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.     

P−B Desulfurization: An Enabling Method for Protein Chemical Synthesis and Site‐Specific Deuteration

Cysteine‐mediated native chemical ligation is a powerful method for protein chemical synthesis. Herein, we report an unprecedentedly mild system (TCEP/NaBH₄ or TCEP/LiBEt₃H; TCEP=tris(2‐carboxyethyl)phosphine) for chemoselective peptide desulfurization to achieve effective protein synthesis via the native chemical ligation–desulfurization approach. This method, termed P−B desulfurization, features usage of common reagents, simplicity of operation, robustness, high yields, clean conversion, and versatile functionality compatibility with complex peptides/proteins. In addition, this method can be used for incorporating deuterium into the peptides after cysteine desulfurization by running the reaction in D₂O buffer. Moreover, this method enables the clean desulfurization of peptides carrying post‐translational modifications, such as phosphorylation and crotonylation. The effectiveness of this method has been demonstrated by the synthesis of the cyclic peptides dichotomin C and E and synthetic proteins, including ubiquitin, γ‐synuclein, and histone H2A.     

The chemical bonding and spectral assignments of rhodium(III)‐catalyzed closo‐dodecaborate complexes: Ab initio study

The chemical bonding and spectral assignments of rhodium(III)‐catalyzed closo‐dodecaborate (RhCD) complex are systematically studied using the density functional theory calculations. It is found that the calculated main bond lengths of framework are in good agreement with experimental X‐ray observation, and the pronounced hybridization of B‐2p and Rh‐4d states is responsible for the structural stability, reflected by the large dissociation energy and HOMO–LUMO energy gap. The AdNDP chemical bonding analysis indicates that the RhCD complex can be stabilized by two H‐bridged 3c‐2e σ‐bonds (B‐H‐Rh triangles). Additionally, the theoretical calculations reproduce well the main experimental IR spectrum, and the characteristic peaks are properly assigned. These results will be helpful for further insight into the unique electronic structure of the species, and provide valuable references for potential applications in novel materials.     

Enzyme‐Instructed Assemblies Enable Mitochondria Localization of Histone H2B in Cancer Cells

Presently, little is known of how the inter‐organelle crosstalk impacts cancer cells owing to the lack of approaches that can manipulate inter‐organelle communication in cancer cells. We found that a negatively charged, enzyme cleavable peptide (MitoFlag) enables the trafficking of histone protein H2B, a nuclear protein, to the mitochondria in cancer cells. MitoFlag interacts with the nuclear location sequence of H2B to block it from entering the nucleus. A protease on the mitochondria cleaves the Flag from the MitoFlag/H2B complex to form assemblies that retain H2B on the mitochondria and facilitate H2B entering the mitochondria. Adding NLS, replacing aspartic acid by glutamic acid residues, or changing the l ‐ to d ‐aspartic acid residue on MitoFlag abolishes the trafficking of H2B into mitochondria of HeLa cells. As the first example of the enzyme‐instructed self‐assembly of a synthetic peptide for trafficking endogenous proteins, this work provides insights for understanding and manipulating inter‐organelle communication in cells.     

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.     

Chemical Synthesis, 3D Structure,and ASIC Binding Site of the Toxin Mambalgin‐2

Mambalgins are a novel class of snake venom components that exert potent analgesic effects mediated through the inhibition of acid‐sensing ion channels (ASICs). The 57‐residue polypeptide mambalgin‐2 (Ma‐2) was synthesized by using a combination of solid‐phase peptide synthesis and native chemical ligation. The structure of the synthetic toxin, determined using homonuclear NMR, revealed an unusual three‐finger toxin fold reminiscent of functionally unrelated snake toxins. Electrophysiological analysis of Ma‐2 on wild‐type and mutant ASIC1a receptors allowed us to identify α‐helix 5, which borders on the functionally critical acidic pocket of the channel, as a major part of the Ma‐2 binding site. This region is also crucial for the interaction of ASIC1a with the spider toxin PcTx1, thus suggesting that the binding sites for these toxins substantially overlap. This work lays the foundation for structure–activity relationship (SAR) studies and further development of this promising analgesic peptide.     

Synthesis and Macrodomain Binding of Mono‐ADP‐Ribosylated Peptides

Mono‐ADP‐ribosylation is a dynamic posttranslational modification (PTM) with important roles in signaling. Mammalian proteins that recognize or hydrolyze mono‐ADP‐ribosylated proteins have been described. We report the synthesis of ADP‐ribosylated peptides from the proteins histone H2B, RhoA and, HNP‐1. An innovative procedure was applied that makes use of pre‐phosphorylated amino acid building blocks. Binding assays revealed that the macrodomains of human MacroD2 and TARG1 exhibit distinct specificities for the different ADP‐ribosylated peptides, thus showing that the sequence surrounding ADP‐ribosylated residues affects the substrate selectivity of macrodomains.     

NMR Spectroscopic Characterization of Isocyano‐ and 1, 1′‐Diisocyanoferrocene. The Molecular Structure of Isocyanoferrocene

Isocyanoferrocene ( 1 ) and 1, 1′‐diisocyanoferrocene ( 2 ) were prepared and studied by ¹H, ¹³C, ¹⁴N and ⁵⁷Fe NMR spectroscopy in order to gain a more complete data set. The NMR data of 1 (chemical shifts and coupling constants) were calculated by DFT methods [B3LYP/6‐311+G(d, p)] and compare favourably with experimental data. The molecular structure of 1 was determined by X‐ray structural analysis, and an almost undistorted ferrocene‐like geometry was found.     

Peptide o‐Aminoanilides as Crypto‐Thioesters for Protein Chemical Synthesis

Fully unprotected peptide o‐aminoanilides can be efficiently activated by NaNO₂ in aqueous solution to furnish peptide thioesters for use in native chemical ligation. This finding enables the convergent synthesis of proteins from readily synthesizable peptide o‐aminoanilides as a new type of crypto‐thioesters. The practicality of this approach is shown by the synthesis of histone H2B from five peptide segments. Purification or solubilization tags, which are sometimes needed to improve the efficiency of protein chemical synthesis, can be incorporated into the o‐aminoanilide moiety, as demonstrated in the preparation of the cyclic protein lactocyclicin Q.     

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.     

1H NMR analysis of O‐methyl‐inositol isomers: a joint experimental and theoretical study

Density functional theory (DFT) calculations of ¹H NMR chemical shifts for l ‐quebrachitol isomers were performed using the B3LYP functional employing the 6‐31G(d,p) and 6‐311 + G(2d,p) basis sets. The effect of the solvent on the B3LYP‐calculated NMR spectrum was accounted for using the polarizable continuum model. Comparison is made with experimental ¹H NMR spectroscopic data, which shed light on the average uncertainty present in DFT calculations of chemical shifts and showed that the best match between experimental and theoretical B3LYP ¹H NMR profiles is a good strategy to assign the molecular structure present in the sample handled in the experimental measurements. Among four plausible O‐methyl‐inositol isomers, the l ‐quebrachitol 2a structure was unambiguously assigned based only on the comparative analysis of experimental and theoretical ¹H NMR chemical shift data. The B3LYP infrared (IR) spectrum was also calculated for the four isomers and compared with the experimental data, with analysis of the theoretical IR profiles corroborating assignment of the 2a structure. Therefore, it is confirmed in this study that a combined experimental/DFT spectroscopic investigation is a powerful tool in structural/conformational analysis studies. Copyright © 2012 John Wiley & Sons, Ltd.     

Selective Inhibition of Lysine‐Specific Demethylase 5A (KDM5A) Using a Rhodium(III) Complex for Triple‐Negative Breast Cancer Therapy

Lysine‐specific demethylase 5A (KDM5A) has recently become a promising target for epigenetic therapy. In this study, we designed and synthesized metal complexes bearing ligands with reported demethylase and p27 modulating activities. The Rh(III) complex 1 was identified as a direct, selective and potent inhibitor of KDM5A that directly abrogate KDM5A demethylase activity via antagonizing the KDM5A‐tri‐/di‐methylated histone 3 protein–protein interaction (PPI) in vitro and in cellulo. Complex 1 induced accumulation of H3K4me3 and H3K4me2 levels in cells, causing growth arrest at G1 phase in the triple‐negative breast cancer (TNBC) cell lines, MDA‐MB‐231 and 4T1. Finally, 1 exhibited potent anti‐tumor activity against TNBC xenografts in an in vivo mouse model, presumably via targeting of KDM5A and hence upregulating p27. Moreover, complex 1 was less toxic compared with two clinical drugs, cisplatin and doxorubicin. To our knowledge, complex 1 is the first metal‐based KDM5A inhibitor reported in the literature. We anticipate that complex 1 may be used as a novel scaffold for the further development of more potent epigenetic agents against cancers, including TNBC.     

Identification of ADP-ribosylated histones by the combined use of high-performance liquid chromatography and electrophoresis

Reversed-phase high-performance liquid chromatography (HPLC) was employed for analysing mono- and oligo(ADP-ribosyl)ated histones. Under the chromatographic conditions described, the ADP-ribosylated histones showed similar retention times to the unmodified histones, although the molecular weight and the charge of the proteins are significantly altered by their modification. The simultaneous elution of unmodified and labelled modified histones was detected by two types of gel electrophoresis and by autoradiography. In addition, the HPLC fractions did not display overlapping ladders of the multiply modified histones, as is commonly seen in one-dimensional electrophoretic analyses of unfractionated material. Hence individual bands could be unambiguously assigned. After in vitro labelling of isolated rat liver nuclei, the following ADP-ribosylated and unmodified histones were identified by HPLC and gel electrophoresis: histone H1(0), four histone H1 subfractions, histone H2A.1, histone H2A.2, oxidized histone H2A.2, histone H2A.X, histone H2A.Z, histone H2B, three histone H3 variants and histone H4.     

Targeted Histone Peptides: Insights into the Spatial Regulation of the Methyltransferase PRC2 by using a Surrogate of Heterotypic Chromatin

Eukaryotic genomes are dynamically regulated through a host of epigenetic stimuli. The substrate for these epigenetic transactions, chromatin, is a polymer of nucleosome building blocks. In native chromatin, each nucleosome can differ from its neighbors as a result of covalent modifications to both the DNA and the histone packaging proteins. The heterotypic nature of chromatin presents a formidable obstacle to biochemical studies seeking to understand the role of context on epigenetic regulation. A chemical approach to the production of heterotypic chromatin that can be used in such studies is introduced. This method involves the attachment of a user‐defined modified histone peptide to a designated nucleosome within the polymer by using a peptide nucleic acid (PNA) targeting compound. This strategy was applied to dissect the effect of chromatin context on the activity of the histone methyltransferase PRC2. The results show that PRC2 can be stimulated to produce histone H3 methylation from a defined nucleation site.     

Tetraphenylethylene Conjugated with a Specific Peptide as a Fluorescence Turn‐On Bioprobe for the Highly Specific Detection and Tracing of Tumor Markers in Live Cancer Cells

Smart molecular probes and flexible methods are attracting remarkable interest for the visualization of cancer‐related biological and chemical events. In this work, a new fluorescence turn‐on probe with dual‐recognition characteristics for the specific imaging of cancer cells is reported. This new bioprobe is rationally designed by linking tetraphenylethylene (TPE), an aggregation‐induced emission (AIE) fluorophore, with the small peptide IHGHHIISVG (referred to as AP2H), a targeting ligand to the broad‐spectrum cancer‐related protein LAPTM4B. The binding of the probe TPE‐AP2H with the target, both in solution and at the cellular level, switches on the fluorescence of TPE because of the inhibition of internal rotations within the TPE framework. Accordingly, this bioprobe allows the real‐time monitoring and subcellular localization of LAPTM4B in cancer cells, with a very high target‐to‐background ratio for the imaging. Furthermore, brighter fluorescence images are detected after incubation of TPE‐AP2H with tumor cells at lower pH values. Thus, this new bioprobe is more advantageous because it can simultaneously target the LAPTM4B protein and sense the characteristic low‐pH environment of tumor cells. In addition, TPE‐AP2H displays high photostability and low cytotoxicity. Therefore, this new bioprobe is promising for the more accurate and reliable imaging of tumor markers in live cancer cells.     

Indirect nuclear 57Fe-13C and 57Fe-1H spin-spin coupling in ferrocenes and cyclopentadienyliron complexes: measurements and DFT calculations

Several 57Fe-labeled ferrocene derivatives and other cyclopentadienyliron complexes were studied by 57Fe and 13C NMR with respect to isotope-induced chemical shifts 1Delta12/13C(57Fe) and the magnitude and sign of coupling constants 1J(57Fe,13C) and 2J(57Fe,1H). The geometries of the complexes were optimized by DFT methods [B3LYP/6-311+G(d,p)] and chemical shifts (GIAO) and coupling constants were calculated at the same level of theory. The trends in calculated 57Fe nuclear shielding agree well with the experimental data and, in the case of coupling constants 1J(57Fe,13C) and 2J(57Fe,1H), both sign and magnitude are correctly reproduced.     

Metabolic‐intermediate complex formation with cytochrome P450: Theoretical studies in elucidating the reaction pathway for the generation of reactive nitroso intermediate

Mechanism‐based inhibition (MBI) of cytochrome P450 (CYP) can lead to drug–drug interactions and often to toxicity. Some aliphatic and aromatic amines can undergo biotransformation reactions to form reactive metabolites such as nitrosoalkanes, leading to MBI of CYPs. It has been proposed that the nitrosoalkanes coordinate with the heme iron, forming metabolic‐intermediate complex (MIC), resulting in the quasi‐irreversible inhibition of CYPs. Limited mechanistic details regarding the formation of reactive nitroso intermediate and its coordination with heme‐iron have been reported. A quantum chemical analysis was performed to elucidate potential reaction pathways for the generation of nitroso intermediate and the formation of MIC. Elucidation of the energy profile along the reaction path, identification of three‐dimensional structures of reactive intermediates and transition states, as well as charge and spin density analyses, were performed using the density functional B3LYP method. The study was performed using Cpd I [iron (IV‐oxo] heme porphine with SH^? as the axial ligand) to represent the catalytic domain of CYP, simulating the biotransformation process. Three pathways: (i) N‐oxidation followed by proton shuttle, (ii) N‐oxidation followed by 1,2‐H shift, and (iii) H‐abstraction followed by rebound mechanism, were studied. It was observed that the proton shuttle pathway was more favorable over the whole reaction leading to reactive nitroso intermediate. This study revealed that the MIC formation from a primary amine is a favorable exothermic process, involving eight different steps and preferably takes place on the doublet spin surface of Cpd I . The rate‐determining step was identified to be the first N‐oxidation of primary amine. © 2012 Wiley Periodicals, Inc.     

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