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  1. Abstract Protein posttranslational modification (PTM) is a biochemical mechanism benefitting cellular adaptation to dynamic intracellular and environmental conditions. Recently, several acylation marks have been identified as new protein PTMs occurring on specific lysine residues in mammalian cells: isobutyrylation, methacrylation, benzoylation, isonicotinylation, and lactylation. These acylation marks were initially discovered to occur on nucleosomal histones, but they potentially occur as prevalent biomarkers on non‐histone proteins as well. The existence of these PTMs is a downstream consequence of metabolism and demonstrates the intimate crosstalk between active cellular metabolites and regulation of protein function. Emerging evidence indicates that these acylation marks on histones affect DNA transcription and are functionally distinct from the well‐studied lysine acetylation. Herein, we discuss enzymatic regulation and metabolic etiology of these acylations, 'reader' proteins that recognize different acylations, and their possible physiological and pathological functions. Several of these modifications correlate with other well‐studied acylations and fine‐tune the regulation of gene expression. Overall, findings of these acylation marks reveal new molecular links between metabolism and epigenetics and open up many questions for future investigation. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. 
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  2. The family of lysine acetyltransferases (KATs) regulates epigenetics and signaling pathways in eukaryotic cells. So far, knowledge of different KAT members contributing to the cellular acetylome is limited, which limits our understanding of biological functions of KATs in physiology and disease. Here, we found that a clickable acyl-CoA reporter, 3-azidopropanoyl CoA (3AZ-CoA), presented remarkable cell permeability and effectively acylated proteins in cells. We rationally engineered the major KAT member, histone acetyltransferase 1 (HAT1), to generate its mutant forms that displayed excellent bio-orthogonal activity for 3AZ-CoA in substrate labeling. We were able to apply the bio-orthogonal enzyme–cofactor pair combined with SILAC proteomics to achieve HAT1 substrate targeting, enrichment, and proteomic profiling in living cells. A total of 123 protein substrates of HAT1 were disclosed, underlining the multifactorial functions of this important enzyme than hitherto known. This study demonstrates the first example of utilizing bio-orthogonal reporters as a chemoproteomic strategy for substrate mapping of individual KAT isoforms in the native biological contexts. 
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  3. Abstract

    Histone acetyltransferases (HATs, also known as lysine acetyltransferases, KATs) catalyze acetylation of their cognate protein substrates using acetyl‐CoA (Ac‐CoA) as a cofactor and are involved in various physiological and pathological processes. Advances in mass spectrometry‐based proteomics have allowed the discovery of thousands of acetylated proteins and the specific acetylated lysine sites. However, due to the rapid dynamics and functional redundancy of HAT activities, and the limitation of using antibodies to capture acetylated lysines, it is challenging to systematically and precisely define both the substrates and sites directly acetylated by a given HAT. Here, we describe a chemoproteomic approach to identify and profile protein substrates of individual HAT enzymes on the proteomic scale. The approach involves protein engineering to enlarge the Ac‐CoA binding pocket of the HAT of interest, such that a mutant form is generated that can use functionalized acyl‐CoAs as a cofactor surrogate to bioorthogonally label its protein substrates. The acylated protein substrates can then be chemoselectively conjugated either with a fluorescent probe (for imaging detection) or with a biotin handle (for streptavidin pulldown and chemoproteomic identification). This modular chemical biology approach has been successfully implemented to identify protein substrates of p300, GCN5, and HAT1, and it is expected that this method can be applied to profile and identify the sub‐acetylomes of many other HAT enzymes. © 2022 Wiley Periodicals LLC.

    Basic Protocol 1: Labeling HAT protein substrates with azide/alkyne‐biotin

    Alternate Protocol: Labeling protein substrates of HATs with azide/alkyne‐TAMRA for in‐gel visualization

    Support Protocol 1: Expression and purification of HAT mutants

    Support Protocol 2: Synthesis of Ac‐CoA surrogates

    Basic Protocol 2: Streptavidin enrichment of biotinylated HAT substrates

    Basic Protocol 3: Chemoproteomic identification of HAT substrates

    Basic Protocol 4: Validation of specific HAT substrates with western blotting

     
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  4. null (Ed.)
    Protein arginine methyltransferases (PRMTs) are essential epigenetic and post-translational regulators in eukaryotic organisms. Dysregulation of PRMTs is intimately related to multiple types of human diseases, particularly cancer. Based on the previously reported PRMT1 inhibitors bearing the diamidine pharmacophore, we performed virtual screening to identify additional amidine-associated structural analogs. Subsequent enzymatic tests and characterization led to the discovery of a top lead K313 (2-(4-((4-carbamimidoylphenyl)amino)phenyl)-1 H -indole-6-carboximidamide), which possessed low-micromolar potency with biochemical IC 50 of 2.6 μM for human PRMT1. Limited selectivity was observed over some other PRMT isoforms such as CARM1 and PRMT7. Molecular modeling and inhibition pattern studies suggest that K313 is a nonclassic noncompetitive inhibitor to PRMT1. K313 significantly inhibited cell proliferation and reduced the arginine asymmetric dimethylation level in the leukaemia cancer cells. 
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  5. null (Ed.)
    Abstract Short-chain acylations of lysine residues in eukaryotic proteins are recognized as essential posttranslational chemical modifications (PTMs) that regulate cellular processes from transcription, cell cycle, metabolism, to signal transduction. Lysine butyrylation was initially discovered as a normal straight chain butyrylation (Knbu). Here we report its structural isomer, branched chain butyrylation, i.e. lysine isobutyrylation (Kibu), existing as a new PTM on nuclear histones. Uniquely, isobutyryl-CoA is derived from valine catabolism and branched chain fatty acid oxidation which is distinct from the metabolism of n-butyryl-CoA. Several histone acetyltransferases were found to possess lysine isobutyryltransferase activity in vitro, especially p300 and HAT1. Transfection and western blot experiments showed that p300 regulated histone isobutyrylation levels in the cell. We resolved the X-ray crystal structures of HAT1 in complex with isobutyryl-CoA that gleaned an atomic level insight into HAT-catalyzed isobutyrylation. RNA-Seq profiling revealed that isobutyrate greatly affected the expression of genes associated with many pivotal biological pathways. Together, our findings identify Kibu as a novel chemical modification mark in histones and suggest its extensive role in regulating epigenetics and cellular physiology. 
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  6. Protein acylation, exemplified by lysine acetylation, is a type of indispensable and widespread protein posttranslational modification in eukaryotes. Functional annotation of various lysine acetyltransferases (KATs) is critical to understanding their regulatory roles in abundant biological processes. Traditional radiometric and immunosorbent assays have found broad use in KAT study but have intrinsic limitations. Designing acyl–coenzyme A (CoA) reporter molecules bearing chemoselective chemical warhead groups as surrogates of the native cofactor acetyl-CoA for bioorthogonal labeling of KAT substrates has come into a technical innovation in recent years. This chemical biology platform equips molecular biologists with empowering tools in acyltransferase activity detection and substrate profiling. In the bioorthogonal labeling, protein substrates are first enzymatically modified with a functionalized acyl group. Subsequently, the chemical warhead on the acyl chain conjugates with either an imaging chromophore or an affinity handle or any other appropriate probes through an orthogonal chemical ligation. This bioorganic strategy reformats the chemically inert acetylation and acylation marks into a chemically maneuverable functionality and generates measurable signals without recourse to radioisotopes or antibodies. It offers ample opportunities for facile sensitive detection of KAT activity with temporal and spatial resolutions as well as allows for chemoproteomic profiling of protein acetylation pertaining to specific KATs of interest on the global scale. We reviewed here the past and current advances in bioorthogonal protein acylations and highlighted their wide-spectrum applications. We also discussed the design of other related acyl-CoA and CoA-based chemical probes and their deployment in illuminating protein acetylation and acylation biology.

     
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  7. Abstract

    The side‐chain acetylation of lysine residues in histones and non‐histone proteins catalyzed by lysine acetyltransferases (KATs) represents a widespread posttranslational modification (PTM) in the eukaryotic cells. Lysine acetylation plays regulatory roles in major cellular pathways inside and outside the nucleus. In particular, KAT‐mediated histone acetylation has an effect on all DNA‐templated epigenetic processes. Aberrant expression and activation of KATs are commonly observed in human diseases, especially cancer. In recent years, the study of KAT functions in biology and disease has greatly benefited from chemical biology tools and strategies. In this Review, we present the past and current accomplishments in the design of chemical biology approaches for the interrogation of KAT activity and function. These methods and probes are classified according to their mechanisms of action and respective applications, with both strengths and limitations discussed.

     
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