A Genetically Encoded Allysine for the Synthesis of Proteins with Site‐Specific Lysine Dimethylation

Using the amber suppression approach, N^ϵ‐(4‐azidobenzoxycarbonyl)‐δ,ϵ‐dehydrolysine, an allysine precursor is genetically encoded in E. coli. Its genetic incorporation followed by two sequential biocompatible reactions allows convenient synthesis of proteins with site‐specific lysine dimethylation. Using this approach, dimethyl‐histone H3 and p53 proteins have been synthesized and used to probe functions of epigenetic enzymes including histone demethylase LSD1 and histone acetyltransferase Tip60. We confirmed that LSD1 is catalytically active toward H3K4me2 and H3K9me2 but inert toward H3K36me2, and methylation at p53 K372 directly activates Tip60 for its catalyzed acetylation at p53 K120.     

Dynamics simulation on the flexibility and backbone motions of HP1 chromodomain bound to free and lysine 9‐methylated histone H3 tails

Histone methylation has emerged as a central epigenetic modification with both activating and repressive roles in eukaryotic chromatin. Drosophila HP1 (heterochromatin‐associated protein 1) is one of the chromodomain proteins that contain the essential aromatic residues as the recognition pocket for lysine methylated histone H3 tail. The aromatic cage indicates that the complex of chromodomain protein binding lysine methylated histone H3 tail can be seen as a typical host–guest system between protein and protein. About 10‐ns molecular dynamics simulations have been carried out in this study to examine how the presence of mono‐, trimethylated lysine 9 histone H3 tail (Me1K9, Me3K9 H3) influences the motions of HP1 protein receptor. The study shows that the conformation of HP1 protein free of H3 tail easily changes, whereas that of HP1 protein bound to methylated H3 tail does not. But the conformation of inserted Me1K9 H3 changes obviously as the Me1K recognition makes hydrogen‐bonded interactions associated with the aromatic cage even more unstable than those in free HP1 protein. The conformational change of Me1K9 H3 is correlated with the motions of HP1 protein. As the recognition factor going from Me1K to Me3K produces a more favorable interaction for aromatic ring, hydrogen‐bonded interactions associated with aromatic cage in Me3K9 H3‐HP1 complex were observed to be much more stable than those in Me1K9 H3‐HP1 complex and free HP1. Because of correlation, the flexibility of Me3K9 H3 decreases. The simulations indicate that both the MeK and the surrounding histone tail sequence are necessary features of recognition which significantly affect the flexibility and backbone motions of HP1 chromodomain. These findings confirm a regulatory mechanism of protein–protein interactions through a trimethylated post‐translational modification. © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2009     

A quantitative analysis of histone methylation and acetylation isoforms from their deuteroacetylated derivatives: application to a series of knockout mutants

The core histones, H2A, H2B, H3 and H4, undergo post‐translational modifications (PTMs) including lysine acetylation, methylation and ubiquitylation, arginine methylation and serine phosphorylation. Lysine residues may be mono‐, di‐ and trimethylated, the latter resulting in an addition of mass to the protein that differs from acetylation by only 0.03639 Da, but that can be distinguished either on high‐performance mass spectrometers with sufficient mass accuracy and mass resolution or via retention times. Here we describe the use of chemical derivatization to quantify methylated and acetylated histone isoforms by forming deuteroacetylated histone derivatives prior to tryptic digestion and bottom‐up liquid chromatography‐mass spectrometric analysis. The deuteroacetylation of unmodified or mono‐methylated lysine residues produces a chemically identical set of tryptic peptides when comparing the unmodified and modified versions of a protein, making it possible to directly quantify lysine acetylation. In this work, the deuteroacetylation technique is used to examine a single histone H3 peptide with methyl and acetyl modifications at different lysine residues and to quantify the relative abundance of each modification in different deacetylase and methylase knockout yeast strains. This application demonstrates the use of the deuteroacetylation technique to characterize modification ‘cross‐talk’ by correlating different PTMs on the same histone tail. Copyright © 2013 John Wiley & Sons, Ltd.     

Traceless Synthesis of Asymmetrically Modified Bivalent Nucleosomes

Nucleosomes carry extensive post‐translational modifications (PTMs), which results in complex modification patterns that are involved in epigenetic signaling. Although two copies of each histone coexist in a nucleosome, they may not carry the same PTMs and are often differently modified (asymmetric). In bivalent domains, a chromatin signature prevalent in embryonic stem cells (ESCs), namely H3 methylated at lysine 4 (H3K4me3), coexists with H3K27me3 in asymmetric nucleosomes. We report a general, modular, and traceless method for producing asymmetrically modified nucleosomes. We further show that in bivalent nucleosomes, H3K4me3 inhibits the activity of the H3K27‐specific lysine methyltransferase (KMT) polycomb repressive complex 2 (PRC2) solely on the same histone tail, whereas H3K27me3 stimulates PRC2 activity across tails, thereby partially overriding the H3K4me3‐mediated repressive effect. To maintain bivalent domains in ESCs, PRC2 activity must thus be locally restricted or reversed.     

Hydroxamic Acid‐Piperidine Conjugate is an Activated Catalyst for Lysine Acetylation under Physiological Conditions

Lysine acylation of proteins is an essential chemical reaction for posttranslational modification and as a means of protein modification in various applications. N,N‐Dimethyl‐4‐aminopyridine (DMAP) derivatives are widely‐used catalysts for lysine acylation of proteins; however, the DMAP moiety mostly exists in a protonated, and thus deactivated, form under physiological conditions due to its basicity. An alternative catalytic motif furnishing higher acylation activity would further broaden the possible applications of chemical lysine acylation. We herein report that the hydroxamic acid‐piperidine conjugate Ph‐HXA is a more active catalytic motif for lysine acetylation than DMAP under physiological conditions. In contrast to DMAP, the hydroxamic acid moiety is mostly deprotonated under aqueous neutral pH, resulting in a higher concentration of the activated form. The Ph‐HXA catalyst is also more tolerant of deactivation by a high concentration of glutathione than DMAP. Therefore, Ph‐HXA might be a suitable catalytic motif for target protein‐selective and site‐selective acetylation in cells.     

LSD1: more than demethylation of histone lysine residues

Lysine-specific histone demethylase 1 (LSD1) represents the first example of an identified nuclear protein with histone demethylase activity. In particular, it plays a special role in the epigenetic regulation of gene expression, as it removes methyl groups from mono- and dimethylated lysine 4 and/or lysine 9 on histone H3 (H3K4me1/2 and H3K9me1/2), behaving as a repressor or activator of gene expression, respectively. Moreover, it has been recently found to demethylate monomethylated and dimethylated lysine 20 in histone H4 and to contribute to the balance of several other methylated lysine residues in histone H3 (i.e., H3K27, H3K36, and H3K79). Furthermore, in recent years, a plethora of nonhistone proteins have been detected as targets of LSD1 activity, suggesting that this demethylase is a fundamental player in the regulation of multiple pathways triggered in several cellular processes, including cancer progression. In this review, we analyze the molecular mechanism by which LSD1 displays its dual effect on gene expression (related to the specific lysine target), placing final emphasis on the use of pharmacological inhibitors of its activity in future clinical studies to fight cancer.Subject terms: Epigenetics, Histone post-translational modifications     

Isoxazole‐Derived Amino Acids are Bromodomain‐Binding Acetyl‐Lysine Mimics: Incorporation into Histone H4 Peptides and Histone H3

A range of isoxazole‐containing amino acids was synthesized that displaced acetyl‐lysine‐containing peptides from the BAZ2A, BRD4(1), and BRD9 bromodomains. Three of these amino acids were incorporated into a histone H4‐mimicking peptide and their affinity for BRD4(1) was assessed. Affinities of the isoxazole‐containing peptides are comparable to those of a hyperacetylated histone H4‐mimicking cognate peptide, and demonstrated a dependence on the position at which the unnatural residue was incorporated. An isoxazole‐based alkylating agent was developed to selectively alkylate cysteine residues in situ. Selective monoalkylation of a histone H4‐mimicking peptide, containing a lysine to cysteine residue substitution (K12C), resulted in acetyl‐lysine mimic incorporation, with high affinity for the BRD4 bromodomain. The same technology was used to alkylate a K18C mutant of histone H3.     

Aptamer functionalized magnetic graphene oxide nanocomposites for highly selective capture of histones

The binding coverage of aptamer was an important restricted factor for aptamer‐based affinity enrichment strategy for capturing target molecules. Herein, we designed and prepared aptamer functionalized graphene oxide based nanocomposites (GO/NH₂‐NTA/Fe₃O₄/PEI/Au), and the coverage density of aptamer was high to 33.1 nmol/mg. The high aptamer coverage density was contributed to the large surface area of graphene oxide. The successive modification of Nα,Nα‐Bis(carboxymethyl)‐L‐lysine, magnetic nanoparticles, polyethylenimine, and Au nanoparticles ensured the histone purification with fast speed and high purity. Histones could be captured rapidly and specifically from nucleoproteins by our aptamer based purification strategy, while traditional acid‐extraction could not specifically enrich histones. Compared with traditional acid‐extraction method, rapid and efficient discovery of histones and their post‐translational modifications, such as several kinds of methylation at H3.1K9 and H3.1K27, were achieved confidently. It demonstrated that our aptamer functionalized magnetic graphene oxide nanocomposites have a great potential for histone analysis.     

Quantification of Lysine Acetylation and Succinylation Stoichiometry in Proteins Using Mass Spectrometric Data-Independent Acquisitions (SWATH)

Post-translational modification of lysine residues by N_?-acylation is an important regulator of protein function. Many large-scale protein acylation studies have assessed relative changes of lysine acylation sites after antibody enrichment using mass spectrometry-based proteomics. Although relative acylation fold-changes are important, this does not reveal site occupancy, or stoichiometry, of individual modification sites, which is critical to understand functional consequences. Recently, methods for determining lysine acetylation stoichiometry have been proposed based on ratiometric analysis of endogenous levels to those introduced after quantitative per-acetylation of proteins using stable isotope-labeled acetic anhydride. However, in our hands, we find that these methods can overestimate acetylation stoichiometries because of signal interferences when endogenous levels of acylation are very low, which is especially problematic when using MS1 scans for quantification. In this study, we sought to improve the accuracy of determining acylation stoichiometry using data-independent acquisition (DIA). Specifically, we use SWATH acquisition to comprehensively collect both precursor and fragment ion intensity data. The use of fragment ions for stoichiometry quantification not only reduces interferences but also allows for determination of site-level stoichiometry from peptides with multiple lysine residues. We also demonstrate the novel extension of this method to measurements of succinylation stoichiometry using deuterium-labeled succinic anhydride. Proof of principle SWATH acquisition studies were first performed using bovine serum albumin for both acetylation and succinylation occupancy measurements, followed by the analysis of more complex samples of E. coli cell lysates. Although overall site occupancy was low (<1%), some proteins contained lysines with relatively high acetylation occupancy.

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Highly Selective Lysine Acylation in Proteins Using a Lys-His Tag Sequence

Chemical modification of proteins has numerous applications, but it has been challenging to achieve the required high degree of selectivity on lysine amino groups. Recently, we described the highly selective acylation of proteins with an N-terminal Gly-His₆ segment. This tag promoted acylation of the N-terminal N^α-amine resulting in stable conjugates. Herein, we report the peptide sequences His_n-Lys-His_m, which we term Lys-His tags. In combination with simple acylating agents, they facilitate the acylation of the designated Lys N^ϵ-amine under mild conditions and with high selectivity over native Lys residues. We show that the Lys-His tags, which are 7 to 10 amino acids in length and still act as conventional His tags, can be inserted in proteins at the C-terminus or in loops, thus providing high flexibility regarding the site of modification. Finally, the selective and efficient acylation of the therapeutic antibody Rituximab, pure or mixed with other proteins, demonstrates the scope of the Lys-His tag acylation method.     

Comprehensive Profiling for Histone H4of Human Liver Cells Using High Resolution LTQ-Orbitrap Mass Spectrometry

The post translational modifications of histone variants are playing an important role in the structure of chro‐ matin, the regulation of gene activities and the diagnosis of diseases, and conducting in‐depth researches and discovering new sites depend on new and rational analytical methods to some extent. In this work, the combinatorial method of high resolution LTQ‐Orbitrap mass spectrometry and multiple enzymes was employed to identify the post translational modifications (PTMs) of histone H4 of human liver cells. The novel methylation site, argnine 67 (R 67), was observed besides some sites reported previously such as lysine 31 (K 31), lysine 44 (K 44), argnine 55 (R 55) and lysine 59 (K 59) in the global domain. Meanwhile, various combinations of acetylation of lysine 5 (K 5), lysine 8 (K 8), lysine 12 (K 12), lysine 16 (K 16) and methylation of lysine 20 (K 20) in the NH₂‐terminal tails were also identified after the LC‐MS/MS analysis of trypsin, Arg‐C, Glu‐C and chymotrypsin digests.     

Examining histone modification crosstalk using immobilized libraries established from ligation-ready nucleosomes

Chromatin signaling relies on a plethora of posttranslational modifications (PTM) of the histone proteins which package the long DNA molecules of our cells in reoccurring units of nucleosomes. Determining the biological function and molecular working mechanisms of different patterns of histone PTMs requires access to various chromatin substrates of defined modification status. Traditionally, these are achieved by individual reconstitution of single nucleosomes or arrays of nucleosomes in conjunction with modified histones produced by means of chemical biology. Here, we report an alternative strategy for establishing a library of differentially modified nucleosomes that bypasses the need for many individual syntheses, purification and assembly reactions by installing modified histone tails on ligation-ready, immobilized nucleosomes reconstituted in a single batch. Using the ligation-ready nucleosome strategy with sortase-mediated ligation for histone H3 and intein splicing for histone H2A, we generated libraries of up to 280 individually modified nucleosomes in 96-well plate format. Screening these libraries for the effects of patterns of PTMs onto the recruitment of a well-known chromatin factor, HP1 revealed a previously unknown long-range cross-talk between two modifications. H3S28 phosphorylation enhances recruitment of the HP1 protein to the H3K9 methylated H3-tail only in nucleosomal context. Detailed structural analysis by NMR measurements implies negative charges at position 28 to increase nucleosomal H3-tail dynamics and flexibility. Our work shows that ligation-ready nucleosomes enable unprecedented access to the ample space and complexity of histone modification patterns for the discovery and dissection of chromatin regulatory principles.

280 different patterns of histone modifications were installed in preassembled nucleosomes using PTS and SML enabling screening of readout crosstalk.     

NMR assignments and histone specificity of the ING2 PHD finger

The ING2 plant homeodomain (PHD) finger is recruited to the nucleosome through specific binding to histone H3 trimethylated at lysine 4 (H3K4me3). Here, we describe backbone and side chain assignments of the ING2 PHD finger, analyze its binding to the unmodified and modified histone and p53 peptides, and map the histone H3 and H3K4me3 binding sites based on chemical shift perturbation analysis. Copyright © 2009 John Wiley & Sons, Ltd.     

Small Cause,Great Impact: Modification of the Guanidine Group in the RGD Motif Controls Integrin Subtype Selectivity

Due to its unique role as a hydrogen‐bond donor and its positive charge, the guanidine group is an important pharmacophoric group and often used in synthetic ligands. The chemical modification of the guanidine group is often considered to destroy its function. Herein, we show that the N‐methylation, N‐alkylation, or N‐acylation of the guanidine group can be used to modify the receptor subtype specificity of the integrin ligand cilengitide. Using the αvβ6/α5β1‐biselective ligand c(isoDGRkphg) and the αvβ6‐specific ligand c(FRGDLAFp(NMe)K(Ac) as examples, we show that the binding affinities of the ligands can be fine‐tuned by this method to enhance the selectivity for αvβ6. Furthermore, we describe a new strategy for the functionalization of integrin ligands. By introducing longer N‐alkylguanidine and N‐acylguanidine groups, we are able to simultaneously identify a hitherto unknown anchoring point and enhance the subtype selectivity of the ligand.     

Highly Selective Vapor-Phase Acylation of Veratrole over H_3PO_4/TiO_2-ZrO_2: Using Ethyl Acetate as a Green and Efficient Acylating Agent

A simple sol‐gel method with and without surfactant was applied to prepare TiO₂‐ZrO₂ mixed oxides containing Ti and Zr at a molar ratio of 1:1. Several catalysts containing w=15% –35% H₃PO₄ were set up using these mixed oxides. The physical and chemical properties of catalysts were investigated by BET, SEM and pyridine adsorption‐desorption. The catalytic performance of each material was determined for the vapor‐phase acylation of veratrole (1,2‐dimethoxybenzene) to 3,4‐dimethoxyacetophenone (3,4‐DMAP), which was found to be the major product of the reaction of veratrole with ethyl acetate, with alkylated products being the minor products. 2,3‐Dimethoxyacetophenone (2,3‐DMAP) was not detected in the product stream. In the best experimental conditions, the alkylated products were less than 0.7%. This reaction may represent an environmentally friendly alternative to use the ethyl acetate as the acylating reagent. The feed molar ratios of veratrole/ethyl acetate were varied over a wide range of 0.1 to 1, and the optimum feed ratio of veratrole/ethyl acetate was 1:3. Space velocity employed in the veratrole acylation reported as WHSV (veratrole) was 1.2 h^?1. The acylation reactions were carried out in the temperature range of 423 to 673 K and the optimum H₃PO₄ content for acylation was w=15%.     

Stapled Peptides by Late‐Stage C(sp3)−H Activation

Despite the importance of stapled peptides for drug discovery, only few practical processes to prepare cross‐linked peptides have been described; thus the structural diversity of available staple motifs is currently limited. At the same time, C−H activation has emerged as an efficient approach to functionalize complex molecules. Although there are many reports on the C−H functionalization of amino acids, examples of post‐synthetic peptide C−H modification are rare and comprise almost only C(sp²)−H activation. Herein, we report the development of a palladium‐catalyzed late‐stage C(sp³)−H activation method for peptide stapling, affording an unprecedented hydrocarbon cross‐link. This method was first employed to prepare a library of stapled peptides in solution. The compatibility with various amino acids as well as the influence of the size (i ,i +3 and i ,i +4) and length of the staple were investigated. Finally, a simple solid‐phase procedure was also established.