Protein neddylation: beyond cullin-RING ligases

Abstract

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.

Figure 1. NEDD8 Structure

A Structure-based multiple sequence alignment of NEDD8 from indicated species and human ubiquitin, highlighting identical residues (red) and similar residues (yellow). Asterisks signify residues found to be essential for NEDD8 function in S. pombe. B Structural representation of the NEDD8 hydrophobic patches, contributing most of the known interfaces for interaction with binding partners (Table 1). The side chains contributing to the Ile36 patch are shown in blue, the residues of the Ile44 patch in green and the β1/β2-loop in red. Those interfaces are conserved in ubiquitin. Ala72, which is responsible for discrimination between NEDD8 and ubiquitin by the respective E1 enzymes, is indicated. C, D Structural representations of the NEDD8-specific charged surface patches. Acidic patches are depicted in red and basic surfaces in blue. These surfaces might be responsible for interactions that discriminate between NEDD8 and ubiquitin.

Figure 2. The Neddylation and Deneddylation Pathways

A NEDD8 activation by the heterodimeric NEDD8 E1 activating enzyme (NAE; a heterodimer of APPBP1 and UBA3) requires sequential binding of ATP and NEDD8 to the adenylation site of UBA3, where conjugation of AMP activates the C-terminus of NEDD8. NAE rearrangement into a closed conformation allows thiolation of the NEDD8 C-terminus to the NAE active site cysteine with AMP release. NAE then returns to its open conformation and adenylates a second NEDD8, resulting in conformational rearrangement of its ubiquitin-fold domain (UFD), binding of a NEDD8 E2 enzyme such as UBC12 and transfer of NEDD8 onto the E2 active site cysteine. B Neddylation of cullin (left) or other substrates (right). Cullin neddylation by the E3 ligase RBX1 is aided by DCNLs such as DCNL1 and occurs on a single conserved lysine residue. Neddylation of other substrates occurs on multiple lysines and is mediated mostly by RING E3 ligases. The details of the interaction between the NEDD8 E2 enzyme and a non-RBX RING E3 ligase are unclear. C Molecular models of UBCH5~Ub activated by CBL-B (left) and UBC12~NEDD8 activated by RBX1 (right). A linchpin arginine residue in CBL-B, which is conserved in many RING ubiquitin E3 ligases, would clash with a lysine in UBC12 such that CBL-B does not interact with and ubiquitylate UBC12. D Sequence alignment of RING domain E3 ligases that are reported to function as NEDD8 ligases (left) and of NEDD8 E2 enzymes and the ubiquitin E2 UBCH5 (right). The arrow indicates the E2 linchpin residue position and corresponding neighboring residue (red and blue denote side chain electrostatic potential). E Deneddylation of cullin by the eight-subunit COP9 signalosome (CSN) complex (left) and of non-cullin substrates by DEN1 (right). Besides cullin deneddylation, CSN binding to the C-terminal domain of cullins, mainly mediated through CSN2, keeps CRLs such as RBX1 inactive.

Figure 3. Proposed non-cullin Neddylation Substrates

A Multiple auto-neddylation of the HECT domain ubiquitin E3 ligase SMURF1 on multiple lysines is mediated by a dedicated neddylation active site cysteine, which activates the ubiquitylation activity of SMURF1. B Both neddylation and ubiqutiylation of TP53 occur through MDM2 and together with NUB1 lead to the nuclear export and consequent inactivation of mono-ubiquitinated TP53. Neddylation of E2F1 (lower) in its DNA-binding domain is stimulated by methylation and leads to reduced protein levels and impaired transcriptional activity. C Differential regulation of receptor tyrosine kinase signaling by neddylation: EGF-induced c-CBL-mediated neddylation of the EGFR synergizes with ubiquitylation to target EGFR for lysosomal degradation. On the contrary, TGFβ-induced c-CBL-mediated neddylation of the TGFβRII counteracts the destructive effects of ubiquitylation and stabilizes TGFβRII. D Poly-neddylation of histone H4, recognized by RNF168, can function as a signal for amplification of the DNA damage response cascade. E Neddylation in nucleolar stress signaling. The large ribosomal subunit L11 can be neddylated by MDM2, following impaired ribosome assembly, such that it localizes to the nucleolus and evades proteasomal destruction. Deneddylation by DEN1 counteracts this process, but nucleoplasmic localization of L11 also leads to stabilization of TP53 and cell cycle arrest.