Investigating Mechanisms that Control Ubiquitin-Mediated DAF-16/FOXO Protein Turnover
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.
Key wordsFOXO DAF-16 Protein stability Ubiquitylation assay Deubiquitylation (DUB) assay Ubiquitin Proteasome
This work was supported by the NIH NIA 1R56AG047344-01A1 (C.T.M.) and the Glenn Foundation for Medical Research. C.T.M. is the Director of the Glenn Center for Aging Research at Princeton. We thank Dr. Andrea Carrano for critically reading the manuscript.
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