Activity Guided Azide-methyllysine Photo-trapping for Substrate Profiling of Lysine Demethylases.

Reversible post-translational modifications (PTMs) are key to establishing protein-protein and protein-nucleic acid interactions that govern a majority of the signaling pathways in cells. Sequence-specific PTMs are catalyzed by transferases, and their removal is carried out by a class of reverse-acting enzymes termed "detransferases". Currently available chemoproteomic approaches have been valuable in characterizing substrates of transferases. However, proteome-wide cataloging of the substrates of detransferases is challenging, mostly due to the loss of the epitope, rendering immunoprecipitation and activity-based methods ineffective. Herein, we develop a general chemoproteomic strategy called crosslinking-assisted substrate identification (CASI) for systematic characterization of cellular targets of detransferases and successfully apply it to lysine demethylases (KDMs) which catalyze the removal of methyl groups from lysine sidechain in histones to modulate gene transcription. By setting up a targeted azido-methylamino photo-reaction deep inside the active site of KDM4, engineered to carry p-azido phenylalanine, we reveal a novel "demethylome" that has escaped the traditional methods. The proteomic survey led to the identification of a battery of nonhistone substrates of KDM4, extending the biological footprint of KDM4 beyond its canonical functions in gene transcription. A notable finding of KDM4A-mediated demethylation of an evolutionarily conserved lysine residue in eukaryotic translational initiation factor argues for a much broader role of KDM4A in ribosomal processes. CASI, representing a substantive departure from earlier approaches by shifting focus from simple peptide-based probes to employing full-length photo-activatable demethylases, is poised to be applied to >400 human detransferases, many of which have remained poorly understood due to the lack of knowledge about their cellular targets.

Catalytic Space Engineering as a Strategy to Activate C-H Oxidation on 5-Methylcytosine in Mammalian Genome.

Conditional remodeling of enzyme catalysis is a formidable challenge in protein engineering. Herein, we have undertaken a unique active site engineering tactic to command catalytic outcomes. With ten-eleven translocation (TET) enzyme as a paradigm, we show that variants with an expanded active site significantly enhance multistep C-H oxidation in 5-methylcytosine (5mC), whereas a crowded cavity leads to a single-step catalytic apparatus. We further identify an evolutionarily conserved residue in the TET family with a remarkable catalysis-directing ability. The activating variant demonstrated its prowess to oxidize 5mC in chromosomal DNA for potentiating expression of genes including tumor suppressors.

Engineering Methyllysine Writers and Readers for Allele-Specific Regulation of Protein-Protein Interactions.

Protein-protein interactions mediated by methyllysine are ubiquitous in biological systems. Specific perturbation of such interactions has remained a challenging endeavor. Herein, we describe an allele-specific strategy toward an engineered protein-protein interface orthogonal to the human proteome. We develop a methyltransferase (writer) variant that installs aryllysine moiety on histones that can only be recognized by an engineered chromodomain (reader). We establish biochemical integrity of the engineered interface, provide structural evidence for orthogonality and validate its applicability to identify transcriptional regulators. Our approach provides an unprecedented strategy for specific manipulation of the methyllysine interactome.

Allele-Specific Inhibition of Histone Demethylases.

Histone demethylases play a critical role in mammalian gene expression by removing methyl groups from lysine residues in degree- and site-specific manner. To specifically interrogate members and isoforms of this class of enzymes, we have developed demethylase variants with an expanded active site. The mutant enzymes are capable of performing lysine demethylation with wild-type proficiency, but are sensitive to inhibition by cofactor-competitive molecules embellished with a complementary steric "bump". The selected inhibitors show more than 20-fold selectivity over the wild-type demethylase, thus overcoming issues typical to pharmacological and genetic approaches. The mutant-inhibitor pairs are shown to act on a physiologically relevant full-length substrate. By engineering a conserved amino acid to achieve member-specific perturbation, this study provides a general approach for studying histone demethylases in diverse cellular processes.

Complementary Steric Engineering at the Protein-Ligand Interface for Analogue-Sensitive TET Oxygenases.

Ten-eleven translocation (TET) enzymes employ O2, earth-abundant iron, and 2-ketoglutarate (2KG) to perform iterative C-H oxidation of 5-methylcytosine in DNA to control expression of the mammalian genome. Given that more than 60 such C-H oxygenases are present in humans, determining context-dependent functions of each of these enzymes is a pivotal challenge. In an effort to tackle the problem, we developed analogue-sensitive TET enzymes to perturb the activity of a specific member. We rationally engineered the TET2-2KG interface to develop TET2 variants with an expanded active site that can be specifically inhibited by the N-oxalylglycine (NOG) derivatives carrying a complementary steric "bump". Herein, we describe the identification and engineering of a bulky gatekeeper residue for TET proteins, characterize the orthogonal mutant-inhibitor pairs, and show generality of the approach. Employing cell-permeable NOG analogues, we show that the TET2 mutant can be specifically inhibited to conditionally modulate cytosine methylation in chromosomal DNA in intact human cells. Finally, we demonstrate application of the orthogonal mutant-inhibitor pair to probe transcriptional activity of a specific TET member in cells. Our work provides a general platform for developing analogue-sensitive 2KG-dependent oxygenases to unravel their functions in diverse signaling processes.

Specific Acetylation Patterns of H2A.Z Form Transient Interactions with the BPTF Bromodomain.

Post-translational lysine acetylation of histone tails affects both chromatin accessibility and recruitment of multifunctional bromodomain-containing proteins for modulating transcription. The bromodomain- and PHD finger-containing transcription factor (BPTF) regulates transcription but has also been implicated in high gene expression levels in a variety of cancers. In this report, the histone variant H2A.Z, which replaces H2A in chromatin, is evaluated for its affinity for BPTF with a specific recognition pattern of acetylated lysine residues of the N-terminal tail region. Although BPTF immunoprecipitates H2A.Z-containing nucleosomes, a direct interaction with its bromodomain has not been reported. Using protein-observed fluorine nuclear magnetic resonance (PrOF NMR) spectroscopy, we identified a diacetylation of H2A.Z on lysine residues 4 and 11, with the highest affinity for BPTF with a Kd of 780 µM. A combination of subsequent 1H NMR Carr-Purcell-Meiboom-Gill experiments and photo-cross-linking further confirmed the specificity of the diacetylation pattern at lysines 4 and 11. Because of an adjacent PHD domain, this transient interaction may contribute to a higher-affinity bivalent interaction. Further evaluation of specificity toward a set of bromodomains, including two BET bromodomains (Brd4 and BrdT) and two Plasmodium falciparum bromodomains, resulted in one midmicromolar affinity binder, PfGCN5 (Kd = 650 µM). With these biochemical experiments, we have identified a direct interaction of histone H2A.Z with bromodomains with a specific acetylation pattern that further supports the role of H2A.Z in epigenetic regulation.

A rapid mass spectrometric method for the measurement of catalytic activity of ten-eleven translocation enzymes.

Enzymatic methylation at carbon five on cytosine (5mC) in DNA is a hallmark of mammalian epigenetic programming and is critical to gene regulation during early embryonic development. It has recently been shown that dynamic erasure of 5mC by three members of the ten-eleven translocation (TET) family plays a key role in cellular differentiation. TET enzymes belong to Fe (II)- and 2-ketoglutarate (2KG) dependent dioxygenases that successively oxidize 5mC to 5-hydroxymethyl cytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5CaC), thus providing a chemical basis for the removal of 5mC which once was thought to be a permanent mark in mammalian genome. Since then a wide range of biochemical assays have been developed to characterize TET activity. Majority of these methods require multi-step processing to detect and quantify the TET-mediated oxidized products. In this study, we have developed a MALDI mass spectrometry based method that directly measures the TET activity with high sensitivity while eliminating the need for any intermediate processing steps. We applied this method to the measurement of enzymatic activity of TET2 and 3, Michaleis-Menten parameters (KM and kcat) of TET-2KG pairs and inhibitory concentration (IC50) of known small-molecule inhibitors of TETs. We further demonstrated the suitability of the assay to analyze chemoenzymatic labeling of 5hmC by ß-glucosyltransferase, highlighting the potential for broad application of our method in deconvoluting the functions of novel DNA demethylases.

Engineering Biological C-H Functionalization Leads to Allele-Specific Regulation of Histone Demethylases.

Oxidative C-H hydroxylation of methyl groups, followed by their removal from DNA, RNA, or histones, is an epigenetic process critical to transcriptional reprogramming and cell fate determination. This reaction is catalyzed by Fe(II)-dependent dioxygenases using the essential metabolite 2-ketoglutarate (2KG) as a cofactor. Given that the human genome encodes for more than 60 2KG-dependent dioxygenases, assigning their individual functions remains a significant challenge. Here we describe a protein-ligand interface engineering approach to break the biochemical degeneracy of these enzymes. Using histone lysine demethylase 4 as a proof-of-concept, we show that the enzyme active site can be expanded to employ bulky 2KG analogues that do not sensitize wild-type demethylases. We establish the orthogonality, substrate specificity, and catalytic competency of the engineered demethylation apparatus in biochemical assays. We further demonstrate demethylation of cognate substrates in physiologically relevant settings. Our results provide a paradigm for rapid and conditional manipulation of histone demethylases to uncloak their isoform-specific functions.

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-ß-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.

Quantitative determination of artificial sweeteners and sucrose in energy drinks and mango juice available in Dhaka city.

Energy drinks and mango juice are popular beverages. Apart from the natural ingredients and some additives present in these drinks, sugar is an important component of both. It has been established that, other than providing sweetness, sugars are potent to bring about health consequences for their consumers. Sweeteners, both artificial (aspartame, sodium cyclamate, and saccharin) and natural (sucrose), were our centers of interest. This study aimed to determine the presence and levels of these sweeteners in energy drinks and mango juice. Spectrophotometric methods were used to determine the concentration of the mentioned sugars. For this purpose, a total of 42 samples of 7 different brands were collected from different locations in Dhaka city, Bangladesh. The methods were found to be linear over the concentration range of 10-26 µg/mL (r2=0.9989), 137-320 µg/mL (r2=0.9891), 2.5-24 µg/mL (r2=0.9915) and 2354-2784 µg/mL (r2=0.9985) for aspartame, sodium cyclamate, saccharin, and sucrose, respectively. Mango juice contained a relatively lower amount of saccharin compared to energy drinks. In the case of aspartame, one brand of energy drinks had the least amount. Moreover, both energy drinks and mango juice had a similar content of sodium cyclamate, but one brand of mango juice had a relatively low content of sodium cyclamate.

Nitric oxide inhibits ten-eleven translocation DNA demethylases to regulate 5mC and 5hmC across the genome.

DNA methylation at cytosine bases of eukaryotic DNA (5-methylcytosine, 5mC) is a heritable epigenetic mark that can regulate gene expression in health and disease. Enzymes that metabolize 5mC have been well-characterized, yet the discovery of endogenously produced signaling molecules that regulate DNA methyl-modifying machinery have not been described. Herein, we report that the free radical signaling molecule nitric oxide (NO) can directly inhibit the Fe(II)/2-OG-dependent DNA demethylases ten-eleven translocation (TET) and human AlkB homolog 2 (ALKBH2). Physiologic NO concentrations reversibly inhibited TET and ALKBH2 demethylase activity by binding to the mononuclear non-heme iron atom which formed a dinitrosyliron complex (DNIC) preventing cosubstrates (2-OG and O2) from binding. In cancer cells treated with exogenous NO, or cells endogenously synthesizing NO, there was a global increase in 5mC and 5-hydroxymethylcytosine (5hmC) in DNA, the substrates for TET, that could not be attributed to increased DNA methyltransferase activity. 5mC was also elevated in NO-producing cell-line-derived mouse xenograft and patient-derived xenograft tumors. Genome-wide DNA methylome analysis of cells chronically treated with NO (10 days) demonstrated enrichment of 5mC and 5hmC at gene-regulatory loci which correlated to changes in the expression of NO-regulated tumor-associated genes. Regulation of DNA methylation is distinctly different from canonical NO signaling and represents a novel epigenetic role for NO.

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.

5-Dihydroxyboryluridine enhances cytosolic penetration of antisense oligonucleotides.

The most significant challenge for nucleic acid drug development is their delivery across the cell membrane. Herein, we harness the reversible binding between boronic acids and cell surface glycans to aid in the cellular delivery of synthetic oligonucleotides. We install the artificial nucleotide 5-dihydroxyboryluridine (5boU) in a site-specific manner within druglike antisense oligonucleotides and demonstrate that these boronate-containing nucleic acids have enhanced cytosolic penetration and splice-correcting activity compared to non-boronate analogs. Strategic incorporation of 5boU is a simple, modular, and potentially general means of enhancing cellular delivery of therapeutic nucleic acids.

Bioorthogonal profiling of protein methylation using azido derivative of S-adenosyl-L-methionine.

Protein methyltransferases (PMTs) play critical roles in multiple biological processes. Because PMTs often function in vivo through forming multimeric protein complexes, dissecting their activities in the native contexts is challenging but relevant. To address such a need, we envisioned a Bioorthogonal Profiling of Protein Methylation (BPPM) technology, in which a SAM analogue cofactor can be utilized by multiple rationally engineered PMTs to label substrates of the corresponding native PMTs. Here, 4-azidobut-2-enyl derivative of S-adenosyl-L-methionine (Ab-SAM) was reported as a suitable BPPM cofactor. The resultant cofactor-enzyme pairs were implemented to label specifically the substrates of closely related PMTs (e.g., EuHMT1 and EuHMT2) in a complex cellular mixture. The BPPM approach, coupled with mass spectrometric analysis, enables the identification of the nonhistone targets of EuHMT1/2. Comparison of EuHMT1/2's methylomes indicates that the two human PMTs, although similar in terms of their primary sequences, can act on the distinct sets of nonhistone targets. Given the conserved active sites of PMTs, Ab-SAM and its use in BPPM are expected to be transferable to other PMTs for target identification.

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 ProSeAM's 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.

Hydrophobic cavity-directed azide-acetyllysine photochemistry for profiling non-histone interacting partners of bromodomain protein 1.

Bromodomain containing protein 1 (BRD1) plays critical roles in chromatin acetylation, gene transcription, erythropoiesis, and brain development. BRD1 is also implicated in several human conditions and is a therapeutic target for cancer. Although, the bromodomain is known to bind acetylated histones, how the function of BRD1 is regulated via non-histone acetylation is unexplored. To identify the non-histone acetylome of BRD1, we develop an R585AzF variant carrying photo responsive 4-azido phenylalanine (AzF) via amber suppressor mutagenesis. We demonstrate biochemical integrity of the AzF-containing analogue and its ability to crosslink non-histone interacting partners present in human cells. Subsequent proteomic experiments led to the identification of the novel BRD1 interactome representing diverse signaling pathways. As a proof-of-concept demonstration, we validated acetylated PDIA1 protein as a bona fide binding partner of BRD1. Our work suggests that BRD1 interacts with additional acetyllysine motifs, beyond those characterized in histone proteins.

Allele-Specific Chemical Rescue of Histone Demethylases Using Abiotic Cofactors.

Closely related protein families evolved from common ancestral genes present a significant hurdle in developing member- and isoform-specific chemical probes, owing to their similarity in fold and function. In this piece of work, we explore an allele-specific chemical rescue strategy to activate a "dead" variant of a wildtype protein using synthetic cofactors and demonstrate its successful application to the members of the alpha-ketoglutarate (αKG)-dependent histone demethylase 4 (KDM4) family. We show that a mutation at a specific residue in the catalytic site renders the variant inactive toward the natural cosubstrate. In contrast, αKG derivatives bearing appropriate stereoelectronic features endowed the mutant with native-like demethylase activity while remaining refractory to a set of wild type dioxygenases. The orthogonal enzyme-cofactor pairs demonstrated site- and degree-specific lysine demethylation on a full-length chromosomal histone in the cellular milieu. Our work offers a strategy to modulate a specific histone demethylase by identifying and engineering a conserved phenylalanine residue, which acts as a gatekeeper in the KDM4 subfamily, to sensitize the enzyme toward a novel set of αKG derivatives. The orthogonal pairs developed herein will serve as probes to study the role of degree-specific lysine demethylation in mammalian gene expression. Furthermore, this approach to overcome active site degeneracy is expected to have general application among all human αKG-dependent dioxygenases.

Engineering a methyllysine reader with photoactive amino acid in mammalian cells.

Methyllysine sites in proteins are recognized by an array of reader domains that mediate protein-protein interactions for controlling cellular processes. Herein, we engineer a chromodomain, an essential methyllysine reader, to carry 4-azido-l-phenylalanine (AzF) via amber suppressor mutagenesis and demonstrate its potential to bind and crosslink methylated proteins in human cells. We further develop a first-of-its kind chromodomain variant bearing two AzF units with enhanced crosslinking potential suitable for profiling the transient methyllysine interactome.

Engineering bromodomains with a photoactive amino acid by engaging 'Privileged' tRNA synthetases.

Site-specific placement of unnatural amino acids, particularly those responsive to light, offers an elegant approach to control protein function and capture their fleeting 'interactome'. Herein, we have resurrected 4-(trifluoromethyldiazirinyl)-phenylalanine, an underutilized photo-crosslinker, by introducing several key features including easy synthetic access, site-specific incorporation by 'privileged' synthetases and superior crosslinking efficiency, to develop photo-crosslinkable bromodomains suitable for 'interactome' profiling.

Synthesis of 5-Dihydroxyboryluridine Phosphoramidite and Its Site-Specific Incorporation into Oligonucleotides for Probing Thymine DNA Glycosylase.

A concise synthetic strategy to 5-dihydroxyboryldexoyuridine (5boU) phosphoramidite has been developed. 5boU was introduced into short oligonucleotides in a site-specific manner, demonstrating compatibility of the boronic acid moiety with standard solid-phase DNA synthesis chemistry. Electrophilic 5boU DNAs inhibited thymine DNA glycosylase, a cancer-relevant DNA-modifying enzyme. We envisage diverse applications of 5boU in organic synthesis, medicinal chemistry, and chemical biology.