Reversible Modification Research Articles

Label-Free Quantitative Phosphoproteomics in the Fission Yeast Schizosaccharomyces pombe.

Protein phosphorylation is a dynamic, reversible posttranslational modification that plays an important role in the regulation of cell signaling. Recently, label-free quantitative (LFQ) phosphoproteomics has become a powerful tool to analyze the phosphorylation of proteins within complex samples. In this chapter, we describe how to apply LFQ phosphoproteomics that is based on Fe-IMAC phosphopeptide enrichment followed by strong anion exchange (SAX) and porous graphitic carbon (PGC) fractionation strategies for identification and quantification of changes in the phosphoproteome in the fission yeast Schizosaccharomyces pombe.

Electric-Field Control of the Local Thermal Conductivity in Charge Transfer Oxides.

Phonons, the collective excitations responsible for heat transport in crystalline insulating solids, lack electric charge or magnetic moment, which complicates their active control via external fields. This presents a significant challenge in designing thermal equivalents of basic electronic circuit elements, such as transistors or diodes. Achieving these goals requires precise and reversible modification of thermal conductivity in materials. In this work, the continuous tuning of local thermal conductivity in charge-transfer SrFeO3-x and La0.6Sr0.4CoO3-x oxides using a voltage-biased Atomic Force Microscopy (AFM)tip at room temperature is demonstrated. This method allows the creation of micron-sized domains with well-defined thermal conductivity, achieving reductions of up to 50%, measured by spatially resolved Frequency Domain Thermoreflectance (FDTR). By optimizing the oxide's chemical composition, the thermal states remain stable under normal atmospheric conditions but can be reverted to their original values through thermal annealing in air. A comparison between Mott-Hubbard and charge-transfer oxides reveals the critical role of redox-active lattice oxygen in ensuring full reversibility of the process. This approach marks a significant step toward fabricating oxide-based tunable microthermal resistances and other elements for thermal circuits.

S-palmitoylation of MAP kinase is essential for fungal virulence.

S-palmitoylation is an important reversible protein post-translational modification in organisms. However, its role in fungi is uncertain. Here, we found the treatment of the rice false fungus Ustilaginoidea virens with S-palmitoylation inhibitor 2 BP resulted in a significant decrease in fungal virulence. Comprehensive identification of S-palmitoylation sites and proteins in U. virens revealed a total of 4,089 S-palmitoylation sites identified among 2,192 proteins and that S-palmitoylated proteins were involved in diverse biological processes. Among the five palmitoyltransferases, UvPfa3 and UvPfa4 were found to regulate the pathogenicity of U. virens. We then performed quantitative proteomic analysis of ∆UvPfa3 and ∆UvPfa4 mutants. Interestingly, S-palmitoylated proteins were significantly enriched in the mitogen-activated protein kinase and autophagy pathways, and MAP kinase UvSlt2 was confirmed to be an S-palmitoylated protein which was palmitoylated by UvPfa4. Mutations of S-palmitoylation sites in UvSlt2 resulted in significantly reduced fungal virulence and decreased kinase enzymatic activity and phosphorylation levels. Simulations of molecular dynamics demonstrated mutation of S-palmitoylation sites in UvSlt2 causing decreased hydrophobic solvent-accessible surface area, thereby weakening the bonding force with its substrate UvRlm1. Taken together, S-palmitoylation promotes U. virens virulence through palmitoylation of MAP kinase UvSlt2 by palmitoyltransferase UvPfa4. This enhances the enzymatic phosphorylation activity of the kinase, thereby increasing hydrophobic solvent-accessible surface area and binding activity between the UvSlt2 enzyme and its substrate UvRlm1. Our studies provide a framework for dissecting the biological functions of S-palmitoylation and reveal an important role for S-palmitoylation in regulating the virulence of the pathogen.IMPORTANCES-palmitoylation is an important post-translational lipid modification of proteins. However, its role in fungi is uncertain. In this study, we found that S-palmitoylation promotes virulence of rice false smut fungus U. virens through palmitoylation of MAP kinase UvSlt2 by palmitoyltransferase UvPfa4. This enhances the enzymatic phosphorylation activity of the kinase, thereby increasing hydrophobic solvent-accessible surface area and binding activity between the UvSlt2 enzyme and its substrate UvRlm1. Our studies provide a framework for dissecting the biological functions of S-palmitoylation and reveal an important role for S-palmitoylation in regulating the virulence of the pathogen. This is the first functional study to reveal the role of S-palmitoylation in fungal virulence.

Recent advances in ubiquitin signals regulating plant membrane trafficking.

Ubiquitination is a reversible post-translational modification involving the attachment of ubiquitin, a 76-amino acid protein conserved among eukaryotes. The protein "ubiquitin" was named after it was found to be ubiquitously expressed in cells. Ubiquitination was first identified as a post-translational modification that mediates energy-consuming protein degradation by the proteasome. After half a century, the manifold functions of ubiquitin are widely recognized to play key roles in diverse molecular pathways and physiological processes. Compared to humans, the number of enzymes related to ubiquitination is almost twice as high in plant species such as Arabidopsis and rice, suggesting that this modification plays a critical role in many aspects of plant physiology including development and environmental stress responses. Here, we summarize and discuss recent knowledge of ubiquitination focusing on the regulation of membrane trafficking in plants. Ubiquitination of plasma membrane localized proteins often leads to endocytosis and vacuolar targeting. In addition to cargo proteins, ubiquitination of membrane trafficking regulators regulates the morphodynamics of the endomembrane system. Thus, throughout this review, we focus on the physiological responses regulated by ubiquitination and their underlying mechanisms to clarify what is already known and what would be interesting to investigate in the future.

Palmitoylation regulates norepinephrine transporter uptake, surface localization, and total expression with pathogenic implications in postural orthostatic tachycardia syndrome.

Postural orthostatic tachycardia syndrome (POTS) is an adrenergic signaling disorder characterized by excessive plasma norepinephrine, postural tachycardia, and syncope. The norepinephrine transporter (NET) modulates adrenergic homeostasis via the reuptake of extracellular catecholamines and is implicated in the pathogenesis of adrenergic and neurological disorders. In this study, we reveal NET is palmitoylated in male Sprague-Dawley rats and Lilly Laboratory Cell Porcine Kidney (LLC-PK1) cells. S-palmitoylation, or the addition of a 16-carbon saturated fatty acid, is a reversible post-translational modification responsible for the regulation of numerous biological mechanisms. We found that LLC-PK1 NET is dynamically palmitoylated, and that inhibition with the palmitoyl acyltransferase (DHHC) inhibitor, 2-bromopalmitate (2BP) results in decreased NET palmitoylation within 90 min of treatment. This result was followed closely by a reduction in transport capacity, cell surface, and total cellular NET expression after 120 min of treatment. Increasing 2BP concentrations and treatment time revealed a nearly complete loss of total NET protein. Co-expression with individual DHHCs revealed a single DHHC enzyme, DHHC1, promoted wild-type (WT) hNET palmitoylation and elevated NET protein levels. The POTS-associated NET mutant, A457P, exhibits dramatically decreased transport capacity and cell surface levels which we have confirmed in the current study. In an attempt to recover A457P NET expression, we co-expressed the A457P variant with DHHC1 to drive expression as seen with the WT protein but instead saw an increase in NET N-terminal immuno-detectable forms and fragments. Elimination of a potential palmitoylation site at cysteine 44 in the N-terminal tail of hNET resulted in a low expression phenotype mimicking the A457P hNET variant. Further investigation of A457P NET palmitoylation and surface expression is necessary, but our preliminary novel findings reveal palmitoylation as a mechanism of NET regulation and suggest that dysregulation of this process may contribute to the pathogenesis of adrenergic disorders like POTS.

Acetylation of Histone H3 in Cancer Progression and Prognosis.

Cancer is a multifactorial disease resulting from both genetic factors and epigenetic changes. Histone acetylation, a post-translational modification, which alters chromatin architecture and regulates gene expression is associated with cancer initiation, development and progression. Aberrations in global histone acetylation levels are observed in various cancer cells and are also associated with patients' tumor aggressiveness. Therefore, histone acetylation may have prognostic utility and serve as a potential biomarker of cancer progression and patients' prognosis. The reversible modification of histones by an acetyl group is versatile. One particular histone can be acetylated on different lysine residues, subsequently resulting in different biological outcomes. Here, we discuss recent findings on the acetylation of the highly conserved histone protein H3 in the context of cancer biology. Specifically, we review the acetylation of particular H3 residues in various cancer types. We further highlight the significance of H3 acetylation levels as a potential cancer biomarker with prognostic implications.

Dynamic landscape of m6A modifications and related post-transcriptional events in muscle-invasive bladder cancer

BackgroundMuscle-invasive bladder carcinoma (MIBC) is a serious and more advanced stage of bladder carcinoma. N6-Methyladenosine (m6A) is a dynamic and reversible modifications that primarily affects RNA stability and alternative splicing. The dysregulation of m6A in MIBC can be potential target for clinical interventions, but there have been limited studies on m6A modifications in MIBC and their associations with post-transcriptional regulatory processes.MethodsPaired tumor and adjacent-normal tissues were obtained from three patients with MIBC following radical cystectomy. The additional paired tissues for validation were obtained from patients underwent transurethral resection. Utilizing Nanopore direct-RNA sequencing, we characterized the m6A RNA methylation landscape in MIBC, with a focus on identifying post-transcriptional events potentially affected by changes in m6A sites. This included an examination of differential transcript usage, polyadenylation signal sites, and variations in poly(A) tail length, providing insights into the broader impact of m6A alterations on RNA processing in MIBC.ResultsThe prognostic-related m6A genes and m6A-risk model constructed by machine learning enables the stratification of high and low-risk patients with precision. A novel m6A modification site in the 3’ untranslated region (3’UTR) of IGLL5 gene were identified, characterized by a lower m6A methylation ratio, elongated poly(A) tails, and a notable bias in transcript usage. Furthermore, we discovered two particular transcripts, VWA1-203 and CEBPB-201. VWA1-203 displayed diminished m6A methylation levels, a truncated 3’UTR, and an elongated poly(A) tail, whereas CEBPB-201 showed opposite trends, highlighting the complex interplay between m6A modifications and RNA processing. Source code was provided on GitHub (https://github.com/lelelililele/Nanopore-m6A-analysis).ConclusionsThe state-of-the-art Nanopore direct-RNA sequencing and machine learning techniques enables comprehensive identification of m6A modification and provided insights into the potential post-transcriptional regulation mechanisms on the development and progression in MIBC.

The O-GlcNAc database: introducing new features and tools developed from community feedback.

O-GlcNAc is a reversible post-translational modification found on serine and threonine residues of nucleocytoplasmic proteins. Four years ago, we released the O-GlcNAc Database( oglcnac.mcw.edu ), a comprehensive catalog of O-GlcNAcylated proteins that has become one of the most cited resources in the field, with hundreds of unique users per month. We are now presenting an updated O-GlcNAc Database, which includes nearly 20,000 O-GlcNAcylated proteins and 48 species, marking substantial growth in data volume and scope. This paper presents the most noteworthy features implemented over the last year, often originating from feedback from the O-GlcNAc community. Among these features, we provide a brief overview of the database content, introduce our new protein viewer mode, and discuss the implementation of subcellular localization information and its applications in the O-GlcNAc score. We also provide an interface to use CytOVS, a tool designed to evaluate and sort O-GlcNAcome datasets derived from MS experiments. In conclusion, this new and improved O-GlcNAc Database represents a significant advancement in providing a comprehensive and expanded resource for researchers in the field of O-GlcNAc biology.

GBMPhos: A Gating Mechanism and Bi-GRU-Based Method for Identifying Phosphorylation Sites of SARS-CoV-2 Infection.

Phosphorylation, a reversible and widespread post-translational modification of proteins, is essential for numerous cellular processes. However, due to technical limitations, large-scale detection of phosphorylation sites, especially those infected by SARS-CoV-2, remains a challenging task. To address this gap, we propose a method called GBMPhos, a novel method that combines convolutional neural networks (CNNs) for extracting local features, gating mechanisms to selectively focus on relevant information, and a bi-directional gated recurrent unit (Bi-GRU) to capture long-range dependencies within protein sequences. GBMPhos leverages a comprehensive set of features, including sequence encoding, physicochemical properties, and structural information, to provide an in-depth analysis of phosphorylation sites. We conducted an extensive comparison of GBMPhos with traditional machine learning algorithms and state-of-the-art methods. Experimental results demonstrate the superiority of GBMPhos over existing methods. The visualization analysis further highlights its effectiveness and efficiency. Additionally, we have established a free web server platform to help researchers explore phosphorylation in SARS-CoV-2 infections. The source code of GBMPhos is publicly available on GitHub.

β-Cyclodextrin-Modified Laser-Induced Graphene Electrode for Detection of N6-Methyladenosine in RNA.

Laser-induced graphene (LIG) possesses characteristics of easy handling, miniaturization, and unique electrical properties. We modified the surface of LIG by electropolymerizing β-cyclodextrin (β-CD), which was used to immobilize antibodies on the electrode surface for highly sensitive detection of targets. N6-methyladenosine (m6A) is the most prevalent reversible modification in mammalian messenger RNA and noncoding RNA, influencing the development of various cancers. Here, β-CD was electropolymerized to immobilize the anti-m6A antibody, which subsequently recognized the target m6A. This was integrated into the catalytic hydrogen peroxide-hydroquinone (H2O2-HQ) redox system using phos-tag-biotin to generate electrochemical signals from streptavidin-modified horseradish peroxidase (SA-HRP). Under optimal conditions, the biosensor exhibited a linear range from 0.1 to 100 nM with a minimum detection limit of 96 pM. The method was successfully applied to the recovery analysis of m6A from HeLa cells through spiking experiments and aims to inspire strategies for point-of-care testing (POCT).

Targeting a key protein-protein interaction surface on mitogen-activated protein kinases by a precision-guided warhead scaffold

For mitogen-activated protein kinases (MAPKs) a shallow surface—distinct from the substrate binding pocket—called the D(ocking)-groove governs partner protein binding. Screening of broad range of Michael acceptor compounds identified a double-activated, sterically crowded cyclohexenone moiety as a promising scaffold. We show that compounds bearing this structurally complex chiral warhead are able to target the conserved MAPK D-groove cysteine via reversible covalent modification and interfere with the protein-protein interactions of MAPKs. The electronic and steric properties of the Michael acceptor can be tailored via different substitution patterns. The inversion of the chiral center of the warhead can reroute chemical bond formation with the targeted cysteine towards the neighboring, but less nucleophilic histidine. Compounds bind to the shallow MAPK D-groove with low micromolar affinity in vitro and perturb MAPK signaling networks in the cell. This class of chiral, cyclic and enhanced 3D shaped Michael acceptor scaffolds offers an alternative to conventional ATP-competitive drugs modulating MAPK signaling pathways.

YkuR functions as a protein deacetylase in Streptococcus mutans

Protein acetylation is a common and reversible posttranslational modification tightly governed by protein acetyltransferases and deacetylases crucial for various biological processes in both eukaryotes and prokaryotes. Although recent studies have characterized many acetyltransferases in diverse bacterial species, only a few protein deacetylases have been identified in prokaryotes, perhaps in part due to their limited sequence homology. In this study, we identified YkuR, encoded by smu_318, as a unique protein deacetylase in Streptococcus mutans. Through protein acetylome analysis, we demonstrated that the deletion of ykuR significantly upregulated protein acetylation levels, affecting key enzymes in translation processes and metabolic pathways, including starch and sucrose metabolism, glycolysis/gluconeogenesis, and biofilm formation. In particular, YkuR modulated extracellular polysaccharide synthesis and biofilm formation through the direct deacetylation of glucosyltransferases (Gtfs) in the presence of NAD+. Intriguingly, YkuR can be acetylated in a nonenzymatic manner, which then negatively regulated its deacetylase activity, suggesting the presence of a self-regulatory mechanism. Moreover, in vivo studies further demonstrated that the deletion of ykuR attenuated the cariogenicity of S. mutans in the rat caries model, substantiating its involvement in the pathogenesis of dental caries. Therefore, our study revealed a unique regulatory mechanism mediated by YkuR through protein deacetylation that regulates the physiology and pathogenicity of S. mutans.

Carboxy-terminal polyglutamylation regulates signaling and phase separation of the Dishevelled protein.

Polyglutamylation is a reversible posttranslational modification that is catalyzed by enzymes of the tubulin tyrosine ligase-like (TTLL) family. Here, we found that TTLL11 generates a previously unknown type of polyglutamylation that is initiated by the addition of a glutamate residue to the free C-terminal carboxyl group of a substrate protein. TTLL11 efficiently polyglutamylates the Wnt signaling protein Dishevelled 3 (DVL3), thereby changing the interactome of DVL3. Polyglutamylation increases the capacity of DVL3 to get phosphorylated, to undergo phase separation, and to act in the noncanonical Wnt pathway. Both carboxy-terminal polyglutamylation and the resulting reduction in phase separation capacity of DVL3 can be reverted by the deglutamylating enzyme CCP6, demonstrating a causal relationship between TTLL11-mediated polyglutamylation and phase separation. Thus, C-terminal polyglutamylation represents a new type of posttranslational modification, broadening the range of proteins that can be modified by polyglutamylation and providing the first evidence that polyglutamylation can modulate protein phase separation.

SUMOylation-induced membrane localization of TRPV1 suppresses proliferation and migration in gastric cancer cells

Gastric cancer (GC) remains a significant health challenge due to its high mortality rate and the limited efficacy of current targeted therapies. A critical barrier in developing more effective treatments is the lack of understanding of specific mechanisms driving GC progression. This study investigates the role of Transient Receptor Potential Vanilloid 1 (TRPV1), a non-selective cation channel known for its high Ca2+ permeability and tumor-suppressive properties in gastrointestinal cancers. Specifically, we explore the impact of SUMOylation—a dynamic and reversible post-translational modification—on TRPV1’s function in GC. We demonstrate that SUMOylation of TRPV1 inhibits cell proliferation and migration in MGC-803 and AGS GC cells. By mutating amino acids near TRPV1’s existing SUMO motif (slKpE), we created a bidirectional SUMO motif (EψKψE) that enhances TRPV1 SUMOylation, resulting in further suppression of GC cell proliferation and migration. In vivo studies support these findings, showing that TRPV1 SUMOylation prevents spontaneous tumorigenesis in a mouse GC model. Further investigation reveals that TRPV1 SUMOylation increases the protein’s membrane expression by inhibiting its interaction with the adaptor-related protein complex 2 mu 1 subunit (AP2M1). This elevated membrane expression leads to increased intracellular Ca2+ influx, activating the AMP-activated protein kinase (AMPK) pathway, which in turn inhibits the proliferation and migration of GC cells.

Phosphorylation of VapB antitoxins affects intermolecular interactions to regulate VapC toxin activity in Mycobacterium tuberculosis.

Toxin-antitoxin modules are present in many bacterial pathogens. The VapBC family is particularly abundant in members of the Mycobacterium tuberculosis complex, with 50 modules present in the M. tuberculosis genome. In type IIA modules, the VapB antitoxin protein binds to and inhibits the activity of the co-expressed cognate VapC toxin protein. VapB proteins may also bind to promoter region sequences and repress the expression of the vapB-vapC operon. Though VapB-VapC interactions can control the amount of free VapC toxin in the bacterial cell, the mechanisms that affect this interaction are poorly understood. Based on our recent finding of Ser/Thr phosphorylation of VapB proteins in M. tuberculosis, we substituted phosphomimetic or phosphoablative amino acids at the phosphorylation sites of two VapB proteins. We found that phosphomimetic substitution of VapB27 and VapB46 resulted in decreased interaction with their respective cognate VapC proteins, whereas phosphoablative substitution did not alter binding. Similarly, we determined that phosphomimetic substitution interfered with VapB binding to promoter region DNA sequences. Both decreased VapB-VapC interaction and decreased VapB repression of vapB-vapC operon transcription would result in increased free VapC in the M. tuberculosis cell. In growth inhibition experiments, M. tuberculosis strains expressing vapB46-vapC46 constructs containing a phosphoablative vapB mutation resulted in lower toxicity compared to a strain expressing native vapB46, whereas similar or greater toxicity was observed in the strain expressing the phosphomimetic vapB mutation. These results identify a novel mechanism by which VapC toxicity activity can be regulated by VapB phosphorylation.IMPORTANCEIntracellular bacterial toxins are present in many bacterial pathogens and have been linked to bacterial survival in response to stresses encountered during infection. The activity of many toxins is regulated by a co-expressed antitoxin protein that binds to and sequesters the toxin protein. The mechanisms by which an antitoxin may respond to stresses to alter toxin activity are poorly understood. Here, we show that antitoxin interactions with its cognate toxin and with promoter DNA required for antitoxin and toxin expression can be altered by Ser/Thr phosphorylation of the antitoxin and, thus, affect toxin activity. This reversible modification may play an important role in regulating toxin activity within the bacterial cell in response to signals generated during infection.

M6A methyltransferase ZC3H13 improves pulmonary fibrosis in mice through regulating Bax expression

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease. N6-methyladenosine (m6A) is a reversible RNA modification that was shown to be associated with IPF development. The present study aimed to explore the function and potential mechanism of the m6A methylation enzyme zinc finger CCCH-type containing 13 (ZC3H13) in IPF. In the study, bioinformatic screening yielded a differentially expressed m6A gene, ZC3H13, which was down-regulated in GEO microarrays, BLM-induced mouse models, and cellular models. Overexpression of ZC3H13 reduced histopathological damage of lung tissues in mice, mitigated fibrosis (including reduced α-SMA, collagen Ⅰ, and Vimentin levels, and elevated E-cadherin levels), decreased lung/body weight ratio and lung hydroxyproline levels, reduced oxidative stress (increased SOD activity and GSH-Px activity and decreased MDA levels), suppressed apoptosis within lung tissues and MLE-12 cells, promoted Bcl-2 expression, and inhibited Bax expression. Bax expression was found to be negatively correlated with ZC3H13 expression by correlation analysis. ZC3H13 could bind Bax mRNA and promote its m6A methylation through reading protein YTHDC1, thereby inhibiting its stability. Bax inhibition ameliorated BLM-induced MLE-12 cell dysfunction and partially abrogated the inhibition of MLE-12 cell function by ZC3H13 downregulation. In conclusion, m6A methyltransferase ZC3H13 impedes lung epithelial cell apoptosis and thus improves pulmonary fibrosis by promoting Bax mRNA m6A methylation and down-regulating Bax expression through reading protein YTHDC1.

Radical-mediated click-clip reactions

Click reactions, which are characterized by rapid, high-yielding, and highly selective coupling of two reaction partners, are powerful tools in synthesis but are rarely reversible. Innovative strategies that reverse such couplings in a precise and on-demand manner, enabling a click-clip sequence, would greatly expand the technique’s versatility. Herein, a click and clip reaction pair is demonstrated by manipulation of a sulfilimine linkage. Phenothiazines and amines are rapidly and quantitatively coupled through oxidative sulfilimine bond formation with N-bromosuccinimide, and the resulting sulfilimine bromides then undergo quantitative reversion to the phenothiazines and amines through photoreduction at 380 nanometers. This protocol enables fabrication of depolymerizable macromolecules and reversible modification of aminosaccharides, demonstrating high selectivity and efficiency for manipulating sulfilimine linkages in complex systems.

Global remodeling of ADP-ribosylation by PARP1 suppresses influenza A virus infection.

ADP-ribosylation is a highly dynamic and fully reversible post-translational modification performed by poly(ADP-ribose) polymerases (PARPs) that modulates protein function, abundance, localization and turnover. Here we show that influenza A virus infection causes a rapid and dramatic upregulation of global ADP-ribosylation that inhibits viral replication. Mass spectrometry defined for the first time the global ADP-ribosylome during infection, creating an infection-specific profile with almost 4,300 modification sites on ~1,080 host proteins, as well as over 100 modification sites on viral proteins. Our data indicate that the global increase likely reflects a change in the form of ADP-ribosylation rather than modification of new targets. Functional assays demonstrated that modification of the viral replication machinery antagonizes its activity and further revealed that the anti-viral activity of PARPs and ADP-ribosylation is counteracted by the influenza A virus protein NS1, assigning a new activity to the primary viral antagonist of innate immunity. We identified PARP1 as the enzyme producing the majority of poly(ADP-ribose) present during infection. Influenza A virus replicated faster in cells lacking PARP1, linking PARP1 and ADP-ribosylation to the anti-viral phenotype. Together, these data establish ADP-ribosylation as an anti-viral innate immune-like response to viral infection antagonized by a previously unknown activity of NS1.

Methyl-Transferase-Like Protein 16 (METTL16): The Intriguing Journey of a Key Epitranscriptomic Player Becoming an Emerging Biological Target.

Key epitranscriptomic players have been increasingly characterized for their structural features and their involvement in several diseases. Accordingly, the design and synthesis of novel epitranscriptomic modulators have started opening a glimmer for drug discovery. m6A is a reversible modification occurring on a specific site and is catalyzed by three sets of proteins responsible for opposite functions. Writers (e.g., methyl-transferase-like protein (METTL) 3/METTL14 complex and METTL16) introduce the methyl group on adenosine N-6, by transferring the methyl group from the methyl donor S-adenosyl-methionine (SAM) to the substrate. Despite the rapidly advancing drug discovery progress on METTL3/METTL14, the METTL16 m6A writer has been marginally explored so far. We herein provide the first comprehensive overview of structural and biological features of METTL16, highlighting the state of the art in the field of its biological and structural characterization. We also showcase initial efforts in the identification of structural templates and preliminary structure-activity relationships for METTL16 modulators.

A circular engineered sortase for interrogating histone H3 in chromatin.

Reversible modification of the histone H3 N-terminal tail is critical in regulating chromatin structure, gene expression, and cell states, while its dysregulation contributes to disease pathogenesis. Understanding the crosstalk between H3 tail modifications in nucleosomes constitutes a central challenge in epigenetics. Here we describe an engineered sortase transpeptidase, cW11, that displays highly favorable properties for introducing scarless H3 tails onto nucleosomes. This approach significantly accelerates the production of both symmetrically and asymmetrically modified nucleosomes. We demonstrate the utility of asymmetrically modified nucleosomes produced in this way in dissecting the impact of multiple modifications on eraser enzyme processing and molecular recognition by a reader protein. Moreover, we show that cW11 sortase is very effective at cutting and tagging histone H3 tails from endogenous histones, facilitating multiplex "cut-and-paste" middle down proteomics with tandem mass tags. This cut-and- paste proteomics approach permits the quantitative analysis of histone H3 modification crosstalk after treatment with different histone deacetylase inhibitors. We propose that these chemoenzymatic tail isolation and modification strategies made possible with cW11 sortase will broadly power epigenetics discovery and therapeutic development.