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Programmed ubiquitin acetylation using genetic code expansion reveals altered ubiquitination patterns.

Authors
  • Lacoursiere, Rachel E1
  • O'Donoghue, Patrick1, 2
  • Shaw, Gary S1
  • 1 Department of Biochemistry, The University of Western Ontario, London, Canada. , (Canada)
  • 2 Department of Chemistry, The University of Western Ontario, London, Canada. , (Canada)
Type
Published Article
Journal
FEBS letters
Publication Date
Apr 01, 2020
Volume
594
Issue
7
Pages
1226–1234
Identifiers
DOI: 10.1002/1873-3468.13702
PMID: 31792955
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

Ubiquitination is a post-translational modification (PTM) capable of being regulated by other PTMs, including acetylation. However, the biological consequences of acetylated ubiquitin (acUb) variants are poorly understood, due to their transient nature in vivo and poor characterization in vitro. Since Ub is known to be acetylated in human cells, we produced all possible acUb variants using genetic code expansion. We also developed a protocol that optimizes acetyl-lysine addition to minimize mistranslated proteins and maximize site-specific acUb protein production. Purified acUb proteins were used in pilot ubiquitination assays and found to be competent with IpaH3CT and RNF8 E3 ligases. Overall, this work provides an optimized method to express and purify all acetyl-lysine variants for ubiquitin and shows these proteins can be used to identify potential unique ubiquitination patterns. © 2019 Federation of European Biochemical Societies.

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