The NEDD8 Conjugation Pathway and Its Relevance in Cancer Biology and Therapy

Abstract

Cancer cells depend on signals that promote cell cycle progression and prevent programmed cell death that would otherwise result from cumulative, aberrant stress. These activities require the temporally controlled destruction of specific intracellular proteins by the ubiquitin-proteasome system (UPS). To a large extent, the control points in this process include a family of E3 ubiquitin ligases called cullin-RING ligases (CRLs). The ligase activity of these multicomponent complexes requires modification of the cullin protein situated at their core with a ubiquitin-like protein called NEDD8. Neddylation results in conformational rearrangements within the CRL, which are necessary for ubiquitin transfer to a substrate. The NEDD8 pathway thus has a critical role in mediating the ubiquitination of numerous CRL substrate proteins involved in cell cycle progression and survival including the DNA replication licensing factor Cdt-1, the NF-κB transcription factor inhibitor pIκBα, and the cell cycle regulators cyclin E and p27. The initial step required for attachment of NEDD8 to a cullin is catalyzed by the E1, NEDD8-activating enzyme (NAE). The first-in-class inhibitor of NAE, MLN4924, has been shown to block the activity of NAE and prevent the subsequent neddylation of cullins. Preclinical studies have demonstrated antitumor activity in various solid tumors and hematological malignancies, and preliminary clinical data have shown the anticipated pharmacodynamic effects in humans. Here, we review the NEDD8 pathway, its importance in cancer, and the therapeutic potential of NAE inhibition.

Keywords: DNA rereplication; NEDD8; cullin-RING ligase; ubiquitin.

Figure 1.

Protein degradation by the UPS is a highly regulated process. Proteasomes degrade proteins that are tagged with polyubiquitin chains. The formation of the polyubiquitin chain is catalyzed by a series of enzymes. In the first step, the E1-activating enzyme, UAE, activates ubiquitin and transfers it via a transthiolation reaction to one of many E2 enzymes. The ubiquitin-charged E2 then collaborates with specific E3 ligases to catalyze the formation of the polyubiquitin chain on the substrate protein recruited by that ligase. There are 3 broad subclasses of E3 ligases: HECT domain, RING finger domain, and U-box E3s (for review, see Nalepa et al.).

Figure 2.

CRLs represent the largest subfamily of RING finger domain E3 ligases. These multisubunit ligases consist of a core cullin protein tightly associated with the RING finger domain–containing protein RBX1/2. Various interchangeable adaptor and receptor components facilitate diverse substrate recognition. The cullin protein must be modified by NEDD8 to activate holoenzyme ubiquitin ligase activity. Neddylation, similar to ubiquitination, is initiated by a specific E1 for NEDD8, NAE. NAE utilizes ATP to form NEDD8 adenylate; the NEDD8 is subsequently transferred to a specific cysteine within NAE, forming a NAE-NEDD8 thioester before transthiolation to the active site cysteine of UBC12 or UBE2F, E2s specific for the NEDD8 pathway. Conjugation of NEDD8 to cullin activates the ubiquitin ligase activity of the CRL, and removal of NEDD8 by the COP9 signalosome facilitates dissociation of CRL components. Unneddylated cullin-RING complexes bind to CAND1 until recruited to form a new CRL. The NAE-specific inhibitor MLN4924 forms a covalent adduct with NEDD8 in a reaction catalyzed by NAE, locking the enzyme in an inactive state.

Figure 3.

MLN4924, a first-in-class inhibitor of NAE.