Characterisation of the mammalian family of DCN-type NEDD8 E3 ligases

Abstract

Cullin-RING ligases (CRL) are ubiquitin E3 enzymes that bind substrates through variable substrate receptor proteins and are activated by attachment of the ubiquitin-like protein NEDD8 to the cullin subunit. DCNs are NEDD8 E3 ligases that promote neddylation. Mammalian cells express five DCN-like (DCNL) proteins but little is known about their specific functions or interaction partners. We found that DCNLs form stable stoichiometric complexes with CAND1 and cullins that can only be neddylated in the presence of a substrate adaptor. These CAND–cullin–DCNL complexes might represent ‘reserve’ CRLs that can be rapidly activated when needed. We further found that all DCNLs interact with most cullin subtypes, but that they are probably responsible for the neddylation of different subpopulations of any given cullin. This is consistent with the fact that the subcellular localization of DCNLs in tissue culture cells differs and that they show unique tissue-specific expression patterns in mice. Thus, the specificity between DCNL-type NEDD8 E3 enzymes and their cullin substrates is only apparent in well-defined physiological contexts and related to their subcellular distribution and restricted expression.

Unanchored tri‐NEDD8 inhibits PARP‐1 to protect from oxidative stress‐induced cell death

“…siRNA-mediated knockdown of DCNL1-5 was carried out as previously described (Keuss et al, 2016). Cell lines were routinely checked for mycoplasma contamination.…”

“…The EMBO Journal 38: e100024 | 2019 of UBE2F, reduced non-cullin neddylation in NEDP1 knockout cells, demonstrating that their formation is UBE2M dependent ( Fig 1H). Knockdown of the NEDD8 co-E3s DCUN1D1-5/DCNL1-5/SCCRO1-5 (Kurz et al, 2005;Keuss et al, 2016), which aid in cullin neddylation, had no effect on the neddylated species ( Fig EV1C), suggesting that the DCNLs are either redundant or that the neddylated species are formed by other mechanisms, possibly independently of an E3. Therefore, as with the in vitro reactions that generate NEDD8 chains, the NEDD8 conjugates in NEDP1 KO cells are also generated by NAE1 and UBE2M.…”

“…Cell lines were routinely checked for mycoplasma contamination. siRNA-mediated knockdown of DCNL1-5 was carried out as previously described (Keuss et al, 2016). The same protocol was followed for siRNA-mediated knockdown of UBE2M (Dharmacon Smart Pool L-004348-00), of UBE2F (Dharmacon Smart Pool L-009081-01), and of Keap1 (Dharmacon Smart Pool L-012453-00).…”

“…The same protocol was followed for siRNA-mediated knockdown of UBE2M (Dharmacon Smart Pool L-004348-00), of UBE2F (Dharmacon Smart Pool L-009081-01), and of Keap1 (Dharmacon Smart Pool L-012453-00). HEK 293 and U2OS Cas9 knockouts were generated in Trex Flp-In Flag-Cas9 cell lines as in Keuss et al (2016) and Munoz et al (2014). Whole-cell lysates were prepared with buffer A: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, 0.5% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 3 mM 1,10-phenanthroline, and 15 mM iodoacetamide (IAA), supplemented with phosStop (Roche) and cOmplete mini protease inhibitor (Sigma).…”

Squamous cell carcinoma–related oncogene (SCCRO) neddylates Cul3 protein to selectively promote midbody localization and activity of Cul3KLHL21 protein complex during abscission

“…Biochemical and in vitro analyses show that SCCRO promotes neddylation by enhancing recruitment of E2ϳNedd8 (Ubc12ϳNedd8) thioester to the complex and optimizes the orientation of proteins in the complex to allow efficient transfer of Nedd8 from E2 to the cullin substrates (8). Similar to other core components, SCCRO promotes the neddylation of all cullin family members, albeit with different efficiency (11,12). Although SCCRO enhances reaction efficacy, it is not required for neddylation in vitro.…”