For this purpose, we employed a previously developed strategy that involves antibody-based enrichment of peptides with the K–GG-remnant produced by tryptic digestion of proteins modified by NEDD8, ubiquitin, or the ubiquitin-like modifier ISG15 (Xu et al., 2010; Emanuele et al., 2011; Kim et al., 2011; Udeshi et al., 2013). auto-neddylation. Furthermore, we characterized SENP8/DEN1 as the protease that counteracts Ubc12 auto-neddylation, and observed aberrant neddylation of Ubc12 and additional NEDD8 conjugation pathway parts in SENP8-deficient cells. Importantly, loss of SENP8 function contributes to build up of CRL substrates and defective cell cycle progression. Thus, our study highlights the importance of SENP8 in keeping proper neddylation levels for CRL-dependent proteostasis. DOI: http://dx.doi.org/10.7554/eLife.24325.001 neddylation substrates in such experiments. Another challenge in the recognition of non-cullin neddylation focuses on is the relatively low large quantity and transient nature of NEDD8 changes events in cells, limiting neddylation detection at an endogenous level by proteomic methods. Like additional protein post-translational modifications (PTMs), neddylation is definitely reversible. COP9 signalosome complex subunit 5 (CSN5), a metallo-protease and component of the eight-subunit COP9 signalosome complex (CSN), is the major cullin deneddylase in human being cells (Lyapina et al., 2001; Cope, 2002). CSN is definitely specific for neddylated cullins (Lingaraju et al., 2014; Cavadini et al., 2016); however, deneddylase(s) controlling non-cullin neddylated substrates have been poorly defined (Number 1A). Recently, a cysteine protease called SENP8 (also known as DEN1 or NEDP1) has been characterized that functions distinctly from CSN in deneddylating primarily non-cullin substrates (Chan et al., 2008; Mergner et al., 2015) as well as hyper-neddylated cullins (Mendoza et al., 2003; Wu et al., 2003). SENP8 selectively interacts with NEDD8 and not ubiquitin (Gan-Erdene et al., 2003; Shen et al., 2005), and also takes on a redundant part in proteolytic control of the precursor form of NEDD8 in conjunction with ubiquitin C-terminal hydrolase isozyme 3 (UCHL3) (Wada et al., 1998; Wu et al., 2003). Criteria defining the unique substrate preferences of CSN and SENP8 are still not clear; however, a previous study showed unique neddylation defects in DEN1null versus CSN5null?larvae, suggesting that the two enzymes have non-overlapping functions (Chan et al., 2008). Moreover, the specific substrates for NEDD8 deconjugation by SENP8, as well as the phenotypic effects of long-term SENP8 depletion, have not been thoroughly profiled in mammalian cells. Open in a separate window Number 1. Expression of a deconjugation-resistant NEDD8 mutant (L73P) stabilizes neddylation of INCB8761 (PF-4136309) cullins and additional non-cullin substrates.(A) Schematics of the regulation of NEDD8 substrates by modification with MMP13 either WT- (remaining panel) or L73P-Nedd8 (right panel), and deneddylation by NEDD8-specific proteases. CSN is the deneddylase responsible for deconjugating NEDD8 from cullin substrates, but proteases regulating deneddylation of non-cullin substrates are mainly uncharacterized. (B) Surface representation of NEDD8 (pdb: 1NDD) and details of its C-terminal tail, showing its proteolytic cleavage site and location of the L73P mutation. (C) Recombinant CRL1/Rbx1 was in vitro neddylated INCB8761 (PF-4136309) by purified His-NEDD8-WT or His-NEDD8-L73P, in the presence of E1 and E2 enzymes and ATP. Reactions were quenched, and recombinant CSN was added at increasing concentrations to monitor the ability of each NEDD8 moiety to be deconjugated from CUL1. OPT (1,10-orthophenatroline, 1 mM) was added to samples containing the highest concentration of CSN (last lane) to completely inhibit CSN activity. (D) FLAG-NEDD8-WT or FLAG-NEDD8-L73P was induced in HeLa-FlpIn-N8 cells using 1 ug/mL doxycycline for 48 hr prior to collection. Whole-cell lysates of untreated or Dox-treated cells were incubated with anti-FLAG beads to purify FLAG-NEDD8-conjugates. Immunoblots of input and IP samples were analyzed for FLAG-NEDD8-altered CUL1 and CUL2. (E) HeLa-FlpIn-N8 cells were treated with or without Dox as with D to induce FLAG-NEDD8-WT or FLAG-NEDD8-L73P, and consequently incubated with or without the of the CRL inhibitor MLN4924 (5 M for 4 hr) before harvesting. Whole-cell components were analyzed for FLAG-NEDD8-conjugated CUL1 and CUL2. (F) INCB8761 (PF-4136309) (remaining panel) Workflow for manifestation and purification of FLAG-NEDD8-WT and FLAG-NEDD8-L73P for MS analysis. (right panel) Percentages of total spectral counts recognized in FLAG-IPs from cells expressing either FLAG-NEDD8-WT (orange bars) or FLAG-NEDD8-L73P (purple bars). The figures in the columns show actual spectral counts. The IPs were performed on lysates from your same quantity of cells. DOI: http://dx.doi.org/10.7554/eLife.24325.002 Figure 1source data 1.NEDD8- modified peptides identified by MS analysis of FLAG-NEDD8 IP samples.DOI: http://dx.doi.org/10.7554/eLife.24325.003 Click here to view.(1.4M, xlsx) To capture and INCB8761 (PF-4136309) enrich for potentially low-level non-cullin neddylated substrates, we employed a strategy to stabilize and capture.