Chemical Synthesis of Phosphorylated Histone H2A at Tyr57 Reveals Insight into the Inhibition Mode of the SAGA Deubiquitinating Module

Jbara, Muhammad; Maity, Suman Kumar; Morgan, Michael T.; Wolberger, Cynthia; Brik, Ashraf

doi:10.1002/anie.201600638

Cited by 67 publications

(47 citation statements)

References 55 publications

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“…The mechanism indicated that Y57ph could inhibit the access of Spt‐Ada‐Gcn5 acetyltransferase (SAGA) complex to nucleosome and lead to the decrease in deubiquitination ability of the complex, and thus the amount of H2B ubiquitination increased, which plays multiple roles in transcription activation (Figure a). Brik and Wolberger's work supported the hypothesis employing the chemically synthesized Y57ph‐H2A and K120ub‐H2B . The result of in vitro deubiquitination assay indicated that Y57ph inhibited access of the SAGA complex to the nucleosome surface.…”

Section: Chemical Protein Synthesis That Advances Research On Histonementioning

confidence: 62%

Chemistry‐Driven Epigenetic Investigation of Histone and DNA Modifications

Sueoka

Koyama

Hayashi

et al. 2018

The Chemical Record

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In the regulation processes of gene expression, genomic DNA and nuclear proteins, including histone proteins, cooperate with each other, leading to the distinctive functions of eukaryotic cells such as pluripotency and differentiation. Chemical modification of histone proteins and DNA has been revealed as one of the major driving forces in the complicated epigenetic regulation system. However, understanding of the precise molecular mechanisms is still limited. To address this issue, researchers have proposed both biological and chemical strategies for the preparation and detection of modified proteins and nucleic acids. In this review, we focus on chemical methods around the field of epigenetics. Chemical protein synthesis has enabled the preparation of site-specifically modified histones and their successful application to various in vitro assays, which have emphasized the significance of posttranslational modifications of interest. We also review the modification-specific chemical reactions against synthetic and genomic DNA, which enabled discrimination of several modified bases at single-base resolution.

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Section: Chemical Protein Synthesis That Advances Research On Histonementioning

confidence: 62%

Chemistry‐Driven Epigenetic Investigation of Histone and DNA Modifications

Sueoka

Koyama

Hayashi

et al. 2018

The Chemical Record

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“…Peptide 3a contains the Dbz moiety at the C‐terminal Thr‐370. As the Dbz peptide precursor 3a could not be converted into the Nbz form by using 4‐nitrophenylchloroformate in DCM, we repeated the reaction in DMF: indeed, Brik and coworkers reported that the use of DMF instead of DCM could convert Dbz into Nbz on a peptide bearing a C‐terminal leucine . Unfortunately, in our case, the use of DMF did not solve the problem, and no Nbz peptide was formed (probably, because of the presence of the β‐branched threonine).…”

Section: Discussionmentioning

confidence: 81%

An explorative study towards the chemical synthesis of the immunoglobulin G1 Fc CH3 domain

Grassi

Roschger

Stanojlović

et al. 2018

Journal of Peptide Science

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Monoclonal antibodies, fusion proteins including the immunoglobulin fragment c (Ig Fc) CH2‐CH3 domains, and engineered antibodies are prominent representatives of an important class of drugs and drug candidates, which are referred to as biotherapeutics or biopharmaceuticals. These recombinant proteins are highly heterogeneous due to their glycosylation pattern. In addition, enzyme‐independent reactions, like deamidation, dehydration, and oxidation of sensitive side chains, may contribute to their heterogeneity in a minor amount. To investigate the biological impact of a spontaneous chemical modification, especially if found to be recurrent in a biotherapeutic, it would be necessary to reproduce it in a homogeneous manner. Herein, we undertook an explorative study towards the chemical synthesis of the IgG1 Fc CH3 domain, which has been shown to undergo spontaneous changes like succinimide formation and methionine oxidation. We used Fmoc‐solid‐phase peptide synthesis (SPPS) and native chemical ligation (NCL) to test the accessibility of large fragments of the IgG1 Fc CH3 domain. In general, the incorporation of pseudoproline dipeptides improved the quality of the crude peptide precursors; however, sequences larger than 44 residues could not be achieved by standard stepwise elongation with Fmoc‐SPPS. In contrast, the application of NCL with cysteine residues, which were either native or introduced ad hoc, allowed the assembly of the C‐terminal IgG1 Fc CH3 sequence 371 to 450. The syntheses reported here show advantages and limitations of the chemical approaches chosen for the preparation of the synthetic IgG1 Fc CH3 domain and will guide future plans towards the synthesis of both the native and selectively modified full‐length domain.

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“…Modeling of H2A-Y57p in the structure of the DUB module bound to a ubiquitinated nucleosome [34] indicated that this newly discovered histone PTM has the potential to alter interactions between the arginine anchor and the acidic patch (Figure 1C). This hypothesis was confirmed through use of a fully synthetic strategy to incorporate phosphotyrosine into histone H2A at Y57 [39]. Nucleosomes containing H2A-Y57p and H2B-Ub made by native chemical ligation [40] were used to confirm that this phosphorylation indeed inhibits DUB module activity in vitro [39].…”

Section: Docking Of the Saga Dub Module On Ubiquitinated Nucleosomesmentioning

confidence: 97%

“…This hypothesis was confirmed through use of a fully synthetic strategy to incorporate phosphotyrosine into histone H2A at Y57 [39]. Nucleosomes containing H2A-Y57p and H2B-Ub made by native chemical ligation [40] were used to confirm that this phosphorylation indeed inhibits DUB module activity in vitro [39]. …”

Section: Docking Of the Saga Dub Module On Ubiquitinated Nucleosomesmentioning

confidence: 97%

Recognition of ubiquitinated nucleosomes

Morgan

Wolberger

2017

Current Opinion in Structural Biology

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Histone ubiquitination plays a non-degradative role in regulating transcription and the DNA damage response. A mechanistic understanding of this chromatin modification has lagged that of small histone modifications because of the technical challenges in preparing ubiquitinated nucleosomes. The recent structure of the DUB module of the SAGA coactivator complex bound to a nucleosome containing monoubiquitinated H2B has provided the first view of how specialized subunits target this enzyme to its substrate. Single particle electron microscopy of the intact SAGA coactivator suggests how the DUB module and histone acetyltransferase module engage a nucleosomal substrate. A cryo EM study of 53BP1 bound to nucleosomes containing ubiquitinated H2A and H4 methylated at K20 extends our understanding of recognition of biologically distinct combinations of chromatin marks through multivalent interactions.

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Chemical Synthesis of Phosphorylated Histone H2A at Tyr57 Reveals Insight into the Inhibition Mode of the SAGA Deubiquitinating Module

Cited by 67 publications

References 55 publications

Chemistry‐Driven Epigenetic Investigation of Histone and DNA Modifications

Chemistry‐Driven Epigenetic Investigation of Histone and DNA Modifications

An explorative study towards the chemical synthesis of the immunoglobulin G1 Fc CH3 domain

Recognition of ubiquitinated nucleosomes

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