Forging Isopeptide Bonds Using Thiol–Ene Chemistry: Site-Specific Coupling of Ubiquitin Molecules for Studying the Activity of Isopeptidases

Valkevich, Ellen M.; Guenette, Robert G.; Sanchez, Nicholas A.; Chen, Yi‐Chen; Ge, Ying; Strieter, Eric R.

doi:10.1021/ja300500a

Cited by 119 publications

(118 citation statements)

References 46 publications

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“…Additionally, the disulfide attachment chemistry used has the attractive feature that the modification can be "erased" through simple reduction, thereby offering the potential to study, in a time-resolved way, the functional and structural consequences of PTM removal. Moreover, it may be possible to adapt the strategy to give nonreducible thioether linkages through use of alternate thiol-directed chemistries (36,37). Indeed, analogs of other PTMs can, in principle, be introduced chemically into preassembled chromatin using known cysteine derivatization routes-for example, lysine methylation (2) and acetylation (38).…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…44,45 The linker connecting the dextran and thiol in D1 is located at the reducing end of the dextran and contains amide and ether moieties. All concentrations of conjugate 2 were measured as total dextran concentration (labeled and unlabeled), and all three batches showed identical same staining patterns and qualitative results.…”

Section: Methodsmentioning

confidence: 99%

“…Glutathione (1.5 equiv), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) initiator 44,45 (2.5 equiv), and probe 1a (5 equiv) were added. The reaction mixture was irradiated with a 365-nm light source for 15 min.…”

Section: Methodsmentioning

confidence: 99%

Cytosolic Uptake of Large Monofunctionalized Dextrans

2018

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Dextrans are a versatile class of polysaccharides with applications that span medicine, cell biology, food science, and consumer goods. Here, we report on a new type of large monofunctionalized dextran that exhibits unusual properties: efficient cytosolic and nuclear uptake. This dextran permeates various human cell types without the use of transfection agents, electroporation, or membrane perturbation. Cellular uptake occurs primarily through active transport via receptor-mediated processes. These monofunctionalized dextrans could serve as intracellular delivery platforms for drugs or other cargos.

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“…Ubiquitin conjugates with a thioether linkage have also been reported [37,38]. The thioether linkage was formed through free-radical thiol-ene click reaction between an allyl group introduced at the C-terminus of donor ubiquitin and the thiol group of a cysteine residue of in the substrate protein (Scheme 15).…”

Section: Preparation Of Ub-proteins With Non-native Linkagesmentioning

confidence: 99%

Chemical Methods for Protein Ubiquitination

Yang

Liu

2014

Protein Ligation and Total Synthesis I

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In eukaryotic cells, many proteins undergo extensive post-translational modifications (PTMs) such as methylation, acetylation, phosphorylation, glycosylation, and ubiquitination. Among these, ubiquitination is a particularly interesting PTM from both structural and functional viewpoints. In ubiquitination, the C-terminal carboxyl group of the small ubiquitin protein is attached to the ε-amine of a lysine residue of a substrate protein through an isopeptide bond. Ubiquitination has been shown to be involved in the regulation of many cellular processes including protein degradation and gene expression. And dysfunction of these processes is implicated in many human diseases. Despite many years of intensive research, a large number of protein ubquitination events remain poorly characterized. The challenge lies with the tremendous difficulties in isolating homogeneously modified proteins from biological samples for structural and functional studies. Enzymatic ubiquitination in vitro often has limited practical value due to the large number of substrate-specific E3 ligases and the difficulties in identifying or isolating these enzymes. Chemical approaches to the preparation of ubiquitinated proteins provide a powerful solution, and the development of such approaches has been the subject of intense research by many research laboratories. This review summarizes the methodological developments of protein chemical ubiquitination in recent years.

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Forging Isopeptide Bonds Using Thiol–Ene Chemistry: Site-Specific Coupling of Ubiquitin Molecules for Studying the Activity of Isopeptidases

Cited by 119 publications

References 46 publications

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

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

Cytosolic Uptake of Large Monofunctionalized Dextrans

Chemical Methods for Protein Ubiquitination

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