Conjugation of the ubiquitin-like (Ubl) protein NEDD8 (neural precursor cell expressed developmentally downregulated protein 8) to target proteins, or neddylation, is a fundamental biological process that controls many key cellular functions, including cell cycle progression, the DNA damage response, and apoptosis. Like ubiquitylation, neddylation is catalyzed through an enzymatic cascade consisting of an E1-activating enzyme complex (NAE1/APPBP1-UBA3), two E2 enzymes (Ubc12/Ube2M and Ube2F) and several E3 enzymes such as Really Interesting New Gene (RING)-box protein 1 (Rbx1) and its close homologue Rbx2 (reviewed in [Soucy et al., 2010; Enchev et al., 2014; Brown and Jackson, 2015]). Additional proteins called DCUN1D1-5 (defective in cullin neddylation, domain containing 1–5) have been described in human cells as cofactors for RBX1 and RBX2, which potentiate neddylation activity (Kim et al., 2008; Kurz et al., 2008; Monda et al., 2013). The most well-studied target for NEDD8 conjugation are the cullins (CUL1,−2,–3,−4A,−4B,−5, and −7), which are the scaffold subunits of the largest family of ubiquitin E3 ligases, the cullin-RING ligases (CRLs). NEDD8 transfer from the E2 to a lysine (Lys) residue within the cullin promotes conformational changes that increase the catalytic activity of the CRL, while also blocking the binding of the CRL exchange factor CAND1 (Duda et al., 2008; Saha and Deshaies, 2008; Pierce et al., 2013). Structural changes induced by NEDD8 conjugation to cullin subunits ultimately contribute to the efficient ubiquitylation of downstream CRL substrates and their degradation by the 26S proteasome.

By inducing CRL activation and assembly in this way, NEDD8 ligation to cullins controls a high proportion of ubiquitylation events in cells (~10–20%) (Soucy et al., 2009), making this pathway an attractive target for pharmacological manipulation of protein turnover or proteostasis. An inhibitor of the NEDD8 E1 enzyme, MLN4924, has been recently developed and shown to potently and rapidly suppress neddylation in cells, thereby inhibiting CRL activity. Significantly, preclinical studies using MLN4924 have demonstrated its antitumor activity in several human tumor xenografts and hematological malignancies (Soucy et al., 2009, 2010; Brownell et al., 2010). While the antitumorigenic activity of MLN4924 has been largely attributed to CRL substrates, it is unknown whether non-cullin targets of neddylation could also play important roles in tumorigenesis.

Although cullins are the primary substrates for NEDD8 conjugation, more recently several non-cullin neddylation targets have been described. Among this list of proteins are p53 (Xirodimas et al., 2004), E2F1 (Loftus et al., 2012; Aoki et al., 2013), ribosomal protein L11 (Sundqvist et al., 2009), Smurf1 (Xie et al., 2014) and Histone H4 (Ma et al., 2013b). In addition, large-scale proteomic surveys have uncovered several other neddylation substrates both within and outside the CRL pathway (Jones et al., 2008; Xirodimas et al., 2008). However, some of these non-cullin substrates remain to be fully validated as true NEDD8 targets, as these studies relied on overexpression of epitope-tagged NEDD8 constructs as bait. Since NEDD8 conjugation can also occur through the ubiquitylation machinery under these conditions (Hjerpe et al., 2012), it can be difficult to assign bona fide 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 et al., 2014; Cavadini et al., 2016); however, deneddylase(s) controlling non-cullin neddylated substrates have been poorly defined (Figure 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 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 DEN1^null versus CSN5^null Drosophila 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 SENP8 depletion, have not been thoroughly profiled in mammalian cells.

Figure 1

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Figure 1—source data 1

NEDD8- modified peptides identified by MS analysis of FLAG-NEDD8 IP samples.

https://doi.org/10.7554/eLife.24325.003

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To capture and enrich for potentially low-level non-cullin neddylated substrates, we employed a strategy to stabilize and trap the ‘neddylated’ state of proteins by using an ectopically expressed, deconjugation-resistant mutant of NEDD8, as previously performed using uncleavable ubiquitin and SUMO modifiers (Békés et al., 2011, 2013). The deconjugation-resistant form of NEDD8 was expressed in cells using a doxycycline (Dox)-inducible expression system to minimize the potential of overexpression artifacts, followed by immuno-affinity purification and identification of neddylated substrates by mass spectrometry (MS). Strikingly, we observed enrichment of not only neddylated cullins using this technique but also several novel non-cullin targets, including the NEDD8 E2-conjugating enzyme, Ubc12. In a series of experiments, we confirmed that Ubc12 becomes auto-neddylated both in vitro and in cells, and that Ubc12 and other NEDD8 pathway components form aberrant NEDD8-conjugates in the absence of deneddylating activity. Moreover, we identified SENP8, but not CSN, as the deconjugating enzyme responsible for reversing aberrant neddylation of non-cullin substrates. Importantly, SENP8 loss-of-function studies showed that SENP8 is required for proper CRL activation and G1/S phase cell cycle progression. Finally, to identify candidate substrates with altered ubiquitylation status that could contribute to this defective G1/S regulation, we performed an unbiased proteomics screen to compare the relative abundance of ubiquitylated peptides in parental versus SENP8 knockout cells. Using this approach, we uncovered several potentially relevant substrates with reduced ubiquitylation and proteasome-mediated degradation in SENP8-deficient cells that have important roles in cell cycle regulation. Therefore, our findings suggest a novel role for SENP8 in the quality control of the NEDD8 conjugation pathway through prevention of aberrant neddylation of pathway components and demonstrate the functional consequences of perturbing this regulation on CRL activation and cell growth control.

In this study, we developed a novel strategy to identify new non-cullin neddylation targets, adding to our current understanding of cellular roles for NEDD8 conjugation. Using a deconjugation-resistant FLAG-NEDD8-L73P mutant, we were able to isolate several NEDD8-conjugated proteins in cells for identification by MS (Figure 1), several of which were also found in previous analyses using ectopically-expressed NEDD8 (Jones et al., 2008; Xirodimas et al., 2008). Our system provides an advantage over previous proteomic approaches in that expression of even relatively low levels of the FLAG-NEDD8-L73P is sufficient to enable stabilization and IP of neddylated proteins for MS analysis, thus avoiding potential artificial effects of NEDD8 overexpression (Xirodimas et al., 2008; Kim et al., 2011; Hjerpe et al., 2012). This feature aided our detection of several NEDD8-conjugates unique to this study, including MCM7, DDB1, Rab14, FEM1C, RNF187 and others (see Figure 1—source data 1 for complete list). Future work will be needed to further validate these as true neddylation substrates and functionally characterize these newly identified NEDD-conjugates in cells. Nevertheless, our MS dataset provides a valuable resource to expand upon previous studies of the NEDD8-modified proteome. Recently, several criteria for validation of bona fide neddylation substrates have been delineated (Enchev et al., 2014). In light of our current findings, we propose that identifying the physiological deneddylase could be added to these criteria to show potential dynamic and reversible neddylation of target substrates.

We identified and characterized the NEDD8 E2 conjugating enzyme Ubc12 as a novel substrate for auto-neddylation both in vitro and in cells (Figure 2). Through a combination of both MS-based analysis and Ubc12 Lys site mutagenesis, we identified the site of Ubc12 NEDD8 conjugation at its extreme N-terminus (K3). This modification is distinct from the site of Ubc12~NEDD8 thioester intermediate formation, occurring when NEDD8 is transferred from the active site of the E1 to the E2 active site Cys residue during the NEDD8 conjugation reaction (Huang et al., 2007). We have also shown that Ubc12 neddylation is sensitive to MLN4924 treatment and is dependent on the catalytic Cys residue of Ubc12. NEDD8 modification at the N-terminus of Ubc12 contributes to reduced DCN1 binding and consequently altered CRL activities, although the mechanism by which this occurs is still unclear. One possibility is that the NEDD8 modification could directly impede the initial interaction between the N-terminally acetylated Ubc12 and the hydrophobic binding pocket of DCN1 (Scott et al., 2011). Alternatively, NEDD8 modification could occur on pre-formed Dcn1-Ubc12 complex to induce a conformational change, leading to dissolution of the complex. Further in vitro analyses using purified components will be required to discriminate between these possibilities. Unexpectedly, from our SILAC-MS analysis (Figure 4), we identified additional components within the NEDD8 pathway that can form NEDD8-conjugates, including the E1 subunits UBA3 (UBE1C) and NAE1 (APPBP1), as well as the DCNLs DCUN1D1 and DCUN1D5. Future structural and biochemical work will be necessary to better understand the functional roles of these individual NEDD8 modifications in regulating the efficiency of NEDD8 conjugation to cullin and non-cullin substrates.

Furthermore, we identified the NEDD8-specific cysteine protease, SENP8, as the deneddylase that deconjugates NEDD8 from Ubc12 and other NEDD8 pathway components in human cells. Generation of CRISPR-derived knockout cell lines allowed us to profile effects of SENP8 depletion on global neddylation changes through MS-based proteomics (Figure 4) and immunoblot analysis of NEDD8 conjugates (Figure 5). These analyses revealed that SENP8 deneddylating activity is important to inhibit the aberrant neddylation of Ubc12 and other NEDD8 pathway components in cells (Figures 4 and 5). We also uncovered several previously uncharacterized non-cullin neddylation substrates in the SENP8 knockout cells (see Figure 4—source data 1 for complete list), complementing our MS analyses using L73P-NEDD8 in Figure 1F.

Several adverse phenotypic consequences occur in SENP8 knockout cells, including decreased efficiency of NEDD8 conjugation to cullins (primarily CUL1 and CUL5), decreased CRL activity, and increased stabilization of CRL substrates, summarized in Figure 8. Accompanying these deficiencies, SENP8 knockout cells also showed aberrant G1/S cell cycle progression and accelerated cell growth (Figure 6). Interestingly, these cell cycle alterations partially phenocopy previously reported defects associated with the loss of either Cul1 (Dealy et al., 1999; Wang et al., 1999; Chen and Li, 2010) or Cul5 function (Burnatowska-Hledin et al., 2001; Buchwalter et al., 2008; Bradley et al., 2010; Ma et al., 2013a; Willis et al., 2017). Through an unbiased proteomic screen in which we enriched for K-ε-GG remnant-containing peptides in MG132-treated cells, we were able to identify several candidate substrates with deregulated ubiquitylation status and stability in the SENP8 knockout cells that could potentially contribute to G1/S progression defects. One prominent example is the SCF^βTrCP substrate Cdc25A. Cdc25A acts as a major regulator of both the G1/S and G2/M cell cycle transitions by dephosphorylating and activating Cyclin E/Cdk2 and Cyclin B/Cdk1 complexes, respectively (Strausfeld et al., 1991; Blomberg and Hoffmann, 1999). Several reports have observed shortening of G1 phase and increased cyclin E- and cyclin A-associated kinase activity following overexpression of Cdc25A (Blomberg and Hoffmann, 1999; Sexl et al., 1999). Therefore, the increased total levels (Figure 7E) and impaired degradation of Cdc25A during interphase (Figure 7F) observed in SENP8 knockout cells could potentially play a significant role in promoting the unscheduled dephosphorylation of CDK complexes and accelerated cell cycle progression phenotypes.

Figure 8

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Other SCF^βTrCP substrates with differential ubiquitylation status in SENP8 knockout cells identified through our SILAC-MS screen (Figure 7B) were β-catenin and UHRF1. Aberrant ubiquitylation and degradation of β-catenin in SENP8-deficient cells may have a significant influence on cell growth by affecting transcription of downstream Wnt target genes such as c-Myc and Cyclin D1 (He et al., 1998; Clevers and Nusse, 2012). UHRF1 (ICBP90), an E2F1 target, also plays a critical role in controlling the G1/S transition by methylating and suppressing certain tumor suppressor genes (Unoki et al., 2004; Jeanblanc et al., 2005; Alhosin et al., 2011; Taylor et al., 2013; Mudbhary et al., 2014). Interestingly, UHRF1 has been shown to bind to the methylated promoter region of the p14^ARF tumor suppressor (Unoki et al., 2004), which we also found was significantly downregulated in SENP8-deficient cells (Figure 7E and F). Whether the elevated UHRF1 levels in SENP8 knockout cells account for this reduced expression of p14^ARF is unclear. However, deregulated p14^ARF levels may have a significant impact on the accelerated G1/S progression in SENP8 knockout cells, as p14^ARF plays a critical role as a tumor suppressor protein by blocking mouse double minute (MDM2)-induced degradation of p53 (Stott et al., 1998), and inhibiting E2F transcriptional activity (Eymin et al., 2001). The combined effects of p14^ARF levels, deregulated CRL substrates identified in our SILAC-MS screen (Figure 7B), and undiscovered substrates (including unknown CUL5 targets, which are largely uncharacterized) are likely to contribute to the deregulated cell cycle progression observed in SENP8-deficient cells.

It is also apparent from our studies that the decreased CRL function accompanying SENP8 loss does not affect all CRL substrates uniformly and is likely to have substrate-specific effects. For example, SENP8 depletion does not entirely phenocopy the effects of complete inhibition of CRL activity following MLN4924 treatment, as we did not see a significant increase in cells with >4C DNA content (indicative of cells undergoing multiple rounds of replication without cell division, or re-replication) or induction of a DNA damage response in SENP8 knockout cells (data not shown). Similarly, using a CSN5-specific inhibitor developed by Novartis, it was recently shown that CSN inhibition does not have pleiotropic effects on all CRL substrates as MLN4924 treatment does (Schlierf et al., 2016), but results in substrate-specific stabilization. This effect was shown to be dependent on the auto-ubiquitylation of unique substrate recognition modules of CRLs following sustained neddylation and activation of CRL complexes. It is likely that SENP8 depletion has similar substrate-specific effects related to the dynamics of CRL activation and substrate-adapter auto-ubiquitylation influenced via aberrant neddylation of Ubc12.

An outstanding question from our study is concerning the biological role for auto-neddylation of NEDD8 pathway components: Is auto-neddylation simply a consequence of prolonged SENP8 depletion, or does it also occur in physiological conditions as a feedback mechanism regulating overstimulation of the NEDD8 pathway? Several recent studies have observed a correlation between overactivation of NEDD8 pathway components, including NEDD8, UBA3, NAE1, and Ubc12, with cancer progression and poor prognosis in human lung cancers (Li et al., 2014), colorectal cancers (Xie et al., 2014), and glioblastomas (Hua et al., 2015). Thus, auto-neddylation within the NEDD8 pathway may represent a deliberate quality control mechanism to limit adverse consequences associated with elevated NEDD8 conjugation and CRL activity. Consistent with this notion, we observed in our own study that NEDD8 conjugation to Ubc12 and other components occurs at defined Lys residues, which are evolutionarily conserved across species. If auto-neddylation of NEDD8 pathway components does in fact occur upon hyper-activation of the neddylation machinery in vivo, this also raises the question of how SENP8 deneddylating activity is regulated under such circumstances. Furthermore, what defines the unique substrate preferences of SENP8 versus those of CSN, the other major deneddylating enzyme in human cells? These issues and others will require further interrogation of SENP8 regulation to resolve. Nevertheless, our study characterizes the SENP8 protease as a key regulator of the NEDD8 pathway, implicating SENP8 as a potential pharmacological target to control CRL-mediated processes in cells.

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Author details

1. Kate E Coleman

   Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, United States

   Contribution

   KEC, Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing—original draft, Writing—review and editing

   Contributed equally with

   Miklós Békés

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0003-4676-4981

2. Miklós Békés

   Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, United States

   Present address

   Nurix Inc, San Francisco, United States

   Contribution

   MB, Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing—review and editing

   Contributed equally with

   Kate E Coleman

   Competing interests

   The authors declare that no competing interests exist.

3. Jessica R Chapman

   Proteomics Laboratory, Division of Advanced Research Technologies, New York University School of Medicine, New York, United States

   Contribution

   JRC, Resources, Data curation, Formal analysis, Methodology, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

4. Sarah B Crist

   Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, United States

   Contribution

   SBC, Data curation, Investigation

   Competing interests

   The authors declare that no competing interests exist.

5. Mathew JK Jones

   Molecular Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, Unites States

   Contribution

   MJKJ, Data curation, Investigation

   Competing interests

   The authors declare that no competing interests exist.

6. Beatrix M Ueberheide

   1. Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, United States
   2. Proteomics Laboratory, Division of Advanced Research Technologies, New York University School of Medicine, New York, United States

   Contribution

   BMU, Formal analysis, Supervision, Funding acquisition, Resources

   Competing interests

   The authors declare that no competing interests exist.

7. Tony T Huang

   Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, United States

   Contribution

   TTH, Formal analysis, Supervision, Funding acquisition, Writing—review and editing

   For correspondence

   tony.huang@nyumc.org

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0001-9291-5002

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