Chemical Synthesis of Ubiquitin, Ubiquitin‐Based Probes, and Diubiquitin

Oualid, Farid El; Merkx, Remco; Ekkebus, Reggy; Hameed, Dharjath S.; Smit, Judith J.; Jong, Annemieke de; Hilkmann, Henk; Sixma, Titia K.; Ovaa, Huib

doi:10.1002/anie.201005995

Cited by 301 publications

(262 citation statements)

References 45 publications

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“…For example, monoubiquitinated α-Synuclein (α-Syn) and histone H2B bearing native isopeptide bonds were prepared and used to shed light on the role of monoubiquitination in regulating α-Syn aggregation and degradation by the proteasome (6, 7) and histone H2B functions within the nucleosomal context (8). Successful synthesis of all Lys-linked di-Ub chains with native isopeptide bonds were also achieved and proved to be instrumental in studies aimed at understanding the structures and dynamics of these chains, as well as their relative sensitivities towards deubiquitinases (9,10). These advances provided access to new structural information about K33-and K6-linked di-Ub chains using NMR and X-ray crystallography, respectively (11,12).…”

mentioning

confidence: 99%

Synthetic polyubiquitinated α-Synuclein reveals important insights into the roles of the ubiquitin chain in regulating its pathophysiology

Haj‐Yahya

Fauvet

Herman-Bachinsky

et al. 2013

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

131

115

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Ubiquitination regulates, via different modes of modifications, a variety of biological processes, and aberrations in the process have been implicated in the pathogenesis of several neurodegenerative diseases. However, our ability to dissect the pathophysiological relevance of the ubiquitination code has been hampered due to the lack of methods that allow site-specific introduction of ubiquitin (Ub) chains to a specific substrate. Here, we describe chemical and semisynthetic strategies for site-specific incorporation of K48-linked di-or tetra-Ub chains onto the side chain of Lys12 of α-Synuclein (α-Syn). These advances provided unique opportunities to elucidate the role of ubiquitination and Ub chain length in regulating α-Syn stability, aggregation, phosphorylation, and clearance. In addition, we investigated the cross-talk between phosphorylation and ubiquitination, the two most common α-Syn pathological modifications identified within Lewy bodies and Parkinson disease. Our results suggest that α-Syn functions under complex regulatory mechanisms involving cross-talk among different posttranslational modifications. eukaryotes is the covalent attachment of ubiquitin (Ub) to proteins. This reversible modification, which regulates a variety of biological processes, such as protein degradation, trafficking, and DNA damage response (1, 2), involves the attachment of the C terminus of Ub mainly to the side chain of a Lys residue in a protein substrate via an isopeptide linkage. The process is catalyzed by three enzymes that act in concert: the Ub-activating enzyme (E1), the Ub-conjugating enzyme (E2), and the Ub ligase (E3). The reaction is repeated, and a second Ub is attached to an internal Lys in the previously conjugated ubiquitin. Several repeats result in the synthesis of a poly-Ub chain that can be of varying lengths and internal linkages. The presence of seven Lys residues as possible anchoring sites within Ub in addition to the N-terminal amine results in a highly complex landscape of diverse Ub bioconjugates, which accounts for the diversity of the Ub signaling (3).Research in the Ub field, which aims at understanding the ubiquitination system at the molecular level, has been hampered by the difficulties of controlling ubiquitination in the cell and challenges associated with the preparation of specific Ub conjugates in vitro. These limitations have inspired the development of novel synthetic strategies to facilitate site-specific ubiquitination of proteins (4, 5). Recent advancements in the field have enabled the synthesis of relatively large amounts of highly complex Ub conjugates of defined covalent structure and provided novel insights into the structural, biochemical, and functional consequences of protein ubiquitination, along with unique opportunities to elucidate the molecular basis of Ub signaling. For example, monoubiquitinated α-Synuclein (α-Syn) and histone H2B bearing native isopeptide bonds were prepared and used to shed light on the role of monoubiquitination in regulating α-Syn aggregation a...

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mentioning

confidence: 99%

Synthetic polyubiquitinated α-Synuclein reveals important insights into the roles of the ubiquitin chain in regulating its pathophysiology

Haj‐Yahya

Fauvet

Herman-Bachinsky

et al. 2013

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

131

115

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“…In particular, δ-thiol Lys building block 89 has been extensively employed by Brik and coworkers to study the ubiquitylation of α-synuclein [104,106], a 140-amino acid presynaptic protein implicated in a number of neurodegenerative diseases, through protein semi-synthesis. Ovaa and coworkers have also reported the incorporation of Lys building block 90 in place of native Lys residues in the 76-amino acid ubiquitin protein sequence to facilitate the synthesis of a library of diubiquitin conjugates using ligation-desulfurization chemistry [105]. The same group recently reported a concise synthetic route to an additional γ-thiol Lys derivative and determined that both γ-and δ-thiol Lys are equally efficient in facilitating the synthesis of diubiquitin conjugates [107].…”

Section: Lysinementioning

confidence: 95%

“…Lys building block 88 was shown to effectively mediate a number of ligations at both the α-and ε-amino groups, and was used in the preparation of side-chain ubiquitylated and biotinylated peptide products [102]. Two δ-thiol Lys derivatives, 89 [104] and 90 [105], have been independently reported for use in side-chain ligation approaches to ubiquitylation. In particular, δ-thiol Lys building block 89 has been extensively employed by Brik and coworkers to study the ubiquitylation of α-synuclein [104,106], a 140-amino acid presynaptic protein implicated in a number of neurodegenerative diseases, through protein semi-synthesis.…”

Section: Lysinementioning

confidence: 99%

Modern Extensions of Native Chemical Ligation for Chemical Protein Synthesis

Malins

Payne

2014

Protein Ligation and Total Synthesis I

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Over the past 20 years, native chemical ligation has facilitated the synthesis of numerous complex peptide and protein targets, with and without post-translational modifications, as well as the design and construction of a variety of engineered protein variants. This powerful methodology has also served as a platform for the development of related chemoselective ligation technologies which have greatly expanded the scope and flexibility of ligation chemistry. This chapter details a number of important extensions of the original native chemical ligation manifold, with particular focus on the application of new methods in the total chemical synthesis of proteins. Topics covered include the development of auxiliary-based ligation methods, the post-ligation manipulation of Cys residues, and the synthesis and utility of unnatural amino acid building blocks (bearing reactive thiol or selenol functionalities) in chemoselective ligation chemistry. Contemporary applications of these techniques to the total chemical synthesis of peptides and proteins are described.

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“…Ovaa et al prepared both δ-and γ-lysine starting from δ-hydroxylysine and L-lysine [74,75]. These two kinds of modified lysine were applied in the synthesis of K48-Ub and K43-Ub and the ligation efficiencies were compared.…”

Section: Ligation At Lysine Sitementioning

confidence: 99%

Postligation-Desulfurization: A General Approach for Chemical Protein Synthesis

Zeng

Wan

2014

Topics in Current Chemistry

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Native chemical ligation, involving regioselective and chemoselective coupling of two unprotected peptide segments, enabled the synthesis of polypeptide with more than 200 amino acids. However, cysteine was indispensable in this synthetic technique in its initial format, which limited its further application. Thus, considerable effort has been put into breaking the restriction of cysteine-containing ligation. As a consequence, postligation-desulfurization, concerning thiol-mediated ligation followed by desulfurization, was developed. This review describes the development and recent progress on the chemical synthesis of peptides and proteins encompassing postligation-desulfurization at alanine, valine, lysine, threonine, leucine, proline, arginine, aspartic acid, glutamate, phenylalanine, glutamine, and tryptophan.

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Chemical Synthesis of Ubiquitin, Ubiquitin‐Based Probes, and Diubiquitin

Cited by 301 publications

References 45 publications

Synthetic polyubiquitinated α-Synuclein reveals important insights into the roles of the ubiquitin chain in regulating its pathophysiology

Synthetic polyubiquitinated α-Synuclein reveals important insights into the roles of the ubiquitin chain in regulating its pathophysiology

Modern Extensions of Native Chemical Ligation for Chemical Protein Synthesis

Postligation-Desulfurization: A General Approach for Chemical Protein Synthesis

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