from 23 chloroplastic proteins, of which only two were previously known to be methylated. These methylproteins are split between the stroma, thylakoids and envelope sub-compartments. They belong to essential metabolic processes, including photosynthesis, and to the chloroplast biogenesis and maintenance machinery (translation, protein import, division). Also, the in silico identification of nine protein methyltransferases that are known or predicted to be targeted to plastids provided a foundation to build the enzymes/substrates relationships that govern methylation in chloroplasts. Thereby, using in vitro methylation assays with chloroplast stroma as a source of methyltransferases we confirmed the methylation sites of two targets, plastid ribosomal protein L11 and the β-subunit of ATP synthase. Furthermore, a biochemical screening of recombinant chloroplastic protein Lys methyltransferases allowed us to identify the enzymes involved in the modification of these substrates. The present study provides a useful resource to build the methyltransferases/methylproteins network and to elucidate the role of protein methylation in chloroplast biology.

Selective Non-nucleoside Inhibitors of Human DNA Methyltransferases Active in Cancer Including in Cancer Stem Cells

DNA methyltransferases (DNMTs) are important enzymes involved in epigenetic control of gene expression and represent valuable targets in cancer chemotherapy. A number of nucleoside DNMT inhibitors (DNMTi) have been studied in cancer, including in cancer stem cells, and two of them (azacytidine and decitabine) have been approved for treatment of myelodysplastic syndromes. However, only a few non-nucleoside DNMTi have been identified so far, and even fewer have been validated in cancer. Through a process of hit-to-lead optimization, we report here the discovery of compound 5 as a potent non-nucleoside DNMTi that is also selective toward other AdoMet-dependent protein methyltransferases. Compound 5 was potent at single-digit micromolar concentrations against a panel of cancer cells and was less toxic in peripheral blood mononuclear cells than two other compounds tested. In mouse medulloblastoma stem cells, 5 inhibited cell growth, whereas related compound 2 showed high cell differentiation. To the best of our knowledge, 2 and 5 are the first non-nucleoside DNMTi tested in a cancer stem cell line.

Molecular Pathways: Protein Methyltransferases in Cancer

The protein methyltransferases (PMT) constitute a large and important class of enzymes that catalyze site-specific methylation of lysine or arginine residues on histones and other proteins. Site-specific histone methylation is a critical component of chromatin regulation of gene transcription-a pathway that is often genetically altered in human cancers. Oncogenic alterations (e.g., mutations, chromosomal translocations, and others) of PMTs, or of associated proteins, have been found to confer unique dependencies of cancer cells on the activity of specific PMTs. Examples of potent, selective small-molecule inhibitors of specific PMTs are reviewed that have been shown to kill cancers cells bearing such oncogenic alterations, while having minimal effect on proliferation of nonaltered cells. Selective inhibitors of the PMTs, DOT1L and EZH2, have entered phase I clinical studies and additional examples of selective PMT inhibitors are likely to enter the clinic soon. The current state of efforts toward clinical testing of selective PMT inhibitors as personalized cancer therapeutics is reviewed here.

Protein Methyltransferase Inhibitors as Personalized Cancer Therapeutics

The protein methyltransferases (PMTs) constitute a class of enzymes that catalyze the methylation of lysine or arginine residues on histones and other proteins. A number of PMTs have been shown to be genetically altered in cancers through, for example: gene amplification, chromosomal translocations, point mutations, and synthetic lethal relationships [Copeland et al. (2013) Oncogene 32: 939-946]. The enzymes DOT1L and EZH2 provide two representative examples of altered PMTs that act as genetic drivers of specific human cancers. The enzymatic activity of DOT1L is associated with a chromosomal translocation that is universally found in patients with MLL-rearranged leukemia [Daigle et al. (2011) Cancer Cell 20: 53-65; Daigle et al. Blood, prepublished June 25, 2013; doi:10.1182/blood-2013-04-497644]. Point mutations at Y641 of EZH2 are found in a subset of non-Hodgkin lymphoma patients; the enzymatic activity of both wild-type and mutant EZH2 are required for pathogenesis in these patients [Sneeringer et al. (2010) Proc. Natl. Acad. Sci. USA 107: 20980-20985; Knutson et al. (2012) Nature Chem. Biol. 8: 890-896]. Also, deletion of the SMARCB1 subunit of the SWI/SNF chromatin-remodeling complex occurs in nearly all malignant rhabdoid tumors (MRTs), a childhood cancer with a particularly poor prognosis. An antagonistic relationship has been demonstrated between the biochemical action on chromatin of the SWI/SNF complex and EZH2 (in the context of the polycomb repressive complex 2), that is relieved in MRTs due to the SMARCB1 deletion. MRTs therefore demonstrate increased reliance on the enzymatic activity of EZH2 for proliferation and we have shown that MRT cells deficient in SMARCB1 are selectively killed by inhibition of EZH2 in cell culture and in mouse xenograft models [Knutson et al. (2013) Proc. Natl. Acad. Sci. USA 110: 7922-7927]. Drug discovery efforts have yielded potent, selective inhibitors of each of these targets that have now transitioned into phase I clinical trials. These inhibitors affect the appropriate histone methyl marks in cells, lead to selective cell killing that is dependent on genetic alteration of the target enzyme, and effect tumor growth inhibition in xenograft models. These data portend the effective use of selective PMT inhibitors as a novel modality for future personalized cancer therapeutics. Disclosures:Copeland:Epizyme: Employment, Equity Ownership.

Abstract PL03-04: Protein methyltransferase inhibitors as personalized cancer therapeutics.

Abstract The protein methyltransferases (PMTs) constitute a class of enzymes that catalyze the methylation of lysine or arginine residues on histones and other proteins. A number of PMTs have been shown to be genetically altered in cancers through, for example, gene amplification, chromosomal translocations, point mutations and synthetic lethal relationships. The enzymes DOT1L and EZH2 provide two representative examples of altered PMTs that act as genetic drivers of specific human cancers. The enzymatic activity of DOT1L is associated with a chromosomal translocation that is universally found in patients with MLL-rearranged leukemia. Point mutations at Y641 of EZH2 are found in a subset of non-Hodgkins lymphoma patients; the enzymatic activity of both wild type and mutant EZH2 are required for pathogenesis in these patients. Also, deletion of the INI1 (SMARCB1 or hSNF5) subunit of the SWI/SNF chromatin-remodeling complex occurs in nearly all malignant rhabdoid tumors (MRTs), a childhood cancer with a particularly poor prognosis. An antagonistic relationship has been demonstrated between the biochemical action on chromatin of the SWI/SNF complex and EZH2 (in the context of the polycomb repressive complex 2), that is relieved in MRTs due to the INI1 deletion. MRTs therefore demonstrate increased reliance on the enzymatic activity of EZH2 for proliferation and we have shown that MRT cells deficient in INI1 are selectively killed by inhibition of EZH2 in cell culture and in mouse xenograft models. Drug discovery efforts have yielded potent, selective inhibitors of each of these targets that have now transitioned into Phase I clinical trials. These inhibitors affect the appropriate histone methyl marks in cells, lead to selective cell killing that is dependent on genetic alteration of the target enzyme and effect tumor growth inhibition in xenograft models. These data portend the effective use of selective PMT inhibitors as a novel modality for future personalized cancer therapeutics. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PL03-04. Citation Format: Robert A. Copeland. Protein methyltransferase inhibitors as personalized cancer therapeutics. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PL03-04.

Abstract A125: Universal homogenous bioluminescent assay to monitor the effect of modulators on various classes of methyltransferases in vitro.

Abstract Methylation/demethylation of DNA and proteins play major roles in modulation of the epigenome and has been implicated in a wide variety of human diseases. Recent biochemical and biological data suggest that the enzymatic activities of several of these enzymes have pathogenic roles in cancer, inflammation, and neurodegenerative diseases. Thus, pharmacological modulation of these enzymes by small molecules will be beneficial in developing novel therapeutics for multiple unmet medical needs. Towards this goal of searching for activators/inhibitors of these enzymes for the development of next generation of drugs, screening assays for these modulators are urgently needed. To address these unmet needs, we have developed a novel assay that monitors the activities of these enzymes and their modulation by small molecules. The assay is bioluminescent based, HTS formatted, and highly sensitive. The assay is universal since it is based on monitoring the formation of the universal product S-adenosylhomocysteine (SAH), i.e., capable of detecting changes in activity of a broad range of methyltransferases such as DNA, protein, and small molecules methyltransferases. In addition, the assay has been validated for all classes of protein methyltransferases (lysine and arginine), and with different types of substrates (small peptides, large proteins, or even nucleosomes). This enables determining the specificity of these enzymes and their substrate requirements. The assay has high signal to background and low C.V. The assay is robust (Z’ value>0.7) and has been validated using various plate densities such as 96-, 384, and 1536-well plates. A strong feature of this assay is its utility with broad range of substrates with no limitations on the use of high concentrations of substrates or the composition of the substrates (short vs. long peptides), thus enabling the generation of kinetic data and determining the mechanism of action of various modulators of methyltransferases of interest. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A125. Citation Format: Said A. Goueli, Kevin Hsaio. Universal homogenous bioluminescent assay to monitor the effect of modulators on various classes of methyltransferases in vitro. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A125.

Defining efficient enzyme–cofactor pairs for bioorthogonal profiling of protein methylation

Protein methyltransferase (PMT)-mediated posttranslational modification of histone and nonhistone substrates modulates stability, localization, and interacting partners of target proteins in diverse cellular contexts. These events play critical roles in normal biological processes and are frequently deregulated in human diseases. In the course of identifying substrates of individual PMTs, bioorthogonal profiling of protein methylation (BPPM) has demonstrated its merits. In this approach, specific PMTs are engineered to process S-adenosyl-L-methionine (SAM) analogs as cofactor surrogates and label their substrates with distinct chemical modifications for target elucidation. Despite the proof-of-concept advancement of BPPM, few efforts have been made to explore its generality. With two cancer-relevant PMTs, EuHMT1 (GLP1/KMT1D) and EuHMT2 (G9a/KMT1C), as models, we defined the key structural features of engineered PMTs and matched SAM analogs that can render the orthogonal enzyme-cofactor pairs for efficient catalysis. Here we have demonstrated that the presence of sulfonium-β-sp(2) carbon and flexible, medium-sized sulfonium-δ-substituents are crucial for SAM analogs as BPPM reagents. The bulky cofactors can be accommodated by tailoring the conserved Y1211/Y1154 residues and nearby hydrophobic cavities of EuHMT1/2. Profiling proteome-wide substrates with BPPM allowed identification of >500 targets of EuHMT1/2 with representative targets validated using native EuHMT1/2 and SAM. This finding indicates that EuHMT1/2 may regulate many cellular events previously unrecognized to be modulated by methylation. The present work, therefore, paves the way to a broader application of the BPPM technology to profile methylomes of diverse PMTs and elucidate their downstream functions.

Abstract 1297: SETD6-mediated deregulation of NF- κB signaling in 4-ABP-induced bladder carcinogenesis.

Abstract Epidemiological data support an etiological role for chemicals from occupational exposure and cigarette smoke with increased risk of bladder cancer. However, epigenetic effects of these chemicals remain to be elucidated. Protein methyltransferases (PMTs) methylate protein lysine and arginine residues and play an important role in regulating gene transcription. Deregulation of SET-domain-containing PMTs has a crucial role in carcinogenesis. SETD6 has been shown to monomethylate RelA (a principal subunit of NF-κB at Lys310). Monomethylated RelA recruits GLP (G9A-like protein), a histone dimethyl transferase to the NF-κB target genes, which results in transcriptional repression. The role of SETD6 in bladder cancer is unknown. We used qPCR and immunoblotting to determine changes in SETD6 at the mRNA and protein level in bladder cancer cells relative to primary urothelial cells. We also found that knockdown of SETD6 significantly decreases the viability and proliferation of bladder cancer cells suggesting that SETD6 may function as an oncogene in bladder cancer. Furthermore, we also found that message and protein levels of SETD6 are induced in primary urothelial cells following treatment of 4-aminobiphenyl (4-ABP), an important constituent of cigarette smoke. Preliminary results indicate that induction may be mediated by 4-ABP-induced ROS. RelAK310Me1 level also increased with 4-ABP treatment showing the increase in SETD6 functional activity with 4-ABP exposure. We also found the decrease in expression of RelB, one of the target genes of RelA. Overall our results show that SETD6 may function as an oncogene in 4-ABP induced bladder cancer by deregulating transactivation property of RelA. These results show for the first time the relevance of PKMTs and epigenetic regulation of NF-κB in carcinogen induced bladder cancer. Supported by RO3 CA136058 (RG). Citation Format: Neelam Mukherjee, Rita Ghosh. SETD6-mediated deregulation of NF- κB signaling in 4-ABP-induced bladder carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1297. doi:10.1158/1538-7445.AM2013-1297

Abstract 4236: Universal homogenous bioluminescent assay to monitor the effect of modulators on various classes of methyltransferases in vitro.

Abstract Methylation/demethylation of DNA and Proteins play major roles in modulation of the epigenome and has been implicated in a wide variety of human diseases. Recent biochemical and biological data suggest that the enzymatic activities of several of these enzymes have pathogenic roles in cancer, inflammation, and neurodegenerative diseases. Thus, pharmacological modulation of these enzymes by small molecules will be beneficial in developing novel therapeutics for multiple unmet medical needs. Towards this goal of searching for activators/inhibitors of these enzymes for the development of next generation of drugs, screening assays for these modulators are urgently needed. To address these unmet needs, we have developed a novel assay that monitors the activities of these enzymes and their modulation by small molecules. The assay is bioluminescent based, HTS formatted and highly sensitive. The assay is universal since it is based on monitoring the formation of the universal product S-adenosylhomocysteine (SAH), i.e., capable of detecting changes in activity of a broad range of methyltransferases such as DNA, protein, and small molecules methyltransferases. In addition, the assay has been validated for all classes of protein methyltransferases (Lysine and Arginine), and with different types of substrates (small peptides, large proteins, or even nucleosomes). This enables determining the specificity of these enzymes and their substrate requirements. The assay has high signal to background and low C.V. The assay is robust (Z’ value > 0.7) and has been validated using various plate densities such as 96-, 384, and 1536-well plates. A strong feature of this assay is its utility with broad range of substrates with no limitations on the use of high concentrations of substrates or the composition of the substrates (short vs. long peptides), thus enabling the generation of kinetic data and determining the mechanism of action of various modulators of methyltransferases of interest. Citation Format: Said A. Goueli, Kevin Hsiao, Hicham Zegzouti. Universal homogenous bioluminescent assay to monitor the effect of modulators on various classes of methyltransferases in vitro. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4236. doi:10.1158/1538-7445.AM2013-4236

Structural Insights of Outer Membrane Protein Methyltransferases from Rickettsia

The observation of correlation between hypermethylation of rickettsial outer membrane protein B (OmpB) and the bacterial virulence has suggested the importance of an enzymatic system for the methylation of OmpB. Two protein lysine methyltransferases (rPKMT1 and rPKMT2) were found to methylate recombinant OmpB fragments through the incorporation of radioactively labeled methyl group from S‐adenosylmethionine (SAM). The two proteins share 45 % identity. LC/MS‐MS analyses showed both rPKMT1 and rPKMT2 methylated at numerous lysine residues of OmpB, but rPKMT1 catalyzed mono‐, di‐ and tri‐methylation of lysine, while rPKMT2 catalyzed exclusively trimethylation of lysine. We used X‐ray crystallography to investigate the structure of the lysine methyltransferases. The crystal structures of rPKMT1 and rPKMT2 and the respective complexes with SAM were determined. These are the first reported crystal structures of outer membrane protein methyltransferases. The crystal structures of rPKMT1 and rPKMT2 are superimposable. Both methyltransferases are dimeric, which was confirmed using dynamic light scattering. Each monomer consists of dimerization, SAM binding, middle, and C‐terminal domains. Comparison of the two structures revealed a number of structural insights of the unusual enzymatic methylation of OmpB.

Bioorthogonal Profiling of Protein Methylation (BPPM) Using an Azido Analog of S‐Adenosyl‐L‐Methionine

Protein methyltransferases (PMTs) utilize S-adenosyl-L-methionine (SAM) as a cofactor and transfer its sulfonium methyl moiety to diverse substrates. These methylation events can lead to meaningful biological outcomes, from transcriptional activation/silencing to cell cycle regulation. This article describes recently developed technology based on protein engineering in tandem with SAM analog cofactors and bioorthogonal click chemistry to unambiguously profile the substrates of a specific PMT. The protocols encapsulate the logic and methods of selectively profiling the substrates of a candidate PMT by (1) engineering the selected PMT to accommodate a bulky SAM analog; (2) generating a proteome containing the engineered PMT; (3) visualizing the proteome-wide substrates of the designated PMT via bioorthogonal labeling with a fluorescent tag; and finally (4) pulling down the proteome-wide substrates for mass spectrometric analysis.

Strategy to Target the Substrate Binding site of SET Domain Protein Methyltransferases

Protein methyltransferases (PMTs) are a novel gene family of therapeutic relevance involved in chromatin-mediated signaling and other biological mechanisms. Most PMTs are organized around the structurally conserved SET domain that catalyzes the methylation of a substrate lysine. A few potent chemical inhibitors compete with the protein substrate, and all are anchored in the channel recruiting the methyl-accepting lysine. We propose a novel strategy to design focused chemical libraries targeting the substrate binding site, where a limited number of warheads each occupying the lysine-channel of multiple enzymes would be decorated by different substituents. A variety of sequence and structure-based approaches used to analyze the diversity of the lysine channel of SET domain PMTs support the relevance of this strategy. We show that chemical fragments derived from published inhibitors are valid warheads that can be used in the design of novel focused libraries targeting other PMTs.

Profiling Protein Methylation with Cofactor Analog Containing Terminal Alkyne Functionality

Enzymatic transmethylation from the cofactor S-adenosyl-L-methionine (SAM) to biological molecules has recently garnered increased attention because of the diversity of possible substrates and implications in normal biology and diseases. To reveal the substrates of protein methyltransferases (PMTs), the present article focuses on an alkyne-containing SAM mimic, Se-adenosyl-L-selenomethionine (ProSeAM), and a cleavable azido-azo-biotin probe to profile the targets of endogenous PMTs in cellular contexts. This article describes the stepwise preparation of cell lysates containing active, endogenous PMTs and subsequent target labeling with ProSeAM. The article continues with the enrichment of the ProSeAM-labeled proteins with the azido-azo biotin probe as a pulldown reagent and the subsequent reductive elution with sodium dithionate for proteomic analysis. The protocols provided here were formulated for ProSeAM as a profiling reagent but can be applied to other terminal-alkyne-containing SAM analog cofactors.