In immunoblot analyses of IP samples, probing with antibodies against Ubc12 and NEDD8 detected a band of similar molecular weight corresponding to di-neddylated Ubc12 in both SENP8 knockout cell lines, but not the parental cells (Figure 3figure supplement 1A). enzyme, as a substrate for auto-neddylation. Furthermore, we characterized SENP8/DEN1 as the protease that counteracts Ubc12 auto-neddylation, and observed aberrant neddylation of Ubc12 and other NEDD8 conjugation pathway components in SENP8-deficient cells. Importantly, loss of SENP8 function contributes to accumulation of CRL substrates and defective cell cycle progression. Thus, our study highlights the importance of SENP8 in maintaining 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 identification of non-cullin neddylation targets is the relatively low abundance and transient nature of NEDD8 modification events in cells, limiting neddylation detection at an endogenous level by proteomic approaches. Like other protein post-translational modifications (PTMs), neddylation is 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 cells (Lyapina et al., 2001; Cope, 2002). CSN is specific for neddylated cullins (Lingaraju Sema3d et al., 2014; Cavadini et al., 2016); however, deneddylase(s) controlling non-cullin neddylated substrates have been poorly defined (Figure 1A). SRI-011381 hydrochloride 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 plays a redundant role in proteolytic processing 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 distinct 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 consequences of long-term SRI-011381 hydrochloride SENP8 depletion, have not been thoroughly profiled in mammalian cells. Open in a separate window Figure 1. Expression of a deconjugation-resistant NEDD8 mutant (L73P) stabilizes neddylation of cullins and other non-cullin substrates.(A) Schematics of the regulation of NEDD8 substrates by modification with either WT- (left panel) or L73P-Nedd8 (right panel), and deneddylation by NEDD8-specific proteases. CSN is the deneddylase responsible for SRI-011381 hydrochloride deconjugating NEDD8 from cullin substrates, but proteases SRI-011381 hydrochloride regulating deneddylation of non-cullin substrates are largely 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 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-modified CUL1 and CUL2. (E) HeLa-FlpIn-N8 cells were treated with or without Dox as in D to induce FLAG-NEDD8-WT or FLAG-NEDD8-L73P, SRI-011381 hydrochloride and subsequently incubated with or without the of the CRL inhibitor MLN4924 (5 M for 4 hr) before harvesting. Whole-cell extracts were analyzed for FLAG-NEDD8-conjugated CUL1 and CUL2. (F) (left panel) Workflow for expression and purification of FLAG-NEDD8-WT and FLAG-NEDD8-L73P for MS analysis. (right panel) Percentages of total spectral counts detected in FLAG-IPs from cells expressing either FLAG-NEDD8-WT (orange bars) or FLAG-NEDD8-L73P (purple bars). The numbers in the columns indicate actual spectral counts. The IPs were performed on lysates from the same number 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.