[en] Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate. Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Masclef, Louis ✱; Maisonneuve-Rosemont Hospital Research Center and Department of Medicine, University of Montréal
Uriarte, Maxime ✱; Université de Liège - ULiège > GIGA > GIGA Molecular & Computational Biology - Gene Expression & Cancer
Ahmed, Oumaima; Maisonneuve-Rosemont Hospital Research Center and Department of Medicine, University of Montréal
Sen Nkwe, Nadine; Maisonneuve-Rosemont Hospital Research Center and Department of Medicine, University of Montréal
Barbour, Haithem; Maisonneuve-Rosemont Hospital Research Center and Department of Medicine, University of Montréal
Iannantuono, Nicholas V G; Maisonneuve-Rosemont Hospital Research Center and Department of Medicine, University of Montréal
Boubekeur, Amina; Maisonneuve-Rosemont Hospital Research Center and Department of Medicine, University of Montréal
Daou, Salima; Maisonneuve-Rosemont Hospital Research Center and Department of Medicine, University of Montréal, Lunenfeld-Tanenbaum Research Institute, Sinai Health System
Affar, El Bachir; Maisonneuve-Rosemont Hospital Research Center and Department of Medicine, University of Montréal, Lunenfeld-Tanenbaum Research Institute, Sinai Health System, el.bachir.affar@umontreal.ca
✱ These authors have contributed equally to this work.
Language :
English
Title :
In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones.
We thank Diana Adjaoud for technical assistance. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (2015-2020), Genome Quebec (2016-2019) and Genome Canada (2016-2019) to E.B.A. E.B.A. is a senior scholar of the Fonds de la Recherche du Québec-Santé (FRQ-S). L.M and N.S.K. have a PhD scholarship from the FRQ-S. H.B had a PhD scholarship from the Ministry of Higher Education and from Scientific Research of Tunisia and the Cole Foundation.We thank Diana Adjaoud for technical assistance. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (2015-2020), Genome Quebec (2016-2019) and Genome Canada (2016-2019) to E.B.A. E.B.A. is a senior scholar of the Fonds de la Recherche du Qu?bec-Sant? (FRQ-S). L.M and N.S.K. have a PhD scholarship from the FRQ-S. H.B had a PhD scholarship from the Ministry of Higher Education and from Scientific Research of Tunisia and the Cole Foundation.
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