Segmental Isotopic Labeling of Ubiquitin Chains To Unravel Monomer‐Specific Molecular Behavior

Castañeda, Carlos; Spasser, Liat; Bavikar, Sudhir N.; Brik, Ashraf; Fushman, David

doi:10.1002/ange.201104649

Cited by 12 publications

(8 citation statements)

References 42 publications

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“…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). More recently, tetra-Ub chains in a free form or anchored to a tripeptide were also synthesized chemically (13)(14)(15).…”

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.

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139

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.

Self Cite

139

115

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“…The preparation of the aforementioned Ub conjugates is usually performed through direct aminolysis of the Ub‐thioester generated through the intein technology . This aminolysis reaction is a relatively slow transformation and therefore, may suffer from heavy hydrolysis, difficult product separation, and low yields.…”

Section: Introductionmentioning

confidence: 88%

New semi‐synthesis of ubiquitin C‐terminal conjugate with 7‐amino‐4‐methylcoumarin

Liang

et al. 2013

Journal of Peptide Science

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The ligation of peptide hydrazides at a Gly site carrying a removal auxiliary was found to be an efficient process. This technology was successfully used for the synthesis of ubiquitin C-terminal conjugates. Recombinant Ub(1-75)-NHNH2 was prepared through the hydrozinolysis of the Ub(1-75)-intein fusion protein. It was ligated with a glycine derivative modified with an acid-sensitive thiol auxiliary. The final acid treatment produced the desired bioactive ubiquitin conjugates in practical quantities. Thus, the method described here extends the protocols of expressed protein ligation.

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“…2017), a condensation reaction of a C-terminal ubiquitin thioester and the ε -amine of a targeted lysine residue (Castaneda et al . 2011a), or a combined recombinant expression–chemical synthesis approach (Castaneda et al . 2011b).…”

Section: Protein Engineering Approaches For Tackling Outstanding Cmentioning

confidence: 99%

A molecular engineering toolbox for the structural biologist

Debelouchina

Muir

2017

Quart. Rev. Biophys.

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Exciting new technological developments have pushed the boundaries of structural biology, and have enabled studies of biological macromolecules and assemblies that would have been unthinkable not long ago. Yet, the enhanced capabilities of structural biologists to pry into the complex molecular world have also placed new demands on the abilities of protein engineers to reproduce this complexity into the test tube. With this challenge in mind, we review the contents of the modern molecular engineering toolbox that allow the manipulation of proteins in a site-specific and chemically well-defined fashion. Thus, we cover concepts related to the modification of cysteines and other natural amino acids, native chemical ligation, intein and sortase-based approaches, amber suppression, as well as chemical and enzymatic bio-conjugation strategies. We also describe how these tools can be used to aid methodology development in X-ray crystallography, nuclear magnetic resonance, cryo-electron microscopy and in the studies of dynamic interactions. It is our hope that this monograph will inspire structural biologists and protein engineers alike to apply these tools to novel systems, and to enhance and broaden their scope to meet the outstanding challenges in understanding the molecular basis of cellular processes and disease.

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Segmental Isotopic Labeling of Ubiquitin Chains To Unravel Monomer‐Specific Molecular Behavior

Cited by 12 publications

References 42 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

New semi‐synthesis of ubiquitin C‐terminal conjugate with 7‐amino‐4‐methylcoumarin

A molecular engineering toolbox for the structural biologist

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