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1.  Pathogenic Bacteria Target NEDD8-Conjugated Cullins to Hijack Host-Cell Signaling Pathways 
PLoS Pathogens  2010;6(9):e1001128.
The cycle inhibiting factors (Cif), produced by pathogenic bacteria isolated from vertebrates and invertebrates, belong to a family of molecules called cyclomodulins that interfere with the eukaryotic cell cycle. Cif blocks the cell cycle at both the G1/S and G2/M transitions by inducing the stabilization of cyclin-dependent kinase inhibitors p21waf1 and p27kip1. Using yeast two-hybrid screens, we identified the ubiquitin-like protein NEDD8 as a target of Cif. Cif co-compartmentalized with NEDD8 in the host cell nucleus and induced accumulation of NEDD8-conjugated cullins. This accumulation occurred early after cell infection and correlated with that of p21 and p27. Co-immunoprecipitation revealed that Cif interacted with cullin-RING ubiquitin ligase complexes (CRLs) through binding with the neddylated forms of cullins 1, 2, 3, 4A and 4B subunits of CRL. Using an in vitro ubiquitylation assay, we demonstrate that Cif directly inhibits the neddylated CUL1-associated ubiquitin ligase activity. Consistent with this inhibition and the interaction of Cif with several neddylated cullins, we further observed that Cif modulates the cellular half-lives of various CRL targets, which might contribute to the pathogenic potential of diverse bacteria.
Author Summary
Among the arsenal of virulence factors used by bacterial pathogens to infect and manipulate their hosts, cyclomodulins are a growing family of bacterial toxins that interfere with the eukaryotic cell-cycle. Cif is one of these cyclomodulins produced by both mammalian and invertebrate pathogenic bacteria. Cif blocks the host cell cycle by inducing the accumulation of two regulators of cell cycle progression: the cyclin-dependent kinase inhibitors p21 and p27. To decipher the mode of action of Cif, we performed yeast two-hybrid screenings. We show that Cif binds to NEDD8 and induce accumulation of neddylated cullins early after infection. Cullins are scaffold components of cullin-RING ubiquitin ligases (CRLs), which ubiquitinate proteins and target them for degradation by the 26S proteasome. We demonstrate that Cif directly inhibits the ubiquitin ligase activity of these CRLs and consequently the targeting of p21 and p27 for ubiquitin-dependent degradation. Targeting at NEDD8 represents a novel strategy for modulation of host cell functions by bacterial pathogens. By inhibiting the most prominent class of ubiquitin-ligases, Cif controls the stability of a cohort of key regulators and impinge on not only cell cycle progression but also on many cellular and biological processes such as immunity, development, transcription, and cell signaling.
doi:10.1371/journal.ppat.1001128
PMCID: PMC2947998  PMID: 20941356
2.  Inhibition of NEDD8 Conjugation Pathway by Novel Molecules: Potential Approaches to Anticancer Therapy 
Molecular oncology  2012;6(3):10.1016/j.molonc.2012.01.003.
Cancer cells can survive through the upregulation of cell cycle and the escape from apoptosis induced by numerous cellular stresses. In the normal cells, these biological cascades depend on scheduled proteolytic degradation of regulatory proteins via the ubiquitin-proteasome pathway. Therefore, interruption of regulated proteolytic pathways leads to abnormal cell-proliferation. Ubiquitin ligases called SCF complex (consisting of Skp-1, cullin, and F-box protein) or CRL (cullin-RING ubiquitin ligase) are predominant in a family of E3 ubiquitin ligases that control a final step in ubiquitination of diverse substrates. To a great extent, the ubiquitin ligase activity of the SCF complex requires the conjugation of NEDD8 to cullins, i.e. scaffold proteins. This review is anticipated to review the downregulation system of NEDD8 conjugation by several factors including a chemical compound such as MLN4924 and protein molecules (e.g. COP9 signalosome, inactive mutant of Ubc12, and NUB1/NUB1L). Since the downregulation of NEDD8 conjugation affects cell cycle progression by inhibiting the ligase activity of SCF complexes, such knowledge in the NEDD8 conjugation pathway will contribute to the more magnificent therapies that selectively suppress tumorigenesis.
doi:10.1016/j.molonc.2012.01.003
PMCID: PMC3826113  PMID: 22306028
Ubiquitination; SCF complex; NEDD8; MLN4924; Ubc12; NUB1
3.  Nedd8 Modification of Cul-1 Activates SCFβTrCP-Dependent Ubiquitination of IκBα 
Molecular and Cellular Biology  2000;20(7):2326-2333.
Regulation of NF-κB occurs through phosphorylation-dependent ubiquitination of IκBα, which is degraded by the 26S proteasome. Recent studies have shown that ubiquitination of IκBα is carried out by a ubiquitin-ligase enzyme complex called SCFβTrCP. Here we show that Nedd8 modification of the Cul-1 component of SCFβTrCP is important for function of SCFβTrCP in ubiquitination of IκBα. In cells, Nedd8-conjugated Cul-1 was complexed with two substrates of SCFβTrCP, phosphorylated IκBα and β-catenin, indicating that Nedd8–Cul-1 conjugates are part of SCFβTrCP in vivo. Although only a minute fraction of total cellular Cul-1 is modified by Nedd8, the Cul-1 associated with ectopically expressed βTrCP was highly enriched for the Nedd8-conjugated form. Moreover, optimal ubiquitination of IκBα required Nedd8 and the Nedd8-conjugating enzyme, Ubc12. The site of Nedd8 ligation to Cul-1 is essential, as SCFβTrCP containing a K720R mutant of Cul-1 only weakly supported IκBα ubiquitination compared to SCFβTrCP containing WT Cul-1, suggesting that the Nedd8 ligation of Cul-1 affects the ubiquitination activity of SCFβTrCP. These observations provide a functional link between the highly related ubiquitin and Nedd8 pathways of protein modification and show how they operate together to selectively target the signal-dependent degradation of IκBα.
PMCID: PMC85397  PMID: 10713156
4.  Characterization of MRFAP1 Turnover and Interactions Downstream of the NEDD8 Pathway* 
Molecular & Cellular Proteomics : MCP  2011;11(3):M111.014407.
The NEDD8-Cullin E3 ligase pathway plays an important role in protein homeostasis, in particular the degradation of cell cycle regulators and transcriptional control networks. To characterize NEDD8-cullin target proteins, we performed a quantitative proteomic analysis of cells treated with MLN4924, a small molecule inhibitor of the NEDD8 conjugation pathway. MRFAP1 and its interaction partner, MORF4L1, were among the most up-regulated proteins after NEDD8 inhibition in multiple human cell lines. We show that MRFAP1 has a fast turnover rate in the absence of MLN4924 and is degraded via the ubiquitin-proteasome system. The increased abundance of MRFAP1 after MLN4924 treatment results from a decreased rate of degradation. Characterization of the binding partners of both MRFAP1 and MORF4L1 revealed a complex protein-protein interaction network. MRFAP1 bound to a number of E3 ubiquitin ligases, including CUL4B, but not to components of the NuA4 complex, including MRGBP, which bound to MORF4L1. These data indicate that MRFAP1 may regulate the ability of MORF4L1 to interact with chromatin-modifying enzymes by binding to MORF4L1 in a mutually exclusive manner with MRGBP. Analysis of MRFAP1 expression in human tissues by immunostaining with a MRFAP1-specific antibody revealed that it was detectable in only a small number of tissues, in particular testis and brain. Strikingly, analysis of the seminiferous tubules of the testis showed the highest nuclear staining in the spermatogonia and much weaker staining in the spermatocytes and spermatids. MRGBP was inversely correlated with MRFAP1 expression in these cell types, consistent with an exchange of MORF4L1 interaction partners as cells progress through meiosis in the testis. These data highlight an important new arm of the NEDD8-cullin pathway.
doi:10.1074/mcp.M111.014407
PMCID: PMC3316733  PMID: 22038470
5.  Inhibition of a NEDD8 Cascade Restores Restriction of HIV by APOBEC3G 
PLoS Pathogens  2012;8(12):e1003085.
Cellular restriction factors help to defend humans against human immunodeficiency virus (HIV). HIV accessory proteins hijack at least three different Cullin-RING ubiquitin ligases, which must be activated by the small ubiquitin-like protein NEDD8, in order to counteract host cellular restriction factors. We found that conjugation of NEDD8 to Cullin-5 by the NEDD8-conjugating enzyme UBE2F is required for HIV Vif-mediated degradation of the host restriction factor APOBEC3G (A3G). Pharmacological inhibition of the NEDD8 E1 by MLN4924 or knockdown of either UBE2F or its RING-protein binding partner RBX2 bypasses the effect of Vif, restoring the restriction of HIV by A3G. NMR mapping and mutational analyses define specificity determinants of the UBE2F NEDD8 cascade. These studies demonstrate that disrupting host NEDD8 cascades presents a novel antiretroviral therapeutic approach enhancing the ability of the immune system to combat HIV.
Author Summary
The APOBEC3 family of editing enzymes catalyzes lethal hypermutation of retroviral genomes to block spread of virus in host. HIV Vif targets APOBEC3 family members for destruction by a cellular ubiquitin ligase containing CUL5. A major goal in the design of the next generation of antiretroviral therapies is to find an inhibitor of Vif so that the activity of the APOBEC3 family of antiretroviral enzymes can be restored. We define a three-enzyme cascade that is required to activate Vif by addition of the ubiquitin-like NEDD8 protein to CUL5. MLN4924, an anti-cancer compound currently in phase 1 clinical trials, inhibits the NEDD8 cascade, blocks the action of Vif, and thus has potent anti-HIV activity. Furthermore, our studies define downstream drug targets in the NEDD8 cascade more selective for inhibition of HIV Vif. We demonstrate pharmacological inhibition of HIV replication through a mechanism that restores the innate immunity provided by APOBEC3 enzymes by targeting a host pathway, providing additional candidates that could be further exploited for therapeutic development. Inhibition of this NEDD8 cascade alone, or in combination with existing antiretroviral drugs could prove to be a useful treatment for HIV.
doi:10.1371/journal.ppat.1003085
PMCID: PMC3531493  PMID: 23300442
6.  Deconjugation of Nedd8 from Cul1 Is Directly Regulated by Skp1-F-box and Substrate, and the COP9 Signalosome Inhibits Deneddylated SCF by a Noncatalytic Mechanism* 
The Journal of Biological Chemistry  2012;287(35):29679-29689.
Background: A detailed description of the kinetics of deneddylation of cullin by CSN has been lacking.
Results: Selected factors and SCF subunits are able to inhibit deneddylation to varying degrees. CSN interferes with SCF-mediated ubiquitination through a noncatalytic mechanism.
Conclusion: Deneddylation of Cul1 by CSN is regulated by F-box protein, substrate, and other factors.
Significance: Our work reported here could facilitate the development of directed therapies.
COP9 signalosome (CSN) mediates deconjugation of the ubiquitin-like protein Nedd8 from the cullin subunits of SCF and other cullin-RING ubiquitin ligases (CRLs). This process is essential to maintain the proper activity of CRLs in cells. Here, we report a detailed kinetic characterization of CSN-mediated deconjugation of Nedd8 from SCF. CSN is an efficient enzyme, with a kcat of ∼1 s−1 and Kmfor neddylated Cul1-Rbx1 of ∼200 nm, yielding a kcat/Km near the anticipated diffusion-controlled limit. Assembly with an F-box-Skp1 complex markedly inhibited deneddylation, although the magnitude varied considerably, with Fbw7-Skp1 inhibiting by ∼5-fold but Skp2-Cks1-Skp1 by only ∼15%. Deneddylation of both SCFFbw7 and SCFSkp2-Cks1 was further inhibited ∼2.5-fold by the addition of substrate. Combined, the inhibition by Fbw7-Skp1 plus its substrate cyclin E was greater than 10-fold. Unexpectedly, our results also uncover significant product inhibition by deconjugated Cul1, which results from the ability of Cul1 to bind tightly to CSN. Reciprocally, CSN inhibits the ubiquitin ligase activity of deneddylated Cul1. We propose a model in which assembled CRL complexes engaged with substrate are normally refractory to deneddylation. Upon consumption of substrate and subsequent deneddylation, CSN can remain stably bound to the CRL and hold it in low state of reduced activity.
doi:10.1074/jbc.M112.352484
PMCID: PMC3436198  PMID: 22767593
Analytical Biochemistry; Enzyme Kinetics; Protein Degradation; Protein-Protein Interactions; Ubiquitin Ligase; CSN; Cop9; Cul1; Nedd8; Deneddylation
7.  UBXN7 docks on neddylated cullin complexes using its UIM motif and causes HIF1α accumulation 
BMC Biology  2012;10:36.
Background
The proteins from the UBA-UBX family interact with ubiquitylated proteins via their UBA domain and with p97 via their UBX domain, thereby acting as substrate-binding adaptors for the p97 ATPase. In particular, human UBXN7 (also known as UBXD7) mediates p97 interaction with the transcription factor HIF1α that is actively ubiquitylated in normoxic cells by a CUL2-based E3 ligase, CRL2. Mass spectrometry analysis of UBA-UBX protein immunoprecipitates showed that they interact with a multitude of E3 ubiquitin-ligases. Conspicuously, UBXN7 was most proficient in interacting with cullin-RING ligase subunits. We therefore set out to determine whether UBXN7 interaction with cullins was direct or mediated by its ubiquitylated targets bound to the UBA domain.
Results
We show that UBXN7 interaction with cullins is independent of ubiquitin- and substrate-binding. Instead, it relies on the UIM motif in UBXN7 that directly engages the NEDD8 modification on cullins. To understand the functional consequences of UBXN7 interaction with neddylated cullins, we focused on HIF1α, a CUL2 substrate that uses UBXD7/p97 as a ubiquitin-receptor on its way to proteasome-mediated degradation. We find that UBXN7 over-expression converts CUL2 to its neddylated form and causes the accumulation of non-ubiquitylated HIF1α. Both of these effects are strictly UIM-dependent and occur only when UBXN7 contains an intact UIM motif. We also show that HIF1α carrying long ubiquitin-chains can recruit alternative ubiquitin-receptors, lacking p97's ATP-dependent segregase activity.
Conclusions
Our study shows that independently of its function as a ubiquitin-binding adaptor for p97, UBXN7 directly interacts with neddylated cullins and causes the accumulation of the CUL2 substrate HIF1α. We propose that by sequestering CUL2 in its neddylated form, UBXN7 negatively regulates the ubiquitin-ligase activity of CRL2 and this might prevent recruitment of ubiquitin-receptors other than p97 to nuclear HIF1α.
doi:10.1186/1741-7007-10-36
PMCID: PMC3349548  PMID: 22537386
cullin; NEDD8; p97; ubiquitin-dependent degradation; UBXD7
8.  Role of Individual Subunits of the Neurospora crassa CSN Complex in Regulation of Deneddylation and Stability of Cullin Proteins 
PLoS Genetics  2010;6(12):e1001232.
The Cop9 signalosome (CSN) is an evolutionarily conserved multifunctional complex that controls ubiquitin-dependent protein degradation in eukaryotes. We found seven CSN subunits in Neurospora crassa in a previous study, but only one subunit, CSN-2, was functionally characterized. In this study, we created knockout mutants for the remaining individual CSN subunits in N. crassa. By phenotypic observation, we found that loss of CSN-1, CSN-2, CSN-4, CSN-5, CSN-6, or CSN-7 resulted in severe defects in growth, conidiation, and circadian rhythm; the defect severity was gene-dependent. Unexpectedly, CSN-3 knockout mutants displayed the same phenotype as wild-type N. crassa. Consistent with these phenotypic observations, deneddylation of cullin proteins in csn-1, csn-2, csn-4, csn-5, csn-6, or csn-7 mutants was dramatically impaired, while deletion of csn-3 did not cause any alteration in the neddylation/deneddylation state of cullins. We further demonstrated that CSN-1, CSN-2, CSN-4, CSN-5, CSN-6, and CSN-7, but not CSN-3, were essential for maintaining the stability of Cul1 in SCF complexes and Cul3 and BTB proteins in Cul3-BTB E3s, while five of the CSN subunits, but not CSN-3 and CSN-5, were also required for maintaining the stability of SKP-1 in SCF complexes. All seven CSN subunits were necessary for maintaining the stability of Cul4-DDB1 complexes. In addition, CSN-3 was also required for maintaining the stability of the CSN-2 subunit and FWD-1 in the SCFFWD-1 complex. Together, these results not only provide functional insights into the different roles of individual subunits in the CSN complex, but also establish a functional framework for understanding the multiple functions of the CSN complex in biological processes.
Author Summary
Protein degradation is precisely controlled in cells. The ubiquitin-mediated protein degradation pathway is highly conserved in eukaryotes, and the activity of ubiquitin ligases is regulated by the Cop9 signalosome (CSN), a multisubunit complex that is evolutionarily conserved from yeast to humans. Determining how the CSN complex functions biologically is crucial for understanding regulation of the ubiquitin-mediated protein degradation pathway. The filamentous fungus N. crassa is commonly used to study protein degradation. Its CSN complex contains seven subunits (CSN-1 to CSN-7). In this study, we generated knockout mutants of individual CSN subunits and observed the phenotypes of each mutant. We demonstrated that six of the seven CSN subunits were essential for cleaving the ubiquitin-like protein Nedd8 from cullin proteins (which act as scaffolds for ubiquitin ligases). In contrast, loss of the CSN-3 subunit had no effect on cullin neddylation. We also found that each CSN subunit had distinct roles in maintaining the stability of key components of cullin-based ubiquitin ligases. In summary, we systematically investigated the unequal contributions of CSN subunits to deneddylation and the maintenance of cullin-based ubiquitin ligases in N. crassa. Our work establishes a framework for understanding the function of CSN subunits in other eukaryotes.
doi:10.1371/journal.pgen.1001232
PMCID: PMC2996332  PMID: 21151958
9.  HECT E3 Ubiquitin Ligase Nedd4-1 Ubiquitinates ACK and Regulates Epidermal Growth Factor (EGF)-Induced Degradation of EGF Receptor and ACK ▿  
Molecular and Cellular Biology  2010;30(6):1541-1554.
ACK (activated Cdc42-associated tyrosine kinase) (also Tnk2) is an ubiquitin-binding protein and plays an important role in ligand-induced and ubiquitination-mediated degradation of epidermal growth factor receptor (EGFR). Here we report that ACK is ubiquitinated by HECT E3 ubiquitin ligase Nedd4-1 and degraded along with EGFR in response to EGF stimulation. ACK interacts with Nedd4-1 through a conserved PPXY WW-binding motif. The WW3 domain in Nedd4-1 is critical for binding to ACK. Although ACK binds to both Nedd4-1 and Nedd4-2 (also Nedd4L), Nedd4-1 is the E3 ubiquitin ligase for ubiquitination of ACK in cells. Interestingly, deletion of the sterile alpha motif (SAM) domain at the N terminus dramatically reduced the ubiquitination of ACK by Nedd4-1, while deletion of the Uba domain dramatically enhanced the ubiquitination. Use of proteasomal and lysosomal inhibitors demonstrated that EGF-induced ACK degradation is processed by lysosomes, not proteasomes. RNA interference (RNAi) knockdown of Nedd4-1, not Nedd4-2, inhibited degradation of both EGFR and ACK, and overexpression of ACK mutants that are deficient in either binding to or ubiquitination by Nedd4-1 blocked EGF-induced degradation of EGFR. Our findings suggest an essential role of Nedd4-1 in regulation of EGFR degradation through interaction with and ubiquitination of ACK.
doi:10.1128/MCB.00013-10
PMCID: PMC2832494  PMID: 20086093
10.  SAG/RBX2 is a novel substrate of NEDD4-1 E3 ubiquitin ligase and mediates NEDD4-1 induced chemosensitization 
Oncotarget  2014;5(16):6746-6755.
Sensitive to apoptosis gene (SAG), also known as RBX2, ROC2, or RNF7, is a RING component of SCF E3 ubiquitin ligases, which regulates cellular functions through ubiquitylation and degradation of many protein substrates. Although our previous studies showed that SAG is transcriptionally induced by redox, mitogen and hypoxia via AP-1 and HIF-1, it is completely unknown whether and how SAG is ubiquitylated and degraded. Here we report that NEDD4-1, a HECT domain-containing E3 ubiquitin ligase, binds via its HECT domain directly with SAG's C-terminal RING domain and ubiquitylates SAG for proteasome-mediated degradation. Consistently, SAG protein half-life is shortened or extended by NEDD4-1 overexpression or silencing, respectively. We also found that SAG bridges NEDD4-1 via its C-terminus and CUL-5 via its N-terminus to form a NEDD4-1/SAG/CUL-5 tri-complex. Biologically, NEDD4-1 overexpression sensitizes cancer cells to etoposide-induced apoptosis by reducing SAG levels through targeted degradation. Thus, SAG is added to a growing list of NEDD4-1 substrates and mediates its biological function.
PMCID: PMC4196160  PMID: 25216516
E3 ubiquitin ligase; NEDD4-1 E3 ligase; Protein ubiquitylation and degradation; SAG E3 ligase
11.  Stealing the spotlight: CUL4-DDB1 ubiquitin ligase docks WD40-repeat proteins to destroy 
Cell Division  2007;2:5.
Recent investigation of Cullin 4 (CUL4) has ushered this class of multiprotein ubiquitin E3 ligases to center stage as critical regulators of diverse processes including cell cycle regulation, developmental patterning, DNA replication, DNA damage and repair, and epigenetic control of gene expression. CUL4 associates with DNA Damage Binding protein 1 (DDB1) to assemble an ubiquitin E3 ligase that targets protein substrates for ubiquitin-dependent proteolysis. CUL4 ligase activity is also regulated by the covalent attachment of the ubiquitin-like protein NEDD8 to CUL4, or neddylation, and the COP9 signalosome complex (CSN) that removes this important modification. Recently, multiple WD40-repeat proteins (WDR) were found to interact with DDB1 and serve as the substrate-recognition subunits of the CUL4-DDB1 ubiquitin ligase. As more than 150–300 WDR proteins exist in the human genome, these findings impact a wide array of biological processes through CUL4 ligase-mediated proteolysis. Here, we review the recent progress in understanding the mechanism of CUL4 ubiquitin E3 ligase and discuss the architecture of CUL4-assembled E3 ubiquitin ligase complexes by comparison to CUL1-based E3s (SCF). Then, we will review several examples to highlight the critical roles of CUL4 ubiquitin ligase in genome stability, cell cycle regulation, and histone lysine methylation. Together, these studies provide insights into the mechanism of this novel ubiquitin ligase in the regulation of important biological processes.
doi:10.1186/1747-1028-2-5
PMCID: PMC1805432  PMID: 17280619
12.  The Steady-State Repertoire of Human SCF Ubiquitin Ligase Complexes Does Not Require Ongoing Nedd8 Conjugation* 
Molecular & Cellular Proteomics : MCP  2010;10(5):M110.006460.
The human genome encodes 69 different F-box proteins (FBPs), each of which can potentially assemble with Skp1-Cul1-RING to serve as the substrate specificity subunit of an SCF ubiquitin ligase complex. SCF activity is switched on by conjugation of the ubiquitin-like protein Nedd8 to Cul1. Cycles of Nedd8 conjugation and deconjugation acting in conjunction with the Cul1-sequestering factor Cand1 are thought to control dynamic cycles of SCF assembly and disassembly, which would enable a dynamic equilibrium between the Cul1-RING catalytic core of SCF and the cellular repertoire of FBPs. To test this hypothesis, we determined the cellular composition of SCF complexes and evaluated the impact of Nedd8 conjugation on this steady-state. At least 42 FBPs assembled with Cul1 in HEK 293 cells, and the levels of Cul1-bound FBPs varied by over two orders of magnitude. Unexpectedly, quantitative mass spectrometry revealed that blockade of Nedd8 conjugation led to a modest increase, rather than a decrease, in the overall level of most SCF complexes. We suggest that multiple mechanisms including FBP dissociation and turnover cooperate to maintain the cellular pool of SCF ubiquitin ligases.
doi:10.1074/mcp.M110.006460
PMCID: PMC3098594  PMID: 21169563
13.  Cul4B regulates neural progenitor cell growth 
BMC Neuroscience  2012;13:112.
Background
Cullin ubiquitin ligases are activated via the covalent modification of Cullins by the small ubiquitin-like protein nedd8 in a process called neddylation. Genetic mutations of cullin-4b (cul4b) cause a prevalent type of X-linked intellectual disability (XLID) in males, but the physiological function of Cul4B in neuronal cells remains unclear.
Results
There are three major isoforms of Cul4B (1, 2, and 3) in human and rodent tissues. By examining the endogenous Cul4B isoforms in the brain, this study demonstrates that Cul4B-1 and Cul4B-2 isoforms are unneddylated and more abundant in the brain whereas the lesser species Cul4B-3 that misses the N-terminus present in the other two isoforms is neddylated. The data suggest that the N-terminus of Cul4B inhibits neddylation in the larger isoforms. Immunostaining of human NT-2 cells also shows that most Cul4B is unneddylated, especially when it is localized in the process in G0-synchronized cells. This study demonstrates that Cul4B accumulates during mitosis and downregulation of Cul4B arrests NPCs and NT-2 cells in the G2/M phase of the cell cycle. In both human and rodent brain tissues, Cul4B-positive cells accumulate β-catenin in the dentate subgranular zone and the subventricular zone. These Cul4B-positive cells also co-express the MPM-2 mitotic epitope, suggesting that Cul4B is also necessary for mitosis progression in vivo.
Conclusions
This study provides first evidence that unneddylated Cul4B isoforms exist in the brain and are necessary for mitosis progression in NPCs. The data suggest that unneddylated Cul4B isoforms specifically inhibits β-catenin degradation during mitosis. Furthermore, unneddylated Cul4B may play a role in addition to cell cycle since it is exclusively localized to the processes in starved NT-2 cells. Further analyses of the different isoforms of Cul4B will help understand the cognitive deficits in Cul4B-linked XLID and give insights into drug and biomarker discoveries.
doi:10.1186/1471-2202-13-112
PMCID: PMC3506489  PMID: 22992378
Cullin; Neurogenesis; Ubiquitination; Neddylation; Mental retardation; β-catenin
14.  A Targeted Proteomic Analysis of the Ubiquitin-Like Modifier Nedd8 and Associated Proteins 
Journal of proteome research  2008;7(3):1274-1287.
Nedd8 is a small ubiquitin-like protein that can be conjugated to substrate–proteins in a process known as neddylation. Although neddylation plays a critical regulatory role in cell proliferation and development, the spectrum of Nedd8 substrates and its interaction network remain poorly understood. To explore the neddylation pathway at the proteome level, we have affinity purified Nedd8 modified and associated proteins from HEK293 cells stably expressing GST-Nedd8 and employed LC–MS/MS for subsequent protein identification. A total of 496 GST-Nedd8 modified and associated proteins have been identified, including all of the eight cullin family members (i.e., Cul-1, -2, -3, -4A, -4B, -5, -7, and Parc) that are involved in the neddylation and ubiquitin-proteasome degradation pathway. In addition, a group of proteins involved in transcription, DNA repair and replication, cell cycle regulation and chromatin organization, and remodeling have been copurified and identified. Apart from protein identification, the neddylation sites of cullins were determined by MS/MS analysis, which agree well with previous mutagenesis studies. Furthermore, MS analyses revealed that Nedd8 K11, K22, K48, and K60 can form chains in vivo, whereas Nedd8 K22 and K48 can be neddylated in vitro. These results present the first molecular evidence for in vitro and in vivo polyneddylation, suggesting that chain formation of ubiquitin and ubiquitin-like proteins may be a general phenomenon for these modifications. Although much remains to be explored for the biological significance of the observations, this work provides critically important information regarding Nedd8 chain assembly and its interaction network. The vast amount of proteomic information obtained here can provide clues on the biological role of Nedd8 and lay the foundation for an in-depth analysis of the regulation of the Nedd8 pathway.
doi:10.1021/pr700749v
PMCID: PMC2676899  PMID: 18247557
Nedd8; neddylation; ubiquitin-like protein; cullin-containing ubiquitin ligase; ubiquitination; Nedd8 interaction network; affinity purification; polyneddylation; mass spectrometry
15.  NEDD8 links Cullin–Ring ubiquitin Ligase function to the p97 pathway 
Summary
The AAA ATPase p97 and its UBA-UBX cofactors are thought to extract ubiquitinated proteins from membranes or protein complexes as a prelude to their degradation. However, ubiquitinated targets have not yet been identified for many cofactors, leaving their biological function unclear. Previous analysis has linked the p97 pathway to Cullin-RING ubiquitin Ligases (CRLs); here we demonstrate that the p97 cofactor UBXD7 mediates the p97-CRL interaction through its conserved ubiquitin-interacting motif (UIM). UBXD7, and its yeast ortholog Ubx5, associate only with the active, NEDD8- or Rub1-modified form of cullins. Disruption of the Ubx5 UIM motif results in a loss of CRL binding and consequently impedes degradation of a Cul3 substrate. These results uncover an unexpected and conserved role for NEDD8 in linking CRL ubiquitin ligase function to the p97 pathway.
doi:10.1038/nsmb.2269
PMCID: PMC3348432  PMID: 22466964
16.  Neddylation and CAND1 Independently Stimulate SCF Ubiquitin Ligase Activity in Candida albicans 
Eukaryotic Cell  2012;11(1):42-52.
SCF (Skp1–cullin/Cdc53–F-box protein) ubiquitin ligases bind substrates via the variable F-box protein and, in conjunction with the RING domain protein Rbx1 and the ubiquitin-conjugating enzyme Ubc3/Cdc34, catalyze substrate ubiquitination. The cullin subunit can be modified covalently by conjugation of the ubiquitin-like protein Rub1/NEDD8 (neddylation) or bound noncovalently by the protein CAND1 (cullin-associated, neddylation-dissociated). Expression of the Candida albicans CAND1 gene homolog CaTIP120 in Saccharomyces cerevisiae is toxic only in the presence of CaCdc53, consistent with a specific interaction between CaTip120 and CaCdc53. To genetically analyze this system in C. albicans, we deleted the homologs of RUB1/NEDD8, TIP120/CAND1, and the deneddylase gene JAB1, and we also generated a temperature-sensitive allele of the essential CaCDC53 gene by knock-in site-directed mutagenesis. Deletion of CaRUB1 and CaTIP120 caused morphological, growth, and protein degradation phenotypes consistent with a reduction in SCF ubiquitin ligase activity. Furthermore, the double Carub1−/− Catip120−/− mutant was more defective in SCF activity than either individual deletion mutant. These results indicate that CAND1 stimulates SCF ubiquitin ligase activity and that it does so independently of neddylation. Our data do not support a role for CAND1 in the protection of either the F-box protein or cullin from degradation but are consistent with the suggested role of CAND1 in SCF complex remodeling.
doi:10.1128/EC.05250-11
PMCID: PMC3255936  PMID: 22080453
17.  Disruption of the COP9 Signalosome Csn2 Subunit in Mice Causes Deficient Cell Proliferation, Accumulation of p53 and Cyclin E, and Early Embryonic Death 
Molecular and Cellular Biology  2003;23(19):6790-6797.
Csn2 (Trip15/Cops2/Alien) encodes the second subunit of the COP9 signalosome (CSN), an eight-subunit heteromeric complex homologous to the lid subcomplex of the 26S proteasome. CSN is a regulator of SCF (Skp1-cullin-F-box protein)ubiquitin ligases, mostly through the enzymatic activity that deconjugates the ubiquitin-like protein Nedd8 from the SCF Cul1 component. In addition, CSN associates with protein kinase activities targeting p53, c-Jun, and IκB for phosphorylation. Csn2 also interacts with and regulates a subset of nuclear hormone receptors and is considered a novel corepressor. We report that targeted disruption of Csn2 in mice caused arrest of embryo development at the peri-implantation stage. Csn2−/− blastocysts failed to outgrow in culture and exhibited a cell proliferation defect in inner cell mass, accompanied by a slight decrease in Oct4. In addition, lack of Csn2 disrupted the CSN complex and resulted in a drastic increase in cyclin E, supporting a role for CSN in cooperating with the SCF-ubiquitin-proteasome system to regulate protein turnover. Furthermore, Csn2−/− embryos contained elevated levels of p53 and p21, which may contribute to premature cell cycle arrest of the mutant.
doi:10.1128/MCB.23.19.6790-6797.2003
PMCID: PMC193936  PMID: 12972599
18.  The CUL1 C-Terminal Sequence and ROC1 Are Required for Efficient Nuclear Accumulation, NEDD8 Modification, and Ubiquitin Ligase Activity of CUL1 
Molecular and Cellular Biology  2000;20(21):8185-8197.
Members of the cullin and RING finger ROC protein families form heterodimeric complexes to constitute a potentially large number of distinct E3 ubiquitin ligases. We report here that the highly conserved C-terminal sequence in CUL1 is dually required, both for nuclear localization and for modification by NEDD8. Disruption of ROC1 binding impaired nuclear accumulation of CUL1 and decreased NEDD8 modification in vivo but had no effect on NEDD8 modification of CUL1 in vitro, suggesting that ROC1 promotes CUL1 nuclear accumulation to facilitate its NEDD8 modification. Disruption of NEDD8 binding had no effect on ROC1 binding, nor did it affect nuclear localization of CUL1, suggesting that nuclear localization and NEDD8 modification of CUL1 are two separable steps, with nuclear import preceding and required for NEDD8 modification. Disrupting NEDD8 modification diminishes the IκBα ubiquitin ligase activity of CUL1. These results identify a pathway for regulation of CUL1 activity—ROC1 and the CUL1 C-terminal sequence collaboratively mediate nuclear accumulation and NEDD8 modification, facilitating assembly of active CUL1 ubiquitin ligase. This pathway may be commonly utilized for the assembly of other cullin ligases.
PMCID: PMC86428  PMID: 11027288
19.  Targeted silencing of Jab1/Csn5 in human cells downregulates SCF activity through reduction of F-box protein levels 
BMC Biochemistry  2006;7:1.
Background
SCF ubiquitin ligases target numerous proteins for ubiquitin dependent proteolysis, including p27 and cyclin E. SCF and other cullin-RING ligases (CRLs) are regulated by the ubiquitin-like protein Nedd8 that covalently modifies the cullin subunit. The removal of Nedd8 is catalyzed by the Jab1/MPN domain metalloenzyme (JAMM) motif within the Csn5 subunit of the Cop9 Signalosome.
Results
Here, we conditionally knock down Csn5 expression in HEK293 human cells using a doxycycline-inducible shRNA system. Cullin levels were not altered in CSN-deficient human cells, but the levels of multiple F-box proteins were decreased. Molecular analysis indicates that this decrease was due to increased Cul1- and proteasome-dependent turnover. Diminished F-box levels resulted in reduced SCF activity, as evidenced by accumulation of two substrates of the F-box protein Fbw7, cyclin E and c-myc, in Csn5-depleted cells.
Conclusion
We propose that deneddylation of Cul1 is required to sustain optimal activity of SCF ubiquitin ligases by repressing 'autoubiquitination' of F-box proteins within SCF complexes, thereby rescuing them from premature degradation.
doi:10.1186/1471-2091-7-1
PMCID: PMC1360668  PMID: 16401342
20.  Caspase-1 Promotes Epstein-Barr Virus Replication by Targeting the Large Tegument Protein Deneddylase to the Nucleus of Productively Infected Cells 
PLoS Pathogens  2013;9(10):e1003664.
The large tegument proteins of herpesviruses contain N-terminal cysteine proteases with potent ubiquitin and NEDD8-specific deconjugase activities, but the function of the enzymes during virus replication remains largely unknown. Using as model BPLF1, the homologue encoded by Epstein-Barr virus (EBV), we found that induction of the productive virus cycle does not affect the total level of ubiquitin-conjugation but is accompanied by a BPLF1-dependent decrease of NEDD8-adducts and accumulation of free NEDD8. Expression of BPLF1 promotes cullin degradation and the stabilization of cullin-RING ligases (CRLs) substrates in the nucleus, while cytoplasmic CRLs and their substrates are not affected. The inactivation of nuclear CRLs is reversed by the N-terminus of CAND1, which inhibits the binding of BPLF1 to cullins and prevents efficient viral DNA replication. Targeting of the deneddylase activity to the nucleus is dependent on processing of the catalytic N-terminus by caspase-1. Inhibition of caspase-1 severely impairs viral DNA synthesis and the release of infectious virus, pointing a previously unrecognized role of the cellular response to danger signals triggered by EBV reactivation in promoting virus replication.
Author Summary
Viruses rely on the host cell for replication and have evolved sophisticated strategies to manipulate and harness the cellular metabolic pathways and defense responses. A better knowledge of these viral strategies will provide new targets for antiviral therapies. The N-terminus of the large tegument proteins of herpesviruses encodes an ubiquitin and NEDD8-specific deconjugase, but the function of the enzyme during virus replication is largely unknown. Here we report that, endogenously expressed BPLF1, the homolog encoded by Epstein-Barr virus (EBV), promotes a dramatic decrease of NEDD8-conjugates and the accumulation of free NEDD8 in cells entering the productive virus cycle. BPLF1 exerts its deneddylase activity in the nucleus, which promotes the accumulation of cullin-RING ligase (CRL) substrates that are required for efficient virus replication. Targeting of the viral enzyme to the nucleus is dependent on processing of the catalytic N-terminus by caspase-1. Inhibition of caspase-1 severely impairs viral DNA synthesis and the release of infectious virus, pointing to an unexpected role of the cellular response to danger signals triggered by EBV reactivation in promoting virus replication.
doi:10.1371/journal.ppat.1003664
PMCID: PMC3795028  PMID: 24130483
21.  The Cullin3 Ubiquitin Ligase Functions as a Nedd8-bound Heterodimer 
Molecular Biology of the Cell  2007;18(3):899-909.
Cullins are members of a family of scaffold proteins that assemble multisubunit ubiquitin ligase complexes to confer substrate specificity for the ubiquitination pathway. Cullin3 (Cul3) forms a catalytically inactive BTB-Cul3-Rbx1 (BCR) ubiquitin ligase, which becomes functional upon covalent attachment of the ubiquitin homologue neural-precursor-cell-expressed and developmentally down regulated 8 (Nedd8) near the C terminus of Cul3. Current models suggest that Nedd8 activates cullin complexes by providing a recognition site for a ubiquitin-conjugating enzyme. Based on the following evidence, we propose that Nedd8 activates the BCR ubiquitin ligase by mediating the dimerization of Cul3. First, Cul3 is found as a neddylated heterodimer bound to a BTB domain-containing protein in vivo. Second, the formation of a Cul3 heterodimer is mediated by a Nedd8 molecule, which covalently attaches itself to one Cul3 molecule and binds to the winged-helix B domain at the C terminus of the second Cul3 molecule. Third, complementation experiments revealed that coexpression of two distinct nonfunctional Cul3 mutants can rescue the ubiquitin ligase function of the BCR complex. Likewise, a substrate of the BCR complex binds heterodimeric Cul3, suggesting that the Cul3 complex is active as a dimer. These findings not only provide insight into the architecture of the active BCR complex but also suggest assembly as a regulatory mechanism for activation of all cullin-based ubiquitin ligases.
doi:10.1091/mbc.E06-06-0542
PMCID: PMC1805106  PMID: 17192413
22.  Cand1 Promotes Assembly of New SCF Complexes Through Dynamic Exchange of F-box Proteins 
Cell  2013;153(1):206-215.
SUMMARY
The modular SCF ubiquitin ligases feature a large family of substrate receptors that enable recognition of diverse targets. However, how the repertoire of SCF complexes is sustained remains unclear. Real-time measurements of formation and disassembly indicate that SCFFbxw7 is extraordinarily stable but, in the Nedd8-deconjugated state, is rapidly disassembled by the cullin-binding protein Cand1. Binding and ubiquitylation assays show that Cand1 is a protein exchange factor that accelerates the rate at which Cul1–Rbx1 equilibrates with multiple F-box Protein–Skp1 modules. Depletion of Cand1 from cells impedes recruitment of new F-box proteins to pre-existing Cul1 and profoundly alters the cellular landscape of SCF complexes. We suggest that catalyzed protein exchange may be a general feature of dynamic macromolecular machines and propose a hypothesis for how substrates, Nedd8, and Cand1 collaborate to regulate the cellular repertoire of SCF complexes.
doi:10.1016/j.cell.2013.02.024
PMCID: PMC3656483  PMID: 23453757
23.  The NEDD8 modification pathway in plants 
NEDD8, in plants and yeasts also known as RELATED TO UBIQUITIN (RUB), is an evolutionarily conserved 76 amino acid protein highly related to ubiquitin. Like ubiquitin, NEDD8 can be conjugated to and deconjugated from target proteins, but unlike ubiquitin, NEDD8 has not been reported to form chains similar to the different polymeric ubiquitin chains that have a role in a diverse set of cellular processes. NEDD8-modification is best known as a post-translational modification of the cullin subunits of cullin-RING E3 ubiquitin ligases. In this context, structural analyses have revealed that neddylation induces a conformation change of the cullin that brings the ubiquitylation substrates into proximity of the interacting E2 conjugating enzyme. In turn, NEDD8 deconjugation destabilizes the cullin RING ligase complex allowing for the exchange of substrate recognition subunits via the exchange factor CAND1. In plants, components of the neddylation and deneddylation pathway were identified based on mutants with defects in auxin and light responses and the characterization of these mutants has been instrumental for the elucidation of the neddylation pathway. More recently, there has been evidence from animal and plant systems that NEDD8 conjugation may also regulate the behavior or fate of non-cullin substrates in a number of ways. Here, the current knowledge on NEDD8 processing, conjugation and deconjugation is presented, where applicable, in the context of specific signaling pathways from plants.
doi:10.3389/fpls.2014.00103
PMCID: PMC3968751  PMID: 24711811
CAND1; COP9 signalosome (CSN); cullin; E3 ubiquitin ligase; F-BOX PROTEIN (FBP); NEDD8; RELATED TO UBIQUITIN (RUB); ubiquitin
24.  The NEDD8 Conjugation Pathway and Its Relevance in Cancer Biology and Therapy 
Genes & Cancer  2010;1(7):708-716.
Cancer cells depend on signals that promote cell cycle progression and prevent programmed cell death that would otherwise result from cumulative, aberrant stress. These activities require the temporally controlled destruction of specific intracellular proteins by the ubiquitin-proteasome system (UPS). To a large extent, the control points in this process include a family of E3 ubiquitin ligases called cullin-RING ligases (CRLs). The ligase activity of these multicomponent complexes requires modification of the cullin protein situated at their core with a ubiquitin-like protein called NEDD8. Neddylation results in conformational rearrangements within the CRL, which are necessary for ubiquitin transfer to a substrate. The NEDD8 pathway thus has a critical role in mediating the ubiquitination of numerous CRL substrate proteins involved in cell cycle progression and survival including the DNA replication licensing factor Cdt-1, the NF-κB transcription factor inhibitor pIκBα, and the cell cycle regulators cyclin E and p27. The initial step required for attachment of NEDD8 to a cullin is catalyzed by the E1, NEDD8-activating enzyme (NAE). The first-in-class inhibitor of NAE, MLN4924, has been shown to block the activity of NAE and prevent the subsequent neddylation of cullins. Preclinical studies have demonstrated antitumor activity in various solid tumors and hematological malignancies, and preliminary clinical data have shown the anticipated pharmacodynamic effects in humans. Here, we review the NEDD8 pathway, its importance in cancer, and the therapeutic potential of NAE inhibition.
doi:10.1177/1947601910382898
PMCID: PMC3092238  PMID: 21779466
NEDD8; ubiquitin; cullin-RING ligase; DNA rereplication
25.  Association of SAP130/SF3b-3 with Cullin-RING ubiquitin ligase complexes and its regulation by the COP9 signalosome 
BMC Biochemistry  2008;9:1.
Background
Cullin-RING ubiquitin E3 ligases (CRLs) are regulated by modification of an ubiquitin-like protein, Nedd8 (also known as Rub1) on the cullin subunit. Neddylation is shown to facilitate E3 complex assembly; while un-neddylated cullins are bound by CAND1 that prevents recruitment of the substrates. The level of Nedd8 modification is critically dependent on the COP9 signalosome (CSN), an eight-subunit protein complex containing Nedd8 isopeptidase activity.
Results
We report isolation of SAP130 (SF3b-3) as a CSN1 interacting protein. SAP130 is homologous to DDB1, and is a component of SF3b RNA splicing complex and STAGA/TFTC transcription complexes, but its specific function within these complexes is unknown. We show that SAP130 can interact with a variety of cullin proteins. It forms tertiary complexes with fully assembled CRL E3 complexes such as SCFSkp2, Elongin B/C -Cul2- VHL and Cul4-DDB complex by binding to both N-terminal and C-terminal domain of cullins. SAP130 preferentially associates with neddylated cullins in vivo. However knock-down of CAND1 abolished this preference and increased association of SAP130 with Cul2. Furthermore, we provide evidence that CSN regulates SAP130-Cul2 interaction and SAP130-associated polyubiquitinating activity.
Conclusion
SAP130 is a cullin binding protein that is likely involved in the Nedd8 pathway. The association of SAP130 with various cullin member proteins such as Cul1, Cul2 and Cul4A is modulated by CAND1 and CSN. As an established component of transcription and RNA processing complexes, we hypothesis that SAP130 may link CRL mediated ubiquitination to gene expression.
doi:10.1186/1471-2091-9-1
PMCID: PMC2265268  PMID: 18173839

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