Determining the Effects of Neddylation on Cullin-RING Ligase-Dependent Protein Ubiquitination.
cullin-RING ligases
in vitro assay
neddylation
protein purification
ubiquitination
Journal
Current protocols
ISSN: 2691-1299
Titre abrégé: Curr Protoc
Pays: United States
ID NLM: 101773894
Informations de publication
Date de publication:
Mar 2022
Mar 2022
Historique:
entrez:
22
3
2022
pubmed:
23
3
2022
medline:
25
3
2022
Statut:
ppublish
Résumé
As the largest family of ubiquitin (Ub) E3 ligases, cullin-RING ligases (CRLs) play crucial roles in various cellular processes, and their activities are tightly regulated by orchestrated mechanisms. Neddylation, the conjugation of a Ub-like protein NEDD8 to a target protein such as the cullin, represents a key regulatory mechanism for CRLs. Biochemical and structural studies of a few CRLs have revealed that cullin neddylation alters the CRL conformation and activates CRL-dependent protein ubiquitination. Here, using CUL2-RING ligase (CRL2) as an example, we describe our protocols for the preparation of recombinant CUL2 with or without NEDD8 conjugation, which is further used to quantitatively determine the effect of neddylation on CRL2-dependent protein ubiquitination in vitro. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and purification of CUL2•RBX1 from Escherichia coli Support Protocol: Further purification of CUL2•RBX1 with additional chromatography on an FPLC system Basic Protocol 2: Reconstitution of cullin neddylation for quantitative ubiquitination assay in vitro.
Identifiants
pubmed: 35316580
doi: 10.1002/cpz1.401
pmc: PMC8969890
mid: NIHMS1785719
doi:
Substances chimiques
Cullin Proteins
0
Ubiquitin
0
Ubiquitin-Protein Ligases
EC 2.3.2.27
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
e401Subventions
Organisme : American Heart Association-American Stroke Association
ID : 20CDA35270030 - XING LIU
Pays : United States
Organisme : NIGMS NIH HHS
ID : R35 GM138016
Pays : United States
Organisme : NIH HHS
ID : R35GM138016
Pays : United States
Informations de copyright
© 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.
Références
Cell. 2013 Mar 28;153(1):206-15
pubmed: 23453757
Sci Rep. 2021 May 27;11(1):11224
pubmed: 34045610
Mol Cell. 2009 Feb 27;33(4):483-95
pubmed: 19250909
Biochem Biophys Res Commun. 1998 Oct 29;251(3):688-92
pubmed: 9790970
Protein Sci. 2021 Nov;30(11):2338-2345
pubmed: 34459035
Annu Rev Biochem. 2009;78:363-97
pubmed: 19489724
EMBO Rep. 2013 Dec;14(12):1050-61
pubmed: 24232186
J Biol Chem. 2003 Aug 1;278(31):28882-91
pubmed: 12759363
Mol Cell. 2008 Oct 10;32(1):21-31
pubmed: 18851830
Curr Protoc Mol Biol. 2008 Jul;Chapter 10:Unit 10.8
pubmed: 18633991
Nature. 2020 Feb;578(7795):461-466
pubmed: 32051583
Mol Cell. 2018 Mar 1;69(5):773-786.e6
pubmed: 29499133
Nat Rev Mol Cell Biol. 2005 Jan;6(1):9-20
pubmed: 15688063
Nat Rev Mol Cell Biol. 2015 Jan;16(1):30-44
pubmed: 25531226
Curr Protoc Pharmacol. 2016 Jun 01;73:A.3A.1-A.3A.32
pubmed: 27248579
Curr Protoc Protein Sci. 2019 Jun;96(1):e84
pubmed: 30706993
Mol Cell. 2009 Oct 9;36(1):39-50
pubmed: 19818708
Biochem Biophys Res Commun. 2000 Apr 21;270(3):1093-6
pubmed: 10772955
Curr Protoc Mol Biol. 2012 Jan;Chapter 10:Unit 10.2A.
pubmed: 22237856
Cell. 2017 Nov 30;171(6):1326-1339.e14
pubmed: 29103612
Mol Cell. 2008 Jan 18;29(1):23-35
pubmed: 18206966
Methods Enzymol. 2005;398:125-42
pubmed: 16275325
Structure. 2013 Jan 8;21(1):42-53
pubmed: 23201271
Adv Exp Med Biol. 2020;1217:33-46
pubmed: 31898220
Curr Protoc. 2022 Mar;2(3):e401
pubmed: 35316580
Mol Cell. 2003 Dec;12(6):1427-37
pubmed: 14690597