Abstract
Nedd8 is a small ubiquitin-like protein (9 kDa) covalently attached to a conserved lysine residue of a cullin protein which is part of cullin-RING ligases (CRLs). CRLs are major E3 ligases important for protein ubiquitination in the ubiquitin-proteasome pathway (UPP). The activity of CRLs is regulated by cycles of neddylation (CulA-N8, ~98 kDa) and deneddylation (CulA ~89 kDa). The COP9 signalosome (CSN) and Deneddylase A (DenA) are capable of cleaving the isopeptide bond between Nedd8 and CullinA. In contrast to the single protein DenA, CSN is an eight subunit multiprotein complex. Protein crude extracts of different Aspergillus nidulans csn deletion strains were mixed with recombinant CSN subunits expressed and purified from Escherichia coli (E. coli). Western hybridization experiments using anti-CulA or anti-Nedd8 antibodies could show the ratio of neddylated vs. deneddylated CulA. Using the deneddylation assay, we could show that CsnE is the last subunit joining a 7-subunit pre-assembled CSN in vitro and only then CSN can perform cullin deneddylation by the metalloprotease subunit CsnE. This assay is a fast and non-expensive method, which visualizes enzyme activity for deneddylating proteins. It might be also useful for testing the activity of other isopeptidases removing post-translational modifications from substrates in Aspergillus nidulans (A. nidulans) or other organisms.
Keywords: Deneddylation, COP9 signalosome (CSN), DEN1, Western hybridization, Nedd8
Materials and Reagents
Equipment
Software
Procedure
Note: The preparation of buffers, solutions and SDS gels is explained in Recipes section.
Representative data
Figure 7 shows a typical result of an in vitro deneddylation assay similar to those shown in Beckmann et al. (2015). Usually most cullins are deneddylated in wild type. This is visible in the western hybridization and quantification of crude extracts from wild type (wt) where the Nedd8 antibody gives a weak signal at ~100 kDa, with CulA hybridization a strong signal for deneddylated CulA (CulA, **) and a slight signal for neddylated CulA (CulA-Nedd8, *) (Figure 7). In contrast, crude extracts of csn deletion strains show increased neddylated CulA amounts, which cannot be complemented by the addition of the respective recombinant CSN subunit to the deletion strain. The only exception where CulA is mostly deneddylated like in wild type is when recombinant CsnE is added to csnE deletion crude extract. Only then the CSN seems to be active. Signals were quantified relative to the loading control and for Nedd8 relative to wild type. For CulA signals the ratio of neddylated CulA to non-neddylated CulA were calculated. Figure 7. An example for in vitro deneddylation assay: CsnE can restore deneddylation activity of CSN. Western hybridization of A. nidulans crude extracts incubated with and without recombinant CSN proteins. A. The membrane was incubated with α-Nedd8 antibody resulting in a weak signal for wt and ΔcsnE + CsnE and strong signals for all other csn deletion strains with or without recombinant protein at ~100 kDa. α-Tub antibody was used as loading control. B. Detection with α-CulA underlined the result from (A) where the ratio of neddylated CulA (*) to deneddylated CulA (**) in ΔcsnE + recombinant CsnE was similar to the wild type and inverted in comparison to all other samples. Results were quantified using BIO1D software (Peqlab).
Notes
Users of this protocol should have experience in or collaborative groups for protein purification using the ÄKTA system. For reliable data, at least three biological repetitions should be performed. This is also important for calculating error bars. Mean values and standard deviation were calculated using Excel with the formula below: With σ as standard deviation, xi representing each value of the dataset, x̄ arithmetic mean value, n total number of data points, Σ the sum of (xi - x̄)2 of all data points.
Recipes
Acknowledgments
This protocol was adapted from a previously published study (Beckmann et al., 2015) and precursor experiments (Christmann et al., 2013; Harting et al., 2013; von Zeska Kress et al., 2012). We thank Dr. Elena Beckmann for her support and advice and Gabriele Heinrich for excellent technical assistance. We also thank Sabine Reen and Josua Schinke for careful proofreading the manuscript. This research has been supported by the Deutsche Forschungsgemeinschaft (DFG) within the SFB860.
References
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