Background
The enzymes of the NEDDylation pathway play a pivotal role in the activation of the largest class of ubiquitin E3 ligases called Cullin-RING-Ligases (CRLs). Akin to ubiquitylation three classes of enzymes are involved in the process of mammalian NEDDylation; E1 activating enzyme (APP-BP1/UBA3 heterodimer), E2 conjugating enzymes (UBE2M or UBE2F) and E3 ligases (Meyer-Schaller et al. 2009) including the Domain Containing Like Protein 1 (DCNL1) and Ring Box 1 (RBX1) heterodimer (Morimoto et al. 2003; Huang et al. 2011). The APP-BP1/UBA3 heterodimer is a member of the NEDD8 E1-activating enzyme family and cloning of the human genes coding for these proteins were first described by Chow et al. (1996) and Osaka et al. (1998). The APP-BP1 (Amyloid Precursor Protein Binding Protein 1) gene has been mapped to 16q22 by high resolution fluorescence in situ hybridization (Chow et al. 1996). APP-B1 is the regulatory subunit of the E1 whose catalytic partner is UBA3. The two proteins form a complex in vitro and a thioester linkage with NEDD8 suggesting that the APP-BP1/UBA3 complex functions as an E1-like enzyme for the activation of NEDD8 (Osaka et al. 1998). The heterodimeric structure of APP1-BP1/UBA3 has been determined through co-crystallization with NEDD8 and ATP (Walden et al. 2003). The structure consists of an E1-specific domain organised around a catalytic cysteine and a domain involved in E2 recognition which coordinates protein binding and drives the E1's reactions. This ATP-dependent activation of NEDD8 enables its transfer via a transthiolation reaction to either of the NEDD8 E2 conjugating enzymes UBE2M or UBE2F. Subsequently the NEDD8 is conjugated onto the cullin subunit of the CRL. NEDDylation of CRLs trigger a structural change within the C-terminus of the CRL E3 complex which is necessary for the efficient ubiquitylation of itssubstrates (Duda et al. 2008). Substrates of the CRLs play important roles in pathways controlling tumour cell growth. Thus a potent and selective inhibitor (MLN4924) of APP-BP1/UBA3 which disrupts CRL mediated protein turnover has been developed (Bruzzese et al. 2012). Treatment of human tumour cells in vitro with MLN4924 leads to apoptotic death by the de-regulation of S-phase DNA synthesis (Soucy et al. 2009). Senescence was identified as another mechanism of action for MLN4924 in suppressing tumour cell growth through the inhibition of SKP1-Cullin-F-box proteins (SCF) E3 ubiquitin ligases and accumulation of p21 in tumour cell lines (Jia et al. 2011). MLN4924 is now undergoing clinical trials for the treatment of various hematological malignancies.
References
Bruzzese FJ, Milhollen MA, Gavin JM, Josephine HR, Brownell JE (2012) Identification and application of NEDD8 E1 inhibitors. Methods Mol Biol 832, 577-588.
Chow N, Korenberg JR, Chen XN, Neve RL (1996) APP-BP1, a novel protein that binds to the carboxyl-terminal region of the amyloid precursor protein. J Biol Chem 271, 11339-11346.
Duda DM, Borg LA, Scott DC, Hunt HW, Hammel M, et al. (2008) Structural insights into NEDD8 activation of cullin-RING ligases: conformational control of conjugation. Cell 134, 995-1006.
Huang G, Kaufman AJ, Ramanathan Y, Singh B (2011) SCCRO (DCUN1D1) promotes nuclear translocation and assembly of the neddylation E3 complex. J Biol Chem 286, 10297-10304.
Jia L, Li H, Sun Y (2011) Induction of p21-dependent senescence by an NAE inhibitor, MLN4924, as a mechanism of growth suppression. Neoplasia 13, 561-569.
Meyer-Schaller N, Chou YC, Sumara I, Martin DD, Kurz T, et al. (2009) The human Dcn1-like protein DCNL3 promotes Cul3 neddylation at membranes. Proc Natl Acad Sci USA 106, 12365-12370.
Morimoto M, Nishida T, Nagayama Y, Yasuda H (2003) Nedd8-modification of Cul1 is promoted by Roc1 as a Nedd8-E3 ligase and regulates its stability. Biochem Biophys Res Commun 301, 392-398.
Osaka F, Kawasaki H, Aida N, Saeki M, Chiba T, et al. (1998) A new NEDD8-ligating system for cullin-4A. Genes Dev 12, 2263-2268.
Soucy TA, Smith PG, Milhollen MA, Berger AJ, Gavin JM, et al. (2009) An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature 458, 732-736.
Walden H, Podgorski MS, Schulman BA (2003) Insights into the ubiquitin transfer cascade from the structure of the activating enzyme for NEDD8. Nature 422, 330-334.