Convergent Chemical Synthesis of Histone H2B Protein for the Site‐Specific Ubiquitination at Lys34

Siman, Peter; Karthikeyan, Subramanian Vedhanarayanan; Nikolov, Miroslav; Fischle, Wolfgang; Brik, Ashraf

doi:10.1002/ange.201303844

Cited by 42 publications

(22 citation statements)

References 38 publications

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“…When the reaction pH was kept near 6.5, this lactam formation side reaction was suppressed. Lactam formation at a peptide-Lysthioester has also been reported by Brik et al [16] Suppression of lactam formation at pH 6.5 in our hands may be due to the high (7 mm) concentrations of reacting peptide segments used, which would favor the intermolecular ligation reaction over the intramolecular lactam side reaction. The reaction was worked up by adding excess sodium 2-mercaptoethanesulfonate (MESNA), and the desired ligation product Nle 59 -134-thioalkylester 5 was isloated in 53 % yield.…”

supporting

confidence: 62%

Total Chemical Synthesis of the Enzyme Sortase A_ΔN59 with Full Catalytic Activity

et al. 2014

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supporting

confidence: 62%

Total Chemical Synthesis of the Enzyme Sortase A_ΔN59 with Full Catalytic Activity

et al. 2014

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“…To determine which residues in ubiquitin are critical for H3K79 methylation cross-talk, we set out to develop a streamlined methodology for the rapid preparation of ubiquitylated nucleosomes. Current synthetic routes for histone ubiquitylation involve multistep protocols and are performed at the histone level, making the preparation of multiple ubiquitin nucleosomal substrates a formidable challenge (10,23,24,27,28).…”

Section: Resultsmentioning

confidence: 99%

Identification of a functional hotspot on ubiquitin required for stimulation of methyltransferase activity on chromatin

Holt

David

Pollock

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Ubiquitylation of histone H2B at lysine 120 (H2B-Ub) plays a critical role in transcriptional elongation, chromatin conformation, as well as the regulation of specific histone H3 methylations. Herein, we report a strategy for the site-specific chemical attachment of ubiquitin to preassembled nucleosomes. This allowed expedited structure-activity studies into how H2B-Ub regulates H3K79 methylation by the methyltransferase human Dot1. Through an alanine scan of the ubiquitin surface, we identified a functional hotspot on ubiquitin that is required for the stimulation of human Dot1 in vitro. Importantly, this result was validated in chromatin from isolated nuclei by using a synthetic biology strategy that allowed selective incorporation of the hotspot-deficient ubiquitin mutant into H2B. The ubiquitin hotspot additionally impacted the regulation of ySet1-mediated H3K4 methylation but was not required for H2B-Ub-induced impairment of chromatin fiber compaction. These data demonstrate the utility of applying chemical ligation technologies to preassembled chromatin and delineate the multifunctionality of ubiquitin as a histone posttranslational modification.H istone posttranslational modifications (PTMs) modulate chromatin structure and function either by directly altering the intrinsic physical properties of the chromatin fiber or by nucleating the recruitment and activity of a host of transacting nuclear factors (1-3). The chemical diversity, differential dynamics, and sheer number (currently over 100) (4, 5) of these PTMs, along with their combinatorial occurrence at the level of the nucleosome, create a complex and nonstatic molecular architecture in which all chromatin-related processes function. A central challenge in the field of epigenetics is to disentangle how distinct chromatin states control biochemical outputs, which requires the elucidation of the critical determinants governing histone PTM readout.A particularly fascinating histone PTM is the ubiquitylation of H2B at lysine 120 (H2B-Ub). H2B-Ub is enriched near the 5′ end of highly expressed genes and has been implicated in transcriptional elongation, as well as chromatin structure definition (6-9). Moreover, H2B-Ub directly regulates the H3K4 and H3K79 methyltransferases Set1 and Dot1, respectively (10-13). The mechanistic principles underlying these various phenomena remain poorly understood. At 8.5 kDa, ubiquitin is nearly as large as the histone to which it is linked (13.8 kDa in the case of H2B), increasing the nucleosome surface by as much as 4,800 A 2 (14). Thus, compared with smaller PTMs such as acetylation and methylation, ubiquitin is "information rich" in that it alters the steric and electrostatic properties around its attachment site, as well as presenting a large surface area for the recruitment of binding factors. Structural and biochemical studies of ubiquitin-ligand complexes, including the ubiquitylation of histone H2A at lysine 15, have revealed a canonical binding hotspot on ubiquitin involving a hydrophobic patch centered on Leu8/I...

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“…Importantly, one can extend this strategy to many ligation junctions by employing thiol-containing analogs of other amino acids. Indeed, thiolated derivatives of several residues (leucine, phenylalanine, valine, threonine, aspartic acid, lysine, arginine) (82–88) have been employed in the traceless (semi)synthesis of many proteins, including several modified histones (89, 90). …”

Section: Contemporary Synthesis Of Posttranslationally Modified Histomentioning

confidence: 99%

“…Several latent thioester linkers are now available for Fmoc-SPPS of peptide α-thioesters (94–97). These include linkers based on 2-hydroxy-3-mercaptopropionic acid (94), diaminobenzoyl (95), and hydrazine (96) moieties, all of which have been applied to the (semi)synthesis of modified histones (Table 1) (90, 98, 99). Of these, the hydrazine linker, which yields a peptide hydrazide upon cleavage that is eventually converted into an active thioester via an oxidation/thiolysis process, is rapidly becoming the method of choice for the preparation of peptide α-thioesters by the Fmoc approach due to its ease of use and excellent efficiency (96).…”

Section: Contemporary Synthesis Of Posttranslationally Modified Histomentioning

confidence: 99%

Application of the Protein Semisynthesis Strategy to the Generation of Modified Chromatin

Holt

Muir

2015

Annu. Rev. Biochem.

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Histone proteins are subject to a host of posttranslational modifications (PTMs) that modulate chromatin structure and function. Such control is achieved by the direct alteration of the intrinsic physical properties of the chromatin fiber or by regulating the recruitment and activity of a host of trans-acting nuclear factors. The sheer number of histone PTMs presents a formidable barrier to understanding the molecular mechanisms at the heart of epigenetic regulation of eukaryotic genomes. One aspect of this multifarious problem, namely how to access homogeneously modified chromatin for biochemical studies, is well suited to the sensibilities of the organic chemist. Indeed, recent years have witnessed a critical role for synthetic protein chemistry methods in generating the raw materials needed for studying how histone PTMs regulate chromatin biochemistry. This review focuses on what is arguably the most powerful, and widely employed, of these chemical strategies, namely histone semisynthesis via the chemical ligation of peptide fragments.

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Convergent Chemical Synthesis of Histone H2B Protein for the Site‐Specific Ubiquitination at Lys34

Abstract: Wunschgemäß: Eine Methode zur konvergenten chemischen Synthese des Histonproteins H2B wurde entwickelt, die die Kupplung von an Lys34 ermöglicht (siehe Bild). Die Synthese von H2B mit jeder gewünschten posttranslationalen Modifikation sollte somit in Zukunft möglich sein.

Cited by 42 publications

References 38 publications

Total Chemical Synthesis of the Enzyme Sortase A_ΔN59 with Full Catalytic Activity

Total Chemical Synthesis of the Enzyme Sortase A_ΔN59 with Full Catalytic Activity

Identification of a functional hotspot on ubiquitin required for stimulation of methyltransferase activity on chromatin

Application of the Protein Semisynthesis Strategy to the Generation of Modified Chromatin

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Convergent Chemical Synthesis of Histone H2B Protein for the Site‐Specific Ubiquitination at Lys34

Abstract: Wunschgemäß: Eine Methode zur konvergenten chemischen Synthese des Histonproteins H2B wurde entwickelt, die die Kupplung von an Lys34 ermöglicht (siehe Bild). Die Synthese von H2B mit jeder gewünschten posttranslationalen Modifikation sollte somit in Zukunft möglich sein.

Cited by 42 publications

References 38 publications

Total Chemical Synthesis of the Enzyme Sortase AΔN59 with Full Catalytic Activity

Total Chemical Synthesis of the Enzyme Sortase AΔN59 with Full Catalytic Activity

Identification of a functional hotspot on ubiquitin required for stimulation of methyltransferase activity on chromatin

Application of the Protein Semisynthesis Strategy to the Generation of Modified Chromatin

Contact Info

Product

Resources

About

Total Chemical Synthesis of the Enzyme Sortase A_ΔN59 with Full Catalytic Activity

Total Chemical Synthesis of the Enzyme Sortase A_ΔN59 with Full Catalytic Activity