Thus, careful studies with endogenous NEDD8 are needed to confirm that it indeed has non‐cullin substrates (Enchev et al, 2015). Numerous non‐cullin substrates of NEDD8 have also been reported, but their physiological importance has been called into doubt by the realization that NEDD8 can enter the ubiquitin pathway when overexpressed (Hjerpe et al, 2012a, b). These E3 ubiquitin ligase enzymes are multi‐subunit complexes that are built around a core scaffolding cullin protein the mono‐neddylation of the cullin subunit activates CRLs (Morimoto et al, 2000 Read et al, 2000 Ohh et al, 2002 Petroski & Deshaies, 2005 Enchev et al, 2015). NEDD8 is the ubiquitin‐like modifier that is most similar to ubiquitin, and its best characterized function is the regulation of a class of enzymes that ligate ubiquitin to its substrates (Enchev et al, 2015). Although many similarities exist between ubiquitin and ubiquitin‐like modifiers, free, unanchored chains have not been similarly described for UBLs. Free, non‐conjugated chains of ubiquitin have also been described and play an important role in the immune response (Zeng et al, 2010).
Poly‐ubiquitylation with a lysine‐48‐linked ubiquitin chain, for example, results in the proteasomal degradation of the substrate (Chau et al, 1989). After the initial linkage of the first UBL to its substrate, many ubiquitin/UBLs then form lysine‐linked chains on their substrates to regulate diverse cellular processes (Yau & Rape, 2016). Ubiquitin/UBL is then transferred onto a ε‐amino group of a lysine residue of the substrate, where it forms an isopeptide bond (Goldknopf & Busch, 1977 Hershko et al, 1983 Scheffner et al, 1995). In the final step, this ubiquitin/UBL‐charged E2 is recruited by E3 ligases, which also bind to the substrate (Hershko et al, 1983).
Next, the ubiquitin/UBL is passed from E1 to the active‐site cysteine of an E2‐conjugating enzyme (Hershko et al, 1983). First, an E1 activating enzyme adenylates ubiquitin/UBL at its C‐terminus and then links ubiquitin/UBL to the enzyme's active‐site cysteine via a thioester bond (Ciechanover et al, 1981). The conjugation of ubiquitin/UBLs to a substrate entails three enzymatic steps. UBLs are small proteins of approximately 8 kDa molecular mass that become covalently linked to other proteins via an isopeptide bond, usually on lysine residues (van der Veen & Ploegh, 2012). Many cellular processes are regulated via the post‐translational modification of proteins with ubiquitin and ubiquitin‐like proteins (UBLs). Our data suggest that trimeric, acetylated NEDD8 attenuates PARP‐1 activation after oxidative stress, likely to delay the initiation of PARP‐1‐dependent cell death. Surprisingly, these NEDD8 trimers are additionally acetylated, as shown by mass spectrometry analysis, and their binding to PARP‐1 is reduced by the overexpression of histone de‐acetylases, which rescues PARP‐1 activation. In cells in which Nedp1 is deleted, large amounts of tri‐NEDD8 constitutively form, resulting in inhibition of PARP‐1 and protection from PARP‐1‐dependent cell death. One chain type, an unanchored NEDD8 trimer, specifically bound to the second zinc finger domain of PARP‐1 and attenuated its activation. We show that treatment of cells with H 2O 2 results in the accumulation of NEDD8 chains, likely by directly inhibiting the deneddylase NEDP1. Here, we identify a novel role for NEDD8 in regulating the activity of poly(ADP‐ribose) polymerase 1 (PARP‐1) in response to oxidative stress. NEDD8 is a ubiquitin‐like protein that activates cullin‐RING E3 ubiquitin ligases (CRLs).