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

Abstract

NEDD8 is a ubiquitin‐like protein that activates cullin‐RING E3 ubiquitin ligases (CRLs). 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. We show that treatment of cells with H2O2 results in the accumulation of NEDD8 chains, likely by directly inhibiting the deneddylase NEDP1. One chain type, an unanchored NEDD8 trimer, specifically bound to the second zinc finger domain of PARP‐1 and attenuated its 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. 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. 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.

The Balance between Mono- and NEDD8-Chains Controlled by NEDP1 upon DNA Damage Is a Regulatory Module of the HSP70 ATPase Activity

“…By contrast, we observed a significant increase for the modification of NEDD8 on lysines 11 and 48 (K11/K48, SILAC ratios of 10.6 and 11.2, respectively) ( Figure 2B), indicating that deletion of ULP-3 increases NEDD8 chain formation. We did not detect the modification of components of the NEDD8 machinery including NAE and UBC12, which was previously reported in NEDP1 knockout conditions in human cells and plants (Coleman et al, 2017;Keuss et al, 2019;Mergner et al, 2017). This may be a C. elegans-specific effect or due to the fact that IR was included in our proteomics experiments.…”

“…Unanchored ubiquitin chains have been reported to control several processes, including innate immune response, viral uncoating, and aggresome formation (Banerjee et al, 2014;Hao et al, 2013;Ouyang et al, 2012;Zeng et al, 2010). The presented biochemical data on the role of unanchored NEDD8 chains on HSP70 function along with recent studies proposing substrate independent functions for NEDD8 in the control of poly(ADP-ribose) polymerase 1 (PARP-1) activity (Keuss et al, 2019) suggest that unanchored NEDD8 chains can act as regulatory signals.…”

Development of Nanobodies as first-in-class inhibitors for the NEDP1 deNEDDylating enzyme

“…The observation that similarly to NEDP1 deletion, expression of the Nbs result in increase of protein NEDDylation in cells, suggests that the processing but not the deconjugating activity of NEDP1 is redundant. This notion is further supported from the observations that NEDP1 deletion is not essential in plants, Drosophila , C. elegans and human cells 9,11,12,19,20 and that the defect in asexual development in fungi with NEDP1 (DenA) deletion is independent of the processing activity of NEDP1 10 . The reported proteases with dual specificity for NEDD8 and ubiquitin 1,2 or currently unknown NEDD8 proteases could mediate NEDD8 processing in the absence/inhibition of NEDP1.…”

“…COP9, a zinc metalloprotease, has minimal affinity towards NEDD8 but specifically deconjugates NEDD8 from cullins, the best established target for NEDD8 1,2 . NEDP1 on the other hand displays deNEDDylating activity towards non-cullin substrates 7–18 and its deletion causes a dramatic increase in global protein NEDDylation 11,12,19,20 , with the accumulation of poly-NEDD8 chains through lysines K11/K48 as a prominent effect 11,19 . Importantly, both deNEDDylating activities control the canonical NEDD8 pathway 1,2,19 .…”

Ubiquitin-Like Modifiers: Emerging Regulators of Protozoan Parasites

“…The Gly-Gly signature is a useful feature for the detection of modified peptides in mass spectrometry and is widely used for assigning ubiquitination sites [ 29 , 30 ]. As a result, NEDD8 and ISG15 modifications may be overlooked, obscured by more abundant ubiquitin ones, and require complex interventions to ascertain, such as the use of modified NEDD8 enzymatic machinery [ 34 ] and NEDDylation inhibitors [ 35 ].…”