E. coli-Based Selection and Expression Systems for Discovery, Characterization, and Purification of Ubiquitylated Proteins

Levin-Kravets, Olga; Keren-Kaplan, Tal; Attali, Ilan; Sharon, Itai; Tanner, Neta; Shapira, Dar; Rathi, Ritu; Persaud, Avinash K.; Shohat, Noa; Shusterman, Anna; Prag, Gali

doi:10.1007/978-1-4939-8706-1_11

Cited by 3 publications

(1 citation statement)

References 16 publications

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“…Identification and biochemical characterisation of a complete ubiquitylation pathway in Archaea is consistent with the near universal presence of the proteasome. Moreover, ubiquitylation systems are present in many bacterial lineages, most likely resulting from lateral gene transfer (Levin-Kravets et al, 2018, Barlow et al, 2023. The ubiquitylation system has considerably expanded and diversified in eukaryotes, with a repertoire of over 600, 500 and 100 E3 ligases in the H. sapiens, Arabidopsis thaliana and Saccharomyces cerevisiae respectively.…”

Section: Introductionmentioning

confidence: 99%

Evolution of cullin E3 ubiquitin ligases and function in trypanosomes

Pino¹,

Zoltner²,

Yamada³

et al. 2023

Preprint

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Post-translational modifications (PTMs) modulate protein function, with ubiquitylation a pre-eminent example with major roles in protein turnover. Ubiquitylation utilises a ligase enzyme cascade for conjugation of ubiquitin to client proteins and cullin-RING ligases are amongst the most complex known. We reconstructed evolution of cullin-RING E3 ubiquitin ligases across eukaryotes and experimentally characterised two cullin complexes in trypanosomatids, a taxon highly divergent from animals and fungi. We find considerable diversity within cullins and, in particular, trypanosomatids share only a minority of cullins with other lineages. Furthermore, we identify expansions in cullin client adaptor protein families, novel client adaptors and demonstrate client specificity. Finally we show that ornithine decarboxylase (TbODC), an important target of the drug trypanosome eflornithine, is a substrate for TbCul-A and overturn earlier models for eflornithine specificity. These studies highlight lineage-specific roles for cullin E3s and their contributions towards eukaryotic complexity.

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Section: Introductionmentioning

confidence: 99%

Evolution of cullin E3 ubiquitin ligases and function in trypanosomes

Pino¹,

Zoltner²,

Yamada³

et al. 2023

Preprint

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Split Chloramphenicol Acetyl-Transferase Assay Reveals Self-Ubiquitylation-Dependent Regulation of UBE3B

Levin-Kravets

Kordonsky

Shusterman

et al. 2021

Journal of Molecular Biology

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A Bacterial Platform for Studying Ubiquitination Cascades Anchored by SCF-Type E3 Ubiquitin Ligases

Pu,

Wang,

et al. 2024

Biomolecules

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Ubiquitination is one of the most important post-translational modifications in eukaryotes. The ubiquitination cascade includes ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). The E3 ligases, responsible for substrate recognition, are the most abundant and varied proteins in the cascade and the most studied. SKP1-CUL1-F-Box (SCF)-type E3 ubiquitin ligases are multi-subunit RING (Really Interesting New Gene) E3 ubiquitin ligases, composed of CUL1 (Cullin 1), RBX1 (RING BOX 1), SKP1 (S-phase Kinase-associated Protein 1), and F-box proteins. In vitro ubiquitination assays, used for studying the specific recognition of substrate proteins by E3 ubiquitin ligases, require the purification of all components involved in the cascade, and for assays with SCF-type E3 ligases, additional proteins (several SCF complex subunits). Here, the Duet expression system was used to co-express E1, E2, ubiquitin, ubiquitylation target (substrate), and the four subunits of a SCF-type E3 ligase in E. coli. When these proteins co-exist in bacterial cells, ubiquitination occurs and can be detected by Western Blot. The effectiveness of this bacterial system for detecting ubiquitination cascade activity was demonstrated by replicating both AtSCFTIR1-mediated and human SCFFBXO28-mediated ubiquitylation in bacteria. This system provides a basic but adaptable platform for the study of SCF-type E3 ubiquitin ligases.

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E. coli-Based Selection and Expression Systems for Discovery, Characterization, and Purification of Ubiquitylated Proteins

Cited by 3 publications

References 16 publications

Evolution of cullin E3 ubiquitin ligases and function in trypanosomes

Evolution of cullin E3 ubiquitin ligases and function in trypanosomes

Split Chloramphenicol Acetyl-Transferase Assay Reveals Self-Ubiquitylation-Dependent Regulation of UBE3B

A Bacterial Platform for Studying Ubiquitination Cascades Anchored by SCF-Type E3 Ubiquitin Ligases

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