Determining the Effects of Neddylation on Cullin-RING Ligase-Dependent Protein Ubiquitination

Abstract

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.

Keywords: cullin-RING ligases; in vitro assay; neddylation; protein purification; ubiquitination.

Figure 1

Schematic diagram summarizing the procedures included in this article for studying the effects of neddylation on CRL‐dependent protein ubiquitination. For the preparation of recombinant CUL2•RBX1, CUL2 and RBX1 are co‐expressed in E. coli and purified through Ni²⁺ affinity chromatography, TEV cleavage for tag removal, StrepII‐tag affinity chromatography, and size exclusion chromatography. To examine the effect of neddylation on CRL activity, CUL2•RBX1 with or without in vitro neddylation is prepared and used for substrate ubiquitination in vitro. The ubiquitination of substrates is monitored over time by western blot (or fluorescent scan), and the ubiquitination rate is quantitatively analyzed.

Figure 2

Expression and purification of CUL2•RBX1. (A) Schematic of the CUL2•RBX1 expression plasmid. (B) Representative image of the Coomassie blue–stained gel showing the CUL2 protein band before and after the TEV cleavage. (C) Chromatogram showing the elution profile of CUL2•RBX1 purified from the 1‐ml Strep‐Tactin cartridge. (D) Chromatogram showing the elution profile of CUL2•RBX1 purified from the Superdex 200 Increase 10/300 GL column. (E) Representative image of the Coomassie blue–stained gel showing the purified CUL2•RBX1 product.

Figure 3

Quantitative analysis of the effect of neddylation on CRL2‐dependent protein ubiquitination. (A) CUL2•RBX1 with or without neddylation was used for time course–dependent protein ubiquitination in vitro. Mock samples with no CUL2•RBX1 were included as the negative control. Samples were analyzed by western blot with antibodies against CUL2, the FLAG epitope (for the substrate), and VHL (the substrate receptor). (B) Relative levels of unmodified substrates versus time were plotted and fit with single exponential curves.