Investigating Mechanisms that Control Ubiquitin-Mediated DAF-16/FOXO Protein Turnover.

TitleInvestigating Mechanisms that Control Ubiquitin-Mediated DAF-16/FOXO Protein Turnover.
Publication TypeJournal Article
Year of Publication2019
AuthorsHeimbucher, T, Murphy, CT
JournalMethods Mol Biol
Date Published2019
KeywordsCell Line, Forkhead Transcription Factors, Gene Expression, Humans, Protein Binding, Protein Renaturation, Proteolysis, Recombinant Fusion Proteins, Ubiquitin, Ubiquitination

<p>Protein turnover of FOXO family transcription factors is regulated by the ubiquitin-proteasome system. A complex interplay of factors that covalently attach certain types of ubiquitin chains (E3-ubiquitin ligases), and enzymes that are able to remove ubiquitin conjugates (deubiquitylases), regulate the degradation of FOXO proteins by the proteasome. Here, we describe methods to characterize candidate E3-ubiquitin ligases and deubiquitylases as regulators of the FOXO ubiquitylation status. Our protocol can be utilized to purify and enrich a ubiquitylated FOXO pool from cultured cells under denaturing conditions, which inactivates cellular deubiquitylases and thereby protects ubiquitin conjugates on FOXO proteins. In addition, our method describes how ubiquitylated FOXO proteins can be renatured in a stepwise fashion to serve as substrates for in vitro deubiquitylation (DUB) assays.</p>

Alternate JournalMethods Mol Biol
PubMed ID30414143
PubMed Central IDPMC8428773
Grant ListR56 AG047344 / AG / NIA NIH HHS / United States