Activity‐Based Probes Developed by Applying a Sequential Dehydroalanine Formation Strategy to Expressed Proteins Reveal a Potential α‐Globin‐Modulating Deubiquitinase

Meledin, Roman; Mali, Sachitanand M.; Kleifeld, Oded; Brik, Ashraf

doi:10.1002/ange.201800032

Cited by 21 publications

(5 citation statements)

References 42 publications

(43 reference statements)

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“…In the past decade, our group has been heavily involved in developing chemical methods to prepare various Ub conjugates, which have led to various interesting discoveries in this area. These conjugates include free Ub chains, 29,30 activity-based probes, 21,31,32 assays for deubiquitinases (DUBs), 33 and ubiquitinated proteins. 34,35 More recently, we have prepared labeled tetraubiquitinated αglobin consisting of 472 amino acids, which to date is the largest protein ever made from natural amino acids by chemical synthesis.…”

Section: Chemical Protein Synthesis Applied To Ubiquitinationmentioning

confidence: 99%

The Journey for the Total Chemical Synthesis of a 53 kDa Protein

Sun

Brik

2019

Acc. Chem. Res.

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CONSPECTUS: Chemical protein synthesis has been proved as an efficient way to afford medium-sized proteins with high homogeneity in workable quantities for various biochemical, structural, and functional studies. In particular, chemical protein synthesis has enabled access to proteins that are difficult or impossible to prepare by molecular biology approaches, such as those with post-translational modifications and mirror-image proteins. One prominent example is related to ubiquitination, a well-known modification that mediates a variety of cellular processes (e.g., proteasomal degradation). Ubiquitination is considered as a modification that is difficult to introduce into proteins in a test tube to generate ubiquitin (Ub) conjugates with high homogeneity with respect to the chain length and the anchored Lys residue in workable quantities to perform the biochemical and biophysical studies. Chemical protein synthesis has emerged as a powerful approach to prepare Ub conjugates for studies aiming to understand ubiquitination in great detail and decipher its roles in cell processes. Nevertheless, in order to answer more challenging questions in this field, it has been clear that researchers must also prepare Ub conjugates with increased size and complexity. Employing solid-phase peptide synthesis and chemoselective ligation, chemical protein synthesis offers a powerful way to furnish polypeptides composed of 100−200 residues. However, to synthesize larger proteins such as Ub conjugates, longer and more segments are required. This on the other hand leads to difficulties related to solubility, purification, ligation, and late-stage modifications. These challenges have encouraged us to explore more practical synthetic tools to facilitate the synthesis of complex Ub conjugates. In this Account, we summarize the synthetic tools that we have developed to achieve these goals. These include (1) δ-mercaptolysine-mediated isopeptide chemical ligation, (2) chemical synthesis of Ub building blocks, (3) palladium-mediated deprotection of key side chains during protein synthesis, (4) one-pot ligation and desulfurization, and (5) improving the solubility of peptide segments. The developed chemical toolbox has been a key for our successes in the synthesis of diverse and complex Ub conjugates. In this Account, we describe our approaches for generating various Ub conjugates, including (1) the K48 tetra-Ub chain composed of 304 amino acids, (2) the ubiquitinated histones and their analogues made of >200 amino acids, (3) the di-Ub-SUMO-2 hybrid chain composed of 245 amino acids, and (4) the 53 kDa tetra-Ub-α-globin composed of 472 amino acids, which represents the largest protein composed of natural amino acids ever made using chemical protein synthesis. The last target, Flag-Ub-Ub-Ub-Myc-Ub-(HA-α-globin), was prepared in the labeled form where the proximal Ub and distal Ub in the chain were labeled with Myc and Flag tags, respectively, while the α-globin was labeled with the HA tag. Applying the tetra-Ub-α-globin in proteasomal degrada...

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Section: Chemical Protein Synthesis Applied To Ubiquitinationmentioning

confidence: 99%

The Journey for the Total Chemical Synthesis of a 53 kDa Protein

Sun

Brik

2019

Acc. Chem. Res.

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“…7,8 The disorder of specific DUBs has been implicated in many serious diseases including cancer, inflammation, and neurodegenerative disease, making the DUBs important drug targets. 9 In order to explore the activity of DUBs and to find their specific small-molecule inhibitors, a variety of Ub fluorescent reagents have been developed by conjugating a fluorogenic reporter molecule to the C-terminus of Ub. DUBs can release fluorescent molecules by recognizing Ub and hydrolyzing the amide bond, and the resulting change in fluorescence intensity can be used to characterize the activity of DUBs.…”

mentioning

confidence: 99%

Semisynthesis of Ubiquitin and SUMO-Rhodamine 110-Glycine through Aminolysis of Boc-Protected Thioester Counterparts

Fan

Chu

et al. 2019

J. Org. Chem.

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Ubiquitin (Ub)-based fluorescent reagents are crucial to explore the activity of deubiquitinases (DUBs). Ub-Rho110-G is one of the preferred tools, whereas the current synthetic route is time-consuming. Here, we report a new semisynthetic strategy to produce Ub-Rho110-G through direct aminolysis of Boc-protected Ub-Mesna using bisglycyl-rhodamine 110. We also applied this strategy to synthesize active SUMO2-Rho110-G for the first time. Biochemical analysis demonstrated that semisynthetic Ub or SUMO-Rho110-G can be effectively used for the detection of the activity of DUBs or SUMO-specific enzymes.

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“… 31 Our lab developed a new strategy for the synthesis of Lys-48 and Lys-63 linked di-Ub probes to label different DUBs. 32 Similarly, we also reported the synthesis of ubiquitinated α-globin probe 33 and ubiquitinated H2A probe 15d for labeling USP15 and Calypso/ASX, respectively. Recently, Champak and co-workers reported the semisynthesis of Ub-dehydroalanine (DHA) probe by using selenocysteine as a latent bio-orthogonal electrophile to capture the TRIM-25-associated DUB, ubiquitin-specific protease 15 (USP15).…”

Section: Resultsmentioning

confidence: 60%

On-Demand Detachment of Succinimides on Cysteine to Facilitate (Semi)Synthesis of Challenging Proteins

et al. 2020

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The maleimide group is a widely used reagent for bioconjugation of peptides, proteins, and oligonucleotides employing Michael addition and Diels–Alder cycloaddition reactions. However, the utility of this functionality in chemical synthesis of peptides and proteins remains unexplored. We report, for the first time that Pd II complexes can mediate the efficient removal of various succinimide derivatives in aqueous conditions. Succinimide removal by Pd II was applied for the synthesis of two ubiquitin activity-based probes (Ub-ABPs) employing solid phase chemical ligation (SPCL). SPCL was achieved through a sequential three segment ligation on a polymer support via a maleimide anchor. The obtained probes successfully formed the expected covalent complexes with deubiquitinating enzymes (DUBs) USP2 and USP7, highlighting the use of our new method for efficient preparation of unique synthetic proteins. Importantly, we demonstrate the advantages of our newly developed method for the protection and deprotection of native cysteine with a succinimide group in a peptide fragment derived from thioredoxin-1 (Trx-1) obtained via intein based expression to enable ligation/desulfurization and subsequent disulfide bond formation in a one-pot process.

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Activity‐Based Probes Developed by Applying a Sequential Dehydroalanine Formation Strategy to Expressed Proteins Reveal a Potential α‐Globin‐Modulating Deubiquitinase

Cited by 21 publications

References 42 publications

The Journey for the Total Chemical Synthesis of a 53 kDa Protein

The Journey for the Total Chemical Synthesis of a 53 kDa Protein

Semisynthesis of Ubiquitin and SUMO-Rhodamine 110-Glycine through Aminolysis of Boc-Protected Thioester Counterparts

On-Demand Detachment of Succinimides on Cysteine to Facilitate (Semi)Synthesis of Challenging Proteins

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