Protein neddylation: beyond cullin–RING ligases

Key Points NEDD8 (neural precursor cell expressed developmentally downregulated protein 8) and ubiquitin have the highest sequence and structural similarity among all ubiquitin-like proteins. NEDD8-specific conjugation and de-conjugation pathways exist in all studied eukaryotes, which can discriminate between NEDD8 and other ubiquitin-like proteins through NEDD8-specific interaction domains. Nevertheless, a perturbed ratio of free NEDD8 and ubiquitin or cellular stress can result in the conjugation of NEDD8 through the ubiquitylation machinery onto ubiquitylation substrates. This can lead to mis-assignments of neddylation targets, and most published reports lack sufficient evidence to substantiate the discovery of genuine neddylation substrates. We propose a list of necessary criteria for bona fide neddylation substrates and re-evaluate published studies in the light of these criteria. Cullins are the best-studied and only neddylation targets to date that fulfill all of these criteria. We discuss potential examples of neddylation regulating non-cullin ubiquitin E3 ligases, transcription, ribosomal stress and various signalling pathways. Pharmacological inhibition of neddylation is a promising new direction for cancer therapy. We discuss the potential effects of inhibiting non-cullin, as well as cullin, neddylation. Post-translational modification of proteins by NEDD8 has been mainly characterized in terms of the cullin–RING E3 ligase family. However, recent studies have indicated that there might be non-cullin neddylation targets that require further verification. NEDD8 (neural precursor cell expressed developmentally downregulated protein 8) is a ubiquitin-like protein that activates the largest ubiquitin E3 ligase family, the cullin–RING ligases. Many non-cullin neddylation targets have been proposed in recent years. However, overexpression of exogenous NEDD8 can trigger NEDD8 conjugation through the ubiquitylation machinery, which makes validating potential NEDD8 targets challenging. Here, we re-evaluate studies of non-cullin targets of NEDD8 in light of the current understanding of the neddylation pathway, and suggest criteria for identifying genuine neddylation substrates under homeostatic conditions. We describe the biological processes that might be regulated by non-cullin neddylation, and the utility of neddylation inhibitors for research and as potential therapies. Understanding the biological significance of non-cullin neddylation is an exciting