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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.


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 research prospect primed to reveal fundamental insights.

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Figure 1: NEDD8 structure.
Figure 2: The neddylation and deneddylation pathways.
Figure 3: Proposed non-cullin neddylation substrates.


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The authors thank D. Xirodimas, A. Schreiber and A. Smith for critical reading of the manuscript. R.I.E. is supported by a Marie-Curie post-doctoral fellowship, and work in the Peter laboratory is supported by the European Research Council (ERC), the Swiss National Science Foundation (SNF), and the ETH Zürich. B.A.S. acknowledges the Howard Hughes Medical Institute and National Institutes of Health (NIH) R01GM069530, P30CA021765 and ALSAC for support.

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Cullin–RING ligases

A large family of E3 ligases that are nucleated by a cullin scaffold protein and contain a RING domain. Cullin–RING ligases bring together the ubiquitylation substrate (through substrate-specific adaptors) and the E2 enzyme (through the RING domain RBX subunit) to catalyse the ubiquitylation reaction.

UBA domain

A domain that is structurally characterized by a three-helix bundle and that recognizes the Ile44 hydrophobic patch of ubiquitin.

JAMM motif

A metalloprotease His-X-His-X(10)-Asp motif that coordinates a zinc, present in multiple bacterial, archaeal and eukaryotic enzymes, including the deubiquitylating enzymes RPN11, STAMBPL1 and CSN5.


An AAA+ ATPase hexamer involved in eukaryotic signalling and quality control pathways. It delivers ubiquitylated proteins to the 26S proteasome through numerous adaptors.


A motif that comprises an amphipathic helix with conserved negatively charged residues at its N terminus, a contiguous hydrophobic patch in the middle and a C-terminal Ser residue. The conserved Ala side chain of the UIM inserts in the Ile44 hydrophobic pocket of ubiquitin.


A motif that is similar to the UIM but in which the helix runs in the opposite direction. MIUs are known to recognize Lys63-linked ubiquitin chains.

Parkinson's disease

A neurodegenerative disease characterized by the loss of dopaminergic neurons of the midbrain and accumulation of insoluble inclusions containing α-synuclein amyloid fibrils.

Alzheimer's disease

A neurodegenerative disease of the cerebral cortex characterized by the accumulation of aggregated amyloid-β.

Effector caspase

An activated caspase that is produced from inactive pro-caspases through cleavage by initiator caspases. Effector caspases subsequently cleave protein substrates to induce the apoptotic process.

Initiator caspase

This type of caspase is typically activated in response to particular stimuli; once activated, they cleave effector pro-caspases to activate them.

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Enchev, R., Schulman, B. & Peter, M. Protein neddylation: beyond cullin–RING ligases. Nat Rev Mol Cell Biol 16, 30–44 (2015).

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