In vitro and in cell analysis of chemically synthesized histone H2A with multiple modifications

Hayashi, Gosuke; Sueoka, Takuma; Okamoto, Akimitsu

doi:10.1039/c5cc10555b

Cited by 22 publications

(14 citation statements)

References 34 publications

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“…The cysteine residue of the GGGYC peptide was modified with Alexa Fluor 488-maleimide in 25 mM 3-morpholinopropane-1-sulfonic acid (MOPS) buffer (pH 6.8) for 6 h at room temperature. The product, GGGYC–Alexa488 was purified by reversed phase high performance liquid chromatography (RP-HPLC, Jasco, Tokyo, Japan) and identified by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry, as previously described 36 .…”

Section: Methodsmentioning

confidence: 99%

Microfluidic preparation of anchored cell membrane sheets for in vitro analyses and manipulation of the cytoplasmic face

Izuta

Yamaguchi

Misawa

et al. 2017

Sci Rep

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Molecular networks on the cytoplasmic faces of cellular plasma membranes are critical research topics in biological sciences and medicinal chemistry. However, the selective permeability of the cell membrane restricts the researchers from accessing to the intact intracellular factors on the membrane from the outside. Here, a microfluidic method to prepare cell membrane sheets was developed as a promising tool for direct examination of the cytoplasmic faces of cell membranes. Mammalian cells immobilized on a poly(ethylene glycol)-lipid coated substrate were rapidly and efficiently fractured, with the sheer stress of laminar flow in microchannels, resulting in isolation of the bottom cell membrane sheets with exposed intact cytoplasmic faces. On these faces of the cell membrane sheets, both ligand-induced phosphorylation of receptor tyrosine kinases and selective enzymatic modification of a G-protein coupling receptor were directly observed. Thus, the present cell membrane sheet should serve as a unique platform for studies providing new insights into juxta-membrane molecular networks and drug discovery.

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Section: Methodsmentioning

confidence: 99%

Microfluidic preparation of anchored cell membrane sheets for in vitro analyses and manipulation of the cytoplasmic face

Izuta

Yamaguchi

Misawa

et al. 2017

Sci Rep

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“…The ability to synthesize an entire histone with SPPS and NCL has made it possible to investigate histone PTMs and their readers, writers, and erasers in a contextually accurate manner, both independently and in combinations. For example, the first report of a total synthesis of histone H2A demonstrated that known PTMs S1ph, K75me2, and K119Ac K119Ac PTMs do not dramatically effect nucleosome stability, individually [372] . A recent total NCL synthesis of H2AY57ph and H2BK120ub with subsequent nucleosome formation revealed that the Y57ph mark inhibits de‐ubiquitylation of K120ub by the SAGA) complex [373] .…”

Section: Synthetic Histonesmentioning

confidence: 99%

“…For example, the first report of a total synthesis of histone H2A demonstrated that known PTMs S1ph, K75me2, and K119Ac K119Ac PTMs do not dramatically effect nucleosome stability, individually. [372] A recent total NCL synthesis of H2AY57ph and H2BK120ub with subsequent nucleosome formation revealed that the Y57ph mark inhibits de-ubiquitylation of K120ub by the SAGA) complex. [373] Similarly, the Okamoto group also synthesized a H2A with a Y57ph modification and reconstructed nucleosomes.…”

Section: Chemically Synthesized Histonesmentioning

confidence: 99%

Advances and Opportunities in Epigenetic Chemical Biology

2020

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The study of epigenetics has greatly benefited from the development and application of various chemical biology approaches. In this review, we highlight the key targets for modulation and recent methods developed to enact such modulation. We discuss various chemical biology techniques to study DNA methylation and the post-translational modification of histones as well as their effect on gene expression. Additionally , we address the wealth of protein synthesis approaches to yield histones and nucleosomes bearing epigenetic modifications. Throughout, we highlight targets that present opportunities for the chemical biology community, as well as exciting new approaches that will provide additional insight into the roles of epigenetic marks.

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“…In 2016, Okamoto and coworkers conducted the first chemical synthesis of a variety of post-translationally modified histones H2A through Fmoc based SPPS and sequential NCL utilizing N- acylurea chemistry (Hayashi et al, 2016 ). H2A was divided into three peptide segment and the N-terminal cysteine of segment H2A (47–85) was temporarily protected with thiazolidine (Thz) to avoid self-ligation.…”

Section: The Total Chemical Synthesis Of H2a With Ptmsmentioning

confidence: 99%

Total Chemical Synthesis of Modified Histones

et al. 2018

Front. Chem.

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In the post-genome era, epigenetics has received increasing attentions in recent years. The post-translational modifications (PTMs) of four core histones play central roles in epigenetic regulation of eukaryotic genome by either directly altering the biophysical properties of nucleosomes or by recruiting other effector proteins. In order to study the biological functions and structural mechanisms of these histone PTMs, an obligatory step is to prepare a sufficient amount of homogeneously modified histones. This task cannot be fully accomplished either by recombinant technology or enzymatic modification. In this context, synthetic chemists have developed novel protein synthetic tools and state-of-the-art chemical ligation strategies for the preparation of homologous modified histones. In this review, we summarize the recent advances in the preparation of modified histones, focusing on the total chemical synthesis strategies. The importance and potential of synthetic chemistry for the study of histone code will be also discussed.

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In vitro and in cell analysis of chemically synthesized histone H2A with multiple modifications

Cited by 22 publications

References 34 publications

Microfluidic preparation of anchored cell membrane sheets for in vitro analyses and manipulation of the cytoplasmic face

Microfluidic preparation of anchored cell membrane sheets for in vitro analyses and manipulation of the cytoplasmic face

Advances and Opportunities in Epigenetic Chemical Biology

Total Chemical Synthesis of Modified Histones

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