Gil Blum

Loss of BAP1 function leads to EZH2-dependent transformation

The tumor suppressors BAP1 and ASXL1 interact to form a polycomb deubiquitinase complex that removes monoubiquitin from histone H2A lysine 119 (H2AK119Ub). However, BAP1 and ASXL1 are mutated in distinct cancer types, consistent with independent roles in regulating epigenetic state and malignant transformation. Here we demonstrate that Bap1 loss in mice results in increased trimethylated histone H3 lysine 27 (H3K27me3), elevated enhancer of zeste 2 polycomb repressive complex 2 subunit (Ezh2) expression, and enhanced repression of polycomb repressive complex 2 (PRC2) targets. These findings contrast with the reduction in H3K27me3 levels seen with Asxl1 loss. Conditional deletion of Bap1 and Ezh2 in vivo abrogates the myeloid progenitor expansion induced by Bap1 loss alone. Loss of BAP1 results in a marked decrease in H4K20 monomethylation (H4K20me1). Consistent with a role for H4K20me1 in the transcriptional regulation of EZH2, expression of SETD8—the H4K20me1 methyltransferase—reduces EZH2 expression and abrogates the proliferation of BAP1-mutant cells. Furthermore, mesothelioma cells that lack BAP1 are sensitive to EZH2 pharmacologic inhibition, suggesting a novel therapeutic approach for BAP1-mutant malignancies.

Sinefungin Derivatives as Inhibitors and Structure Probes of Protein Lysine Methyltransferase SETD2

Epigenetic regulation is involved in numerous physiological and pathogenic processes. Among the key regulators that orchestrate epigenetic signaling are over 50 human protein lysine methyltransferases (PKMTs). Interrogation of the functions of individual PKMTs can be facilitated by target-specific PKMT inhibitors. Given the emerging need for such small molecules, we envisioned an approach to identify target-specific methyltransferase inhibitors by screening privileged small-molecule scaffolds against diverse methyltransferases. In this work, we demonstrated the feasibility of such an approach by identifying the inhibitors of SETD2. N-propyl sinefungin (Pr-SNF) was shown to interact preferentially with SETD2 by matching the distinct transition-state features of SETD2s catalytically active conformer. With Pr-SNF as a structure probe, we further revealed the dual roles of SETD2s post-SET loop in regulating substrate access through a distinct topological reconfiguration. Privileged sinefungin scaffolds are expected to have broad use as structure and chemical probes of methyltransferases.

Profiling Genome-Wide Chromatin Methylation with Engineered Posttranslation Apparatus within Living Cells

Protein methyltransferases (PMTs) have emerged as important epigenetic regulators in myriad biological processes in both normal physiology and disease conditions. However, elucidating PMT-regulated epigenetic processes has been hampered by ambiguous knowledge about in vivo activities of individual PMTs particularly because of their overlapping but nonredundant functions. To address limitations of conventional approaches in mapping chromatin modification of specific PMTs, we have engineered the chromatin-modifying apparatus and formulated a novel technology, termed clickable chromatin enrichment with parallel DNA sequencing (CliEn-seq), to probe genome-wide chromatin modification within living cells. The three-step approach of CliEn-seq involves in vivo synthesis of S-adenosyl-L-methionine (SAM) analogues from cell-permeable methionine analogues by engineered SAM synthetase (methionine adenosyltransferase or MAT), in situ chromatin modification by engineered PMTs, subsequent enrichment and sequencing of the uniquely modified chromatins. Given critical roles of the chromatin-modifying enzymes in epigenetics and structural similarity among many PMTs, we envision that the CliEn-seq technology is generally applicable in deciphering chromatin methylation events of individual PMTs in diverse biological settings.

Se-adenosyl-L-selenomethionine cofactor analogue as a reporter of protein methylation.

Posttranslational methylation by S-adenosyl-L-methionine(SAM)-dependent methyltransferases plays essential roles in modulating protein function in both normal and disease states. As such, there is a growing need to develop chemical reporters to examine the physiological and pathological roles of protein methyltransferases. Several sterically bulky SAM analogues have previously been used to label substrates of specific protein methyltransferases. However, broad application of these compounds has been limited by their general incompatibility with native enzymes. Here we report a SAM surrogate, ProSeAM (propargylic Se-adenosyl-l-selenomethionine), as a reporter of methyltransferases. ProSeAM can be processed by multiple protein methyltransferases for substrate labeling. In contrast, sulfur-based propargylic SAM undergoes rapid decomposition at physiological pH, likely via an allene intermediate. In conjunction with fluorescent/affinity-based azide probes, copper-catalyzed azide-alkyne cycloaddition chemistry, in-gel fluorescence visualization and proteomic analysis, we further demonstrated ProSeAMs utility to profile substrates of endogenous methyltransferases in diverse cellular contexts. These results thus feature ProSeAM as a convenient probe to study the activities of endogenous protein methyltransferases.

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

Significance Many proteins undergo various posttranslational modifications for proper functions. One such modification is methylation carried out by enzyme–cofactor pairs of protein methyltransferases (PMTs) and S-adenosyl-L-methionine (SAM). Identification of methylated proteins is quite challenging because of the small size and chemical inertness of the methyl group. To address this challenge, we have synthesized SAM surrogates by replacing SAM’s methyl group with bulky, chemically active functionalities and demonstrated their utility as alternative cofactors of engineered PMTs for substrate labeling. Proteins modified with such chemical moieties are amenable to bioorthogonal reactions for subsequent enrichment and identification. An engineered enzyme–cofactor pair has been successfully used to reveal numerous methylated proteins. 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-sp2 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.

Profiling Substrates of Protein Arginine N-Methyltransferase 3 with S-Adenosyl-l-methionine Analogues

Protein arginine N-methyltransferase 3 (PRMT3) belongs to the family of type I PRMTs and harbors the activity to use S-adenosyl-l-methionine (SAM) as a methyl-donor cofactor for protein arginine labeling. However, PRMT3s functions remain elusive with the lacked knowledge of its target scope in cellular settings. Inspired by the emerging Bioorthogonal Profiling of Protein Methylation (BPPM) using engineered methyltransferases and SAM analogues for target identification, the current work documents the endeavor to systematically explore the SAM-binding pocket of PRMT3 and identify suitable PRMT3 variants for BPPM. The M233G single point mutation transforms PRMT3 into a promiscuous alkyltransferase using sp(2)-β-sulfonium-containing SAM analogues as cofactor surrogates. Here the conserved methionine was defined as a hot spot that can be engineered alone or in combination with nearby residues to render cofactor promiscuity of multiple type I PRMTs. With this promiscuous variant and the matched 4-propargyloxy-but-2-enyl (Pob)-SAM analogue as the BPPM reagents, more than 80 novel proteins were readily uncovered as potential targets of PRMT3 in the cellular context. Subsequent target validation and functional analysis correlated the PRMT3 methylation to several biological processes such as cytoskeleton dynamics, whose roles might be compensated by other PRMTs. These BPPM-revealed substrates are primarily localized but not restricted in cytoplasm, the preferred site of PRMT3. The broad localization pattern may implicate the diverse roles of PRMT3 in the cellular setting. The revelation of PRMT3 targets and the transformative character of BPPM for other PRMTs present unprecedented pathways toward elucidating physiological and pathological roles of diverse PRMTs.

Small-Molecule Inhibitors of SETD8 with Cellular Activity

SETD8/SET8/Pr-SET7/KMT5A is the sole protein lysine methyltransferase (PKMT) known to monomethylate lysine 20 of histone H4 in vivo. SETD8’s methyltransferase activity has been implicated in many essential cellular processes including DNA replication, DNA damage response, transcription modulation, and cell cycle regulation. Developing SETD8 inhibitors with cellular activity is a key step toward elucidating the diverse roles of SETD8 via convenient pharmacological perturbation. From the hits of a prior high throughput screen (HTS), SPS8I1–3 (NSC663284, BVT948, and ryuvidine) were validated as potent SETD8 inhibitors. These compounds contain different structural motifs and inhibit SETD8 via distinct modes. More importantly, these compounds show cellular activity by suppressing the H4K20me1 mark of SETD8 and recapitulate characteristic S/G2/M-phase cell cycle defects as observed for RNAi-mediated SETD8 knockdown. The commonality of SPS8I1–3 against SETD8, together with their distinct structures and mechanisms for SETD8 inhibition, argues for the collective application of these compounds as SETD8 inhibitors.

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. Curr. Protoc. Chem. Biol. 5:67‐88

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. Curr. Protoc. Chem. Biol. 5:45‐66

Kinetic isotope effects reveal early transition state of protein lysine methyltransferase SET8

Significance We developed an MS-based method to determine kinetic isotope effects and binding isotope effects on protein lysine methyltransferase SET8-catalyzed monomethylation. These parameters, coupled with steady-state kinetics and molecular modeling, outlined the reaction path of SET8-catalyzed methylation. Upon the formation of the S-adenosyl-l-methionine–SET8–histone 4 lysine 20 intermediate complex followed by lysine deprotonation, the reaction goes through an early, asymmetrical transition state (TS) with the small engagement of the C-N bond and the partial dissociation of the C-S bond. This TS structure is distinct from the known TS structures of other protein lysine methyltransferases (PKMTs) and thus presents the feasibility to design selective TS analog inhibitors against PKMTs. The developed techniques can also be generally applicable to examining other protein methylation and posttranslational modifications. Protein lysine methyltransferases (PKMTs) catalyze the methylation of protein substrates, and their dysregulation has been linked to many diseases, including cancer. Accumulated evidence suggests that the reaction path of PKMT-catalyzed methylation consists of the formation of a cofactor(cosubstrate)–PKMT–substrate complex, lysine deprotonation through dynamic water channels, and a nucleophilic substitution (SN2) transition state for transmethylation. However, the molecular characters of the proposed process remain to be elucidated experimentally. Here we developed a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method and corresponding mathematic matrix to determine precisely the ratios of isotopically methylated peptides. This approach may be generally applicable for examining the kinetic isotope effects (KIEs) of posttranslational modifying enzymes. Protein lysine methyltransferase SET8 is the sole PKMT to monomethylate histone 4 lysine 20 (H4K20) and its function has been implicated in normal cell cycle progression and cancer metastasis. We therefore implemented the MS-based method to measure KIEs and binding isotope effects (BIEs) of the cofactor S-adenosyl-l-methionine (SAM) for SET8-catalyzed H4K20 monomethylation. A primary intrinsic 13C KIE of 1.04, an inverse intrinsic α-secondary CD3 KIE of 0.90, and a small but statistically significant inverse CD3 BIE of 0.96, in combination with computational modeling, revealed that SET8-catalyzed methylation proceeds through an early, asymmetrical SN2 transition state with the C-N and C-S distances of 2.35–2.40 Å and 2.00–2.05 Å, respectively. This transition state is further supported by the KIEs, BIEs, and steady-state kinetics with the SAM analog Se-adenosyl-l-selenomethionine (SeAM) as a cofactor surrogate. The distinct transition states between protein methyltransferases present the opportunity to design selective transition-state analog inhibitors.