Abstract
Ubiquitylation is a posttranslational modification that utilizes protein-protein binding interactions to regulate cellular processes. In ubiquitin signaling, a vast array of mono- and polyubiquitin modifications to substrate proteins are recognized by a diverse group of ubiquitin-binding proteins. Identifying ubiquitin-binding proteins and characterizing their binding properties is necessary for understanding the structural basis of ubiquitin signaling. This chapter provides a means of studying ubiquitin-binding interactions in vitro by describing how to generate monoubiquitin and K63-linked polyubiquitin chains and perform pull-down assays with ubiquitin-binding proteins, which is of particular relevance for DNA damage and other signaling pathways.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Hurley JH, Lee S, Prag G (2006) Ubiquitin-binding domains. Biochem J 399:361–372. https://doi.org/10.1042/BJ20061138
Randles L, Walters KJ (2012) Ubiquitin and its binding domains. Front Biosci (Landmark Ed) 17:2140–2157. https://doi.org/10.2741/4042
Akutsu M, Dikic I, Bremm A (2016) Ubiquitin chain diversity at a glance. J Cell Sci 129:875–880. https://doi.org/10.1242/jcs.183954
Komander D, Rape M (2012) The ubiquitin code. Annu Rev Biochem 81:203–229. https://doi.org/10.1146/annurev-biochem-060310-170328
Swatek KN, Komander D (2016) Ubiquitin modifications. Cell Res 26:399–422. https://doi.org/10.1038/cr.2016.39
Piotrowski J, Beal R, Hoffman L et al (1997) Inhibition of the 26 S proteasome by polyubiquitin chains synthesized to have defined lengths. J Biol Chem 272:23712–23721. https://doi.org/10.1074/jbc.272.38.23712
Berndsen CE, Wolberger C (2011) A spectrophotometric assay for conjugation of ubiquitin and ubiquitin-like proteins. Anal Biochem 418:102–110. https://doi.org/10.1016/j.ab.2011.06.034
Berndsen CE, Wiener R, Yu IW et al (2013) A conserved asparagine has a structural role in ubiquitin-conjugating enzymes. Nat Chem Biol 9:154–156. https://doi.org/10.1038/nchembio.1159
Pickart CM, Raasi S (2005) Controlled synthesis of polyubiquitin chains. Methods Enzymol 399:21–36. https://doi.org/10.1016/S0076-6879(05)99002-2
Sato Y, Yoshikawa A, Mimura H et al (2009) Structural basis for specific recognition of Lys 63-linked polyubiquitin chains by tandem UIMs of RAP80. EMBO J 28:2461–2468. https://doi.org/10.1038/emboj.2009.160
Brickner JR, Soll JM, Lombardi PM et al (2017) A ubiquitin-dependent signalling axis specific for ALKBH-mediated DNA dealkylation repair. Nature 551:389–393. https://doi.org/10.1038/nature24484
Michel MA, Komander D, Elliott PR (2018) Enzymatic assembly of ubiquitin chains. Methods Mol Biol 1844:73–84. https://doi.org/10.1007/978-1-4939-8706-1_6
Dong KC, Helgason E, Yu C et al (2011) Preparation of distinct ubiquitin chain reagents of high purity and yield. Structure 19:1053–1063. https://doi.org/10.1016/j.str.2011.06.010
Acknowledgments
Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM140410. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Anoh, R., Burke, K.A., Schmelyun, D.P., Lombardi, P.M. (2022). Generation of Monoubiquitin and K63-Linked Polyubiquitin Chains for Protein Interaction Studies. In: Mosammaparast, N. (eds) DNA Damage Responses. Methods in Molecular Biology, vol 2444. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2063-2_16
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2063-2_16
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2062-5
Online ISBN: 978-1-0716-2063-2
eBook Packages: Springer Protocols