H3 Peptide Research Articles

Intact Nucleosomal Context Enables Chromodomain Reader MRG15 to Distinguish H3K36me3 from - Me2

A wealth of in vivo evidence demonstrates the physiological importance of histone H3 trimethylation at lysine 36 (H3K36me3), to which chromodomain-containing proteins, such as MRG15, bind preferentially compared to their dimethyl (H3K36me2) counterparts. However, in vitro studies using isolated H3 peptides have failed to recapitulate a causal interaction. Here, we show that MRG15 can clearly discriminate between synthetic, fully intact model nucleosomes containing H3K36me2 and H3K36me3. MRG15 docking studies, along with experimental observations and nucleosome structure analysis suggest a model where the H3K36 side chain is sequestered in intact nucleosomes via a hydrogen bonding interaction with the DNA backbone, which is abrogated when the third methyl group is added to form H3K36me3. Hence, this mechanism provides a ‘methyl-switch’ for context-dependent reader selectivity. These results highlight the importance of such intra-chromatin interactions in understanding epigenetic regulation, a feature which is absent in commonly-used peptide or histone-only models.

Bacterial citrullinated epitopes generated by Porphyromonas gingivalis infection—a missing link for ACPA production

ObjectivesPorphyromonas gingivalis (P.g.) is discussed to be involved in triggering self-reactive immune responses. The aim of this study was to investigate the autocitrullinated prokaryotic peptidylarginine deiminase (PPAD) from P.g. CH2007...

A Multiplexed Microarray Platform for Targeted Proteomics

A Multiplexed Microarray Platform for Targeted Proteomics

The Eaf3 chromodomain acts as a pH sensor for gene expression by altering its binding affinity for histone methylated-lysine residues.

During gene expression, histone acetylation by histone acetyltransferase (HAT) loosens the chromatin structure around the promoter to allow RNA polymerase II (Pol II) to initiate transcription, while de-acetylation by histone deacetylase (HDAC) tightens the structure in the transcribing region to repress false initiation. Histone acetylation is also regulated by intracellular pH (pHi) with global hypoacetylation observed at low pHi, and hyperacetylation, causing proliferation, observed at high pHi. However, the mechanism underlying the pHi-dependent regulation of gene expression remains elusive. Here, we have explored the role of the chromodomain (CD) of budding yeast Eaf3, a common subunit of both HAT and HDAC that is thought to recognize methylated lysine residues on histone H3. We found that Eaf3 CD interacts with histone H3 peptides methylated at Lys4 (H3K4me, a promoter epigenetic marker) and Lys36 (H3K36me, a coding region epigenetic marker), as well as with many dimethyl-lysine peptides and even arginine-asymmetrically dimethylated peptides, but not with unmethylated, phosphorylated or acetylated peptides. The Eaf3 CD structure revealed an unexpected histidine residue in the aromatic cage essential for binding H3K4me and H3K36me. pH titration experiments showed that protonation of the histidine residue around physiological pH controls the charge state of the aromatic cage to regulate binding to H3K4me and H3K36me. Histidine substitution and NMR experiments confirmed the correlation of histidine pKa with binding affinity. Collectively, our findings suggest that Eaf3 CD functions as a pHi sensor and a regulator of gene expression via its pHi-dependent interaction with methylated nucleosomes.

Rational design and improvement of the dimerization-disrupting peptide selectivity between ROCK-I and ROCK-II kinase isoforms in cerebrovascular diseases.

Human rho-associated coiled-coil forming kinases (ROCKs) ROCK-I and ROCK-II have been documented as attractive therapeutic targets for cerebrovascular diseases. Although ROCK-I and ROCK-II share a high degree of structural conservation and are both present in classic rho/ROCK signaling pathway, their downstream substrates and pathological functions may be quite different. Selective targeting of the two kinase isoforms with traditional small-molecule inhibitors is a great challenge due to their surprisingly high homology in kinase domain (~90%) and the full identity in kinase active site (100%). Here, instead of developing small-molecule drugs to selectively target the adenosine triphosphate (ATP) site of two isoforms, we attempt to design peptide agents to selectively disrupt the homo-dimerization event of ROCK kinases through their dimerization domains which have a relatively low conservation (~60%). Three helical peptides H1, H2, and H3 are split from the kinase dimerization domain, from which the isolated H2 peptide is found to have the best capability to rebind at the dimerization interface. A simulated annealing (SA) iteration method is used to improve the H2 peptide selectivity between ROCK-I and ROCK-II. The method accepts moderate degradation in peptide affinity in order to maximize the affinity difference between peptide binding to the two isoforms. Consequently, hundreds of parallel SA runs yielded six promising peptide candidates with ROCK-I over ROCK-II (I over II [IoII]) calculated selectivity and four promising peptide candidates with ROCK-II over ROCK-I (II over I [IIoI]) calculated selectivity. Subsequent anisotropy assays confirm that the selectivity values range between 13.2-fold and 83.9-fold for IoII peptides, and between 5.8-fold and 21.2-fold for IIoI peptides, which are considerably increased relative to wild-type H2 peptide (2.6-fold for IoII and 2.0-fold for IIoI). The molecular origin of the designed peptide selectivity is also analyzed at structural level; it is revealed that the peptide residues can be classified into conserved, non-conserved, and others, in which the non-conserved residues play a crucial role in defining peptide selectivity, while conserved residues confer stability to kinase-peptide binding.

Investigation of Neutral Losses and the Citrulline Effect for Modified H4 N-Terminal Pentapeptides.

Tandem mass spectrometry is an indispensable tool in proteomics used for protein sequencing and quantitation. On the basis of the sequential fragments usually generated from peptide ions via collision-induced dissociation, electron-transfer dissociation, or a combination of the two, probabilistic database search engines could be used for the identification of the peptides. The correct localization of posttranslational modifications (PTMs) poses a more challenging problem than the general identification of proteins. Histones are involved in the regulation of DNA transcription via the wealth of PTMs on their N-terminal tail. In this study, we analyzed the histone H4 peptide SGRGK incorporating four different posttranslational modifications: citrullination, acetylation, phosphorylation, and arginine methylation at various positions. The pentapeptides model the enzymatic cleavage of the N-terminal tail of human histone H4 protein by LysC protease. Fragmentation of the peptides was investigated using higher-energy collisional dissociation (HCD), electron-transfer dissociation (ETD), and electron-transfer higher-energy collisional dissociation (EThcD) on an ultrahigh resolution and mass accuracy instrument. We found that while all three techniques have their unique characteristics, advantages, and pitfalls, EThcD generated the most fragment ion-rich spectra. Despite potential ambiguities regarding exact fragment identities, full sequence coverage and PTM mapping may also be achievable. We also found novel neutral losses from the charge-reduced precursors characteristic to citrullination in ETD and EThcD which may be used in proteomic applications. N-Terminal acetylation and arginine methylation could also be confirmed by their characteristic neutral losses from the charge-reduced precursors.

Rice Histone Propionylation and Generation of Chemically Derivatized Synthetic H3 and H4 Peptides for Identification of Acetylation Sites and Quantification.

Histone proteins are crucial in the study of chromatin dynamics owing to their wide-ranging implications in the regulation of gene expression. Modifications of histones are integral to these regulatory processes in concert with associated proteins, such as transcription factors and coactivators. One of the biochemical techniques available to enhance analysis of histone proteins is chemical derivatization using propionic anhydride. In this protocol, we describe the use of propionylation to efficiently derivatize acid-extracted histones from rice. We also synthesize H3 and H4 tryptic peptides, thus mimicking the nature of derivatized extracted peptides to aid in identification and quantification using targeted-mass spectrometry. Here we make available the masses of the precursor ions and the retention times (RT) of each synthesized peptide. These provide useful information to facilitate histone data analysis. Lastly, we note that we will distribute these synthetic peptides in nanomolar (nM) concentrations to those who wish to utilize them for assays and further experimental studies.

Microscopic Viscosity of Neuronal Plasma Membranes Measured Using Fluorescent Molecular Rotors: Effects of Oxidative Stress and Neuroprotection.

Molecular mobility in neuronal plasma membranes is a crucial factor in brain function. Microscopic viscosity is an important parameter that determines molecular mobility. This study presents the first direct measurement of the microviscosity of plasma membranes of live neurons. Microviscosity maps were obtained using fluorescence lifetime imaging of environment-sensing dyes termed "molecular rotors". Neurons were investigated both in the basal state and following common neurodegenerative stimuli, excitotoxicity, or oxidative stress. Both types of neurotoxic challenges induced microviscosity decrease in cultured neurons, and oxidant-induced membrane fluidification was counteracted by the wide-spectrum neuroprotectant, the H3 peptide. These results provide new insights into molecular mobility in neuronal membranes, paramount for basic brain function, and suggest that preservation of membrane stability may be an important aspect of neuroprotection in brain insults and neurodegenerative disorders.

Structural basis of nucleosome recognition and modification by MLL methyltransferases.

Methyltransferases of the mixed-lineage leukaemia (MLL) family-which include MLL1, MLL2, MLL3, MLL4, SET1A and SET1B-implement methylation of histone H3 on lysine4 (H3K4), and have critical and distinct roles in the regulation of transcription in haematopoiesis, adipogenesis and development1-6. The C-terminal catalytic SET (Su(var.)3-9, enhancer of zeste and trithorax) domains of MLL proteins are associated with a common set of regulatory factors (WDR5, RBBP5, ASH2L and DPY30) to achieve specific activities7-9. Current knowledge of the regulation of MLL activity is limited to the catalysis of histone H3 peptides, and how H3K4 methyl marks are deposited on nucleosomes is poorly understood. H3K4 methylation is stimulated by mono-ubiquitination of histone H2B on lysine120 (H2BK120ub1), a prevalent histone H2B mark that disrupts chromatin compaction and favours open chromatin structures, but the underlying mechanism remains unknown10-12. Here we report cryo-electron microscopy structures of human MLL1 and MLL3 catalytic modules associated with nucleosome core particles that contain H2BK120ub1 or unmodified H2BK120. These structures demonstrate that the MLL1 and MLL3 complexes both make extensive contacts with the histone-fold and DNA regions of the nucleosome; this allows ease of access to the histone H3 tail, which is essential for the efficient methylation of H3K4. The H2B-conjugated ubiquitin binds directly to RBBP5, orienting the association between MLL1 or MLL3 and the nucleosome. The MLL1 and MLL3 complexes display different structural organizations at the interface between the WDR5, RBBP5 and MLL1 (or the corresponding MLL3) subunits, which accounts for the opposite roles of WDR5 in regulating the activity of the two enzymes. These findings transform our understanding of the structural basis for the regulation of MLL activity at the nucleosome level, and highlight the pivotal role of nucleosome regulation in histone-tail modification.

Methyl-CpG-binding domain 9 (MBD9) is required for H2A.Z incorporation into chromatin at a subset of H2A.Z-enriched regions in the Arabidopsis genome

The SWR1 chromatin remodeling complex, which deposits the histone variant H2A.Z into nucleosomes, has been well characterized in yeast and animals, but its composition in plants has remained uncertain. We used the conserved SWR1 subunit ACTIN RELATED PROTEIN 6 (ARP6) as bait in tandem affinity purification experiments to isolate associated proteins from Arabidopsis thaliana. We identified all 11 subunits found in yeast SWR1 and the homologous mammalian SRCAP complexes, demonstrating that this complex is conserved in plants. We also identified several additional proteins not previously associated with SWR1, including Methyl-CpG-BINDING DOMAIN 9 (MBD9) and three members of the Alfin1-like protein family, all of which have been shown to bind modified histone tails. Since mbd9 mutant plants were phenotypically similar to arp6 mutants, we explored a potential role for MBD9 in H2A.Z deposition. We found that MBD9 is required for proper H2A.Z incorporation at thousands of discrete sites, which represent a subset of the genomic regions normally enriched with H2A.Z. We also discovered that MBD9 preferentially interacts with acetylated histone H4 peptides, as well as those carrying mono- or dimethylated H3 lysine 4, or dimethylated H3 arginine 2 or 8. Considering that MBD9-dependent H2A.Z sites show a distinct histone modification profile, we propose that MBD9 recognizes particular nucleosome modifications via its PHD- and Bromo-domains and thereby guides SWR1 to these sites for H2A.Z deposition. Our data establish the SWR1 complex as being conserved across eukaryotes and suggest that MBD9 may be involved in targeting the complex to specific genomic sites through nucleosomal interactions. The finding that MBD9 does not appear to be a core subunit of the Arabidopsis SWR1 complex, along with the synergistic phenotype of arp6;mbd9 double mutants, suggests that MBD9 also has important roles beyond H2A.Z deposition.

Chemical synthesis of the ubiquitinated form of histone H3 and its effect on DNA methyltransferase 1.

Posttranslational modifications of histone proteins, which form nucleosome cores, play an important role in gene regulation. Ubiquitination is one such modification. We previously reported on the synthesis of ubiquitinated histone H3 with an isopeptide mimetic structure. In this report, we describe the preparation of ubiquitinated histone H3 peptides with a native isopeptide structure, which showed a slightly weaker effect on the enzymatic activity of DNA methyltransferase 1 than the previous ubiquitinated H3 peptide analogs. These findings show that a native structure is important for determining the mechanism of the function, although ubiquitinated H3 peptide analogs can mimic the role of the original ubiquitinated H3. We also report on the successful preparation of the ubiquitinated full length histone H3.

Discovery of novel CBP bromodomain inhibitors through TR-FRET-based high-throughput screening.

The cAMP-responsive element binding protein (CREB) binding protein (CBP) and adenoviral E1A-binding protein (P300) are two closely related multifunctional transcriptional coactivators. Both proteins contain a bromodomain (BrD) adjacent to the histone acetyl transferase (HAT) catalytic domain, which serves as a promising drug target for cancers and immune system disorders. Several potent and selective small-molecule inhibitors targeting CBP BrD have been reported, but thus far small-molecule inhibitors targeting BrD outside of the BrD and extraterminal domain (BET) family are especially lacking. Here, we established and optimized a TR-FRET-based high-throughput screening platform for the CBP BrD and acetylated H4 peptide. Through an HTS assay against an in-house chemical library containing 20 000 compounds, compound DC_CP20 was discovered as a novel CBP BrD inhibitor with an IC50 value of 744.3 nM. This compound bound to CBP BrD with a KD value of 4.01 μM in the surface plasmon resonance assay. Molecular modeling revealed that DC_CP20 occupied the Kac-binding region firmly through hydrogen bonding with the conserved residue N1168. At the celluslar level, DC_CP20 dose-dependently inhibited the proliferation of human leukemia MV4-11 cells with an IC50 value of 19.2 μM and markedly downregulated the expression of the c-Myc in the cells. Taken together, the discovery of CBP BrD inhibitor DC_CP20 provides a novel chemical scaffold for further medicinal chemistry optimization and a potential chemical probe for CBP-related biological function research. In addition, this inhibitor may serve as a promising therapeutic strategy for MLL leukemia by targeting CBP BrD protein.

MORC3 Is a Target of the Influenza A Viral Protein NS1

Microrchidia 3 (MORC3), a human ATPase linked to several autoimmune disorders, has been characterized both as a negative and positive regulator of influenza A virus. Here, we report that the CW domain of MORC3 (MORC3-CW) is targeted by the C-terminal tail of the influenza H3N2 protein NS1. The crystal structure of the MORC3-CW:NS1 complex shows that NS1 occupies the same binding site in CW that is normally occupied by histone H3, a physiological ligand of MORC3-CW. Comparable binding affinities of MORC3-CW to H3 and NS1 peptides and to the adjacent catalytic ATPase domain suggest that the viral protein can compete with the host histone for the association with CW, releasing MORC3 autoinhibition and activating the catalytic function of MORC3. Our structural, biochemical, and cellular analyses suggest that MORC3 might affect the infectivity of influenza virus and therefore has a role in cell immune response.

Synchronous delivery of hydroxyapatite and connective tissue growth factor derived osteoinductive peptide enhanced osteogenesis

In bone tissue engineering, electrospun fibrous scaffolds can provide excellent mechanical support, extracellular matrix mimicking components, such as 3D spacial fibrous environment for cell growth and controlled release of signaling molecules for osteogenesis. Here, a facile strategy comprising the incorporation of an osteogenic inductive peptide H1, derived from the cysteine knot (CT) domain of connective tissue growth factor (CTGF), in the core of Silk Fibroin (SF) was developed for osteogenic induction, synergistically with co-delivering hydroxyapatite (HA) from the shell of poly(l-lactic acid-co-ε-caprolactone) (PLCL). The core-shell nanofibrous structure was confirmed by transmission electron microscopy (TEM). Furthermore, the sustained released H1 has effectively promoted proliferation and osteoblastic differentiation of human induced pluripotent stem cells-derived mesenchymal stem cells (hiPS-MSCs). Moreover, after 8 weeks implantation in mice, this SF-H1/PLCL-HA composite induced bone tissue formation significantly faster than SF/PLCL as indicated by μCT. The present study is the first to demonstrate that release of short hydrophilic peptides derived from CTGF combined with HA potentiated the regenerative capacity for healing critical sized calvarial defect in vivo.

Cover Feature: Recognition of ASF1 by Using Hydrocarbon‐Constrained Peptides (ChemBioChem 7/2019)

The cover feature picture shows how inhibition of the histone H3-ASF1 (anti-silencing function 1) protein–protein interaction (PPI) represents a potential approach for the treatment of numerous cancers. To pursue this goal, a hydrocarbon constraint was used to pre-organize the histone H3 peptide in α-helical conformation; despite conferring protection against proteolysis, this constrained peptide unexpectedly exhibited enthalpy–entropy compensation in comparison to the natural sequence, in its binding of ASF1. More information can be found in the communication by F. Ochsenbein, A. J. Wilson et al. on page 891 in Issue 7, 2019 (DOI: 10.1002/cbic.201800633).

Human H4 tail stimulates HIV-1 integration through binding to the carboxy-terminal domain of integrase

The integration of the retroviral genome into the chromatin of the infected cell is catalysed by the integrase (IN)•viral DNA complex (intasome). This process requires functional association between the integration complex and the nucleosomes. Direct intasome/histone contacts have been reported to modulate the interaction between the integration complex and the target DNA (tDNA). Both prototype foamy virus (PFV) and HIV-1 integrases can directly bind histone amino-terminal tails. We have further investigated this final association by studying the effect of isolated histone tails on HIV-1 integration. We show here that the binding of HIV-1 IN to a peptide derived from the H4 tail strongly stimulates integration catalysis in vitro. This stimulation was not observed with peptide tails from other variants or with alpha-retroviral (RAV) and spuma-retroviral PFV integrases. Biochemical analyses show that the peptide tail induces both an increase in the IN oligomerization state and affinity for the target DNA, which are associated with substantial structural rearrangements in the IN carboxy-terminal domain (CTD) observed by NMR. Our data indicate that the H4 peptide tail promotes the formation of active strand transfer complexes (STCs) and support an activation step of the incoming intasome at the contact of the histone tail.

Experience-based discovery (EBD) of aryl hydrazines as new scaffolds for the development of LSD1/KDM1A inhibitors

Phenelzine was first employed to design new aryl hydrazine-based LSD1 inhibitors based on the experience-based discovery (EBD) strategy. Among these compounds, D8 potently inhibited LSD1 (IC50 = 882.30 nM) in a reversible manner. Compound D8 was selective to LSD1 over MAO-A/B and showed H3K4me2 competitive binding to LSD1. The interaction between H3K4me2 and LSD1 was also confirmed by the Co-IP assay. In LSD1 overexpressed A549 cells, compound D8 dose-dependently induced accumulation of LSD1 substrates H3K4me1/2 and H3K9me1/2, showed cellular target engagement to LSD1 and significantly inhibited cell migration of A549 cells. Docking studies suggested that compound D8 occupied the peptide binding region and therefore blocked the access of the peptide substrate to the FAD, finally leading to the demethylase activity inhibition of LSD1. The findings indicate that aryl hydrazines are new scaffolds for the design of LSD1 inhibitors, the identification of D8 provides further evidence for our previously proposed general principle that fused heterocycles with an amine group are potentially active toward LSD1 by competitive binding to LSD1 with H3 peptide substrates.

Bisubstrate inhibitors to target histone acetyltransferase 1.

Developing selective enzyme inhibitors allows for the expansion of molecular toolboxes to investigate functions and activities of target enzymes. The histone acetyltransferase 1 (HAT1) is among the first histone acetyltransferase (HAT) enzymes that were discovered in the mid-1990s; however, it remains one of the poorly studied enzymes in comparison with the other HATs. Although HAT1 has been linked to various disease states, no inhibitors have been reported to target HAT1. Here, we designed a set of peptide-CoA conjugates as bisubstrate inhibitors of HAT1 with submicromolar potency. In particular, the bisubstrate inhibitor H4K12CoA exhibited a low Ki value of 1.1nM for HAT1. In addition, H4K12CoA was shown to be a competitive inhibitor with respect to both AcCoA and H4 peptide, suggesting a unique kinetic mechanism of HAT1 catalysis. Creating these submicromolar inhibitors offers mechanistic tools to better understand how HAT1 recognizes substrates and cofactors, as well as provides chemical leads to further develop therapeutic agents to target this important enzyme for disease therapy.

Discovery of alkoxy benzamide derivatives as novel BPTF bromodomain inhibitors via structure-based virtual screening

Bromodomain PHD finger transcription factor (BPTF), a bromodomain-containing protein, plays a crucial role in the regulation of downstream gene expression through the specific recognition of lysine acetylation on bulk histones. The dysfunction of BPTF is closely involved with the development and progression of many human diseases, especially cancer. Therefore, BPTF bromodomain has become a promising drug target for epigenetic cancer therapy. However, unlike BET family inhibitors, few BPTF bromodomain inhibitors have been reported. In this study, by integrating docking-based virtual screening with biochemical analysis, we identified a novel selective BPTF bromodomain inhibitor DCB29 with the IC50 value of 13.2 ± 1.6 μM by homogenous time-resolved fluorescence resonance energy transfer (HTRF) assays. The binding between DCB29 and BPTF was confirmed by NMR and SPR. Molecular docking disclosed that DCB29 occupied the pocket of acetylated H4 peptide substrate and provided detailed SAR explanations for its derivatives. Collectively, DCB29 presented great potential as a powerful tool for BPTF-related biological research and further medicinal chemistry optimization.

Towards Unraveling the Histone Code by Fragment Blind Docking.

Histones serve as protein spools for winding the DNA in the nucleosome. High variability of their post-translational modifications result in a unique code system often responsible for the pathomechanisms of epigenetics-based diseases. Decoding is performed by reader proteins via complex formation with the N-terminal peptide tails of histones. Determination of structures of histone-reader complexes would be a key to unravel the histone code and the design of new drugs. However, the large number of possible histone complex variations imposes a true challenge for experimental structure determination techniques. Calculation of such complexes is difficult due to considerable size and flexibility of peptides and the shallow binding surfaces of the readers. Moreover, location of the binding sites is often unknown, which requires a blind docking search over the entire surface of the target protein. To accelerate the work in this field, a new approach is presented for prediction of the structure of histone H3 peptide tails docked to their targets. Using a fragmenting protocol and a systematic blind docking method, a collection of well-positioned fragments of the H3 peptide is produced. After linking the fragments, reconstitution of anchoring regions of the target-bound H3 peptide conformations was possible. As a first attempt of combination of blind and fragment docking approaches, our new method is named fragment blind docking (FBD).