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A bacterial genetic selection system for ubiquitylation cascade discovery.

Levin-Kravets O, Tanner N, Shohat N, Attali I, Keren-Kaplan T, Shusterman A, Artzi S, Varvak A, Reshef Y, Shi X, Zucker O, Baram T, Katina C, Pilzer I, Ben-Aroya S, Prag G
Nat Methods 13 945-952 (2016)
Related entries: 1edu, 1h0a, 5loz

Cited: 12 times
EuropePMC logo PMID: 27694912

Abstract

About one-third of the eukaryotic proteome undergoes ubiquitylation, but the enzymatic cascades leading to substrate modification are largely unknown. We present a genetic selection tool that utilizes Escherichia coli, which lack deubiquitylases, to identify interactions along ubiquitylation cascades. Coexpression of split antibiotic resistance protein tethered to ubiquitin and ubiquitylation target together with a functional ubiquitylation apparatus results in a covalent assembly of the resistance protein, giving rise to bacterial growth on selective media. We applied the selection system to uncover an E3 ligase from the pathogenic bacteria EHEC and to identify the epsin ENTH domain as an ultraweak ubiquitin-binding domain. The latter was complemented with a structure-function analysis of the ENTH-ubiquitin interface. We also constructed and screened a yeast fusion library, discovering Sem1 as a novel ubiquitylation substrate of Rsp5 E3 ligase. Collectively, our selection system provides a robust high-throughput approach for genetic studies of ubiquitylation cascades and for small-molecule modulator screening.

Articles - 5lp0 mentioned but not cited (3)

  1. Structure and evolution of ENTH and VHS/ENTH-like domains in tepsin. Archuleta TL, Frazier MN, Monken AE, Kendall AK, Harp J, McCoy AJ, Creanza N, Jackson LP. Traffic 18 590-603 (2017)
  2. Remodeling Membrane Binding by Mono-Ubiquitylation. Tanner N, Kleifeld O, Nachman I, Prag G. Biomolecules 9 E325 (2019)
  3. The importance of residue-level filtering and the Top2018 best-parts dataset of high-quality protein residues. Williams CJ, Richardson DC, Richardson JS. Protein Sci 31 290-300 (2022)


Reviews citing this publication (2)

  1. Protein Engineering in the Ubiquitin System: Tools for Discovery and Beyond. Zhao B, Tsai YC, Jin B, Wang B, Wang Y, Zhou H, Carpenter T, Weissman AM, Yin J. Pharmacol Rev 72 380-413 (2020)
  2. Lead discovery and chemical biology approaches targeting the ubiquitin proteasome system. Akinjiyan FA, Carbonneau S, Ross NT. Bioorg Med Chem Lett 27 4589-4596 (2017)

Articles citing this publication (7)

  1. Formation of NPR1 Condensates Promotes Cell Survival during the Plant Immune Response. Zavaliev R, Mohan R, Chen T, Dong X. Cell 182 1093-1108.e18 (2020)
  2. Identifying the substrate proteins of U-box E3s E4B and CHIP by orthogonal ubiquitin transfer. Bhuripanyo K, Wang Y, Liu X, Zhou L, Liu R, Duong D, Zhao B, Bi Y, Zhou H, Chen G, Seyfried NT, Chazin WJ, Kiyokawa H, Yin J. Sci Adv 4 e1701393 (2018)
  3. The MALDI-TOF E2/E3 Ligase Assay as Universal Tool for Drug Discovery in the Ubiquitin Pathway. De Cesare V, Johnson C, Barlow V, Hastie J, Knebel A, Trost M. Cell Chem Biol 25 1117-1127.e4 (2018)
  4. An Integrative Synthetic Biology Approach to Interrogating Cellular Ubiquitin and Ufm Signaling. Li C, Han T, Guo R, Chen P, Peng C, Prag G, Hu R. Int J Mol Sci 21 E4231 (2020)
  5. UbiGate: a synthetic biology toolbox to analyse ubiquitination. Kowarschik K, Hoehenwarter W, Marillonnet S, Trujillo M. New Phytol 217 1749-1763 (2018)
  6. An enzyme-mediated protein-fragment complementation assay for substrate screening of sortase A. Li N, Yu Z, Ji Q, Sun J, Liu X, Du M, Zhang W. Biochem Biophys Res Commun 486 257-263 (2017)
  7. Probing protein ubiquitination in live cells. Qin W, Steinek C, Kolobynina K, Forné I, Imhof A, Cardoso MC, Leonhardt H. Nucleic Acids Res 50 e125 (2022)


Related citations provided by authors (2)

  1. Epsin 1 undergoes nucleocytosolic shuttling and its eps15 interactor NH(2)-terminal homology (ENTH) domain, structurally similar to Armadillo and HEAT repeats, interacts with the transcription factor promyelocytic leukemia Zn(2)+ finger protein (PLZF).. Hyman J, Chen H, Di Fiore PP, De Camilli P, Brunger AT J Cell Biol 149 537-46 (2000)
  2. Curvature of clathrin-coated pits driven by epsin.. Ford MG, Mills IG, Peter BJ, Vallis Y, Praefcke GJ, Evans PR, McMahon HT Nature 419 361-6 (2002)

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