Peeling away the layers of ubiquitin signaling complexities with synthetic ubiquitin–protein conjugates

Pham, Grace H.; Strieter, Eric R.

doi:10.1016/j.cbpa.2015.06.001

Cited by 20 publications

(19 citation statements)

References 69 publications

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“…Strategies to generate non‐native linkages benefit from their high‐yields and tunability for regiospecific modifications. There have been a number of success stories involving application of Ub conjugates with non‐native linkages to probe biological systems . Notably, non‐native Ub chains were used to determine the minimal Ub signal required for efficient proteasomal targeting of substrate proteins .…”

Section: Introductionmentioning

confidence: 99%

“…There have been a number of success stories involving application of Ub conjugates with non-native linkages to probe biological systems. [7][8][9][10] Notably, non-native Ub chains were used to determine the minimal Ub signal required for efficient proteasomal targeting of substrate proteins. 11 However, there is still some skepticism toward the use of nonnative mimics of isopeptide bonds for biochemical and biophysical studies to facilitate new discoveries.…”

Section: Introductionmentioning

confidence: 99%

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Comparison of native and non-native ubiquitin oligomers reveals analogous structures and reactivities

et al. 2016

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Ubiquitin (Ub) chains regulate a wide range of biological processes, and Ub chain connectivity is a critical determinant of the many regulatory roles that this post-translational modification plays in cells. To understand how distinct Ub chains orchestrate different biochemical events, we and other investigators have developed enzymatic and non-enzymatic methods to synthesize Ub chains of well-defined length and connectivity. A number of chemical approaches have been used to generate Ub oligomers connected by non-native linkages; however, few studies have examined the extent to which non-native linkages recapitulate the structural and functional properties associated with native isopeptide bonds. Here, we compare the structure and function of Ub dimers bearing native and non-native linkages. Using small-angle X-ray scattering (SAXS) analysis, we show that scattering profiles for the two types of dimers are similar. Moreover, using an experimental structural library and atomistic simulations to fit the experimental SAXS profiles, we find that the two types of Ub dimers can be matched to analogous structures. An important application of non-native Ub oligomers is to probe the activity and selectivity of deubiquitinases. Through steady-state kinetic analyses, we demonstrate that different families of deubiquitinases hydrolyze native and non-native isopeptide linkages with comparable efficiency and selectivity. Considering the significant challenges associated with building topologically diverse native Ub chains, our results illustrate that chains harboring non-native linkages can serve as surrogate substrates for explorations of Ub function.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Comparison of native and non-native ubiquitin oligomers reveals analogous structures and reactivities

et al. 2016

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“…All seven lysine residues, along with the N-terminal methionine, can be the sites linked by the C terminus of another ubiquitin, with ubiquitin-K48 and -K63 being the best-characterized residues involved in polyubiquitination (19,20). Chains of ubiquitins are different in length, pattern, and linkage types (21). Ubiquitination was originally found to mark proteins for degradation by the proteasome (22).…”

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confidence: 99%

TRIM25 Is Required for the Antiviral Activity of Zinc Finger Antiviral Protein

Zheng

Wang

et al. 2017

J Virol

118

168

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Zinc finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses by binding to viral mRNAs and repressing the translation and/or promoting the degradation of target mRNA. In addition, ZAP regulates the expression of certain cellular genes. Here, we report that tripartite motif-containing protein 25 (TRIM25), a ubiquitin E3 ligase, is required for the antiviral activity of ZAP. Downregulation of endogenous TRIM25 abolished ZAP's antiviral activity. The E3 ligase activity of TRIM25 is required for this regulation. TRIM25 mediated ZAP ubiquitination, but the ubiquitination of ZAP itself did not seem to be required for its antiviral activity. Downregulation of endogenous ubiquitin or overexpression of the deubiquitinase OTUB1 impaired ZAP's activity. We provide evidence indicating that TRIM25 modulates the target RNA binding activity of ZAP. These results uncover a mechanism by which the antiviral activity of ZAP is regulated.IMPORTANCE ZAP is a host antiviral factor that specifically inhibits the replication of certain viruses, including HIV-1, Sindbis virus, and Ebola virus. ZAP binds directly to target mRNA, and it represses the translation and promotes the degradation of target mRNA. While the mechanisms by which ZAP posttranscriptionally inhibits target RNA expression have been extensively studied, how its antiviral activity is regulated is not very clear. Here, we report that TRIM25, a ubiquitin E3 ligase, is required for the antiviral activity of ZAP. Downregulation of endogenous TRIM25 remarkably abolished ZAP's activity. TRIM25 is required for ZAP optimal binding to target mRNA. These results help us to better understand how the antiviral activity of ZAP is regulated.

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“…Native chemical ligation (NCL) has been an extremely useful tool to make ubiquitin, ubiquitin-peptide conjugates, ubiquitin dimers and ubiquitin tetramers, as reviewed recently by Pham et al [11]. Chemical synthesis of ubiquitinated peptides was first established by Muir and co-workers and utilizes a ligation auxiliary where the auxiliary group is removed under photolytic conditions; yielding a natively linked Ub-peptide conjugate [12].…”

Section: Chemical Strategiesmentioning

confidence: 99%

“…In addition, ubiquitin-based DUB probes and fluorescent ubiquitin-based enzyme substrates have seen an enormous boost, producing ubiquitin-based tools in all sorts of different flavors [7]. Two other highly investigated areas of synthetic and semi-synthetic ubiquitin-conjugate research focus on ubiquitinated histones [8,9] and ubiquitinated asynuclein [10,11]. We will briefly introduce the current synthetic and semi-synthetic methods that can be applied to generate ubiquitin and Ub-based tools and then focus on three other rapidly developing areas.…”

Section: Introductionmentioning

confidence: 99%

Synthetic and semi-synthetic strategies to study ubiquitin signaling

Tilburg

Elhebieshy

Ovaa

2016

Current Opinion in Structural Biology

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The post-translational modification ubiquitin can be attached to the ɛ-amino group of lysine residues or to a protein's N-terminus as a mono ubiquitin moiety. Via its seven intrinsic lysine residues and its N-terminus, it can also form ubiquitin chains on substrates in many possible ways. To study ubiquitin signals, many synthetic and semi-synthetic routes have been developed for generation of ubiquitin-derived tools and conjugates. The strength of these methods lies in their ability to introduce chemo-selective ligation handles at sites that currently cannot be enzymatically modified. Here, we review the different synthetic and semi-synthetic methods available for ubiquitin conjugate synthesis and their contribution to how they have helped investigating conformational diversity of diubiquitin signals. Next, we discuss how these methods help understanding the ubiquitin conjugation-deconjugation system by recent advances in ubiquitin ligase probes and diubiquitin-based DUB probes. Lastly, we discuss how these methods help studying post-translational modification of ubiquitin itself.

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Peeling away the layers of ubiquitin signaling complexities with synthetic ubiquitin–protein conjugates

Cited by 20 publications

References 69 publications

Comparison of native and non-native ubiquitin oligomers reveals analogous structures and reactivities

Comparison of native and non-native ubiquitin oligomers reveals analogous structures and reactivities

TRIM25 Is Required for the Antiviral Activity of Zinc Finger Antiviral Protein

Synthetic and semi-synthetic strategies to study ubiquitin signaling

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