The COP9 signalosome-mediated deneddylation is stimulated by caspases during apoptosis

In concert with the ubiquitin (Ub) proteasome system (UPS) the COP9 signalosome (CSN) controls the stability of cellular regulators. The CSN interacts with cullin-RING Ub ligases (CRLs) consisting of a specific cullin, a RING protein as Rbx1 and substrate recognition proteins. The Ub-like protein Nedd8 is covalently linked to cullins and removed by the CSN-mediated deneddylation. Cycles of neddylation and deneddylation regulate CRLs. Apoptotic stimuli cause caspase-dependent modifications of the UPS. However, little is known about the CSN during apoptosis. We demonstrate in vitro and in vivo that CSN6 is cleaved most effectively by caspase 3 at D₂₃ after 2–3 h of apoptosis induced by anti-Fas-Ab or etoposide. CSN6 processing occurs in CSN–CRL complexes and is followed by the cleavage of Rbx1, the direct interaction partner of CSN6. Caspase-dependent cutting of Rbx1 is accompanied by decrease of neddylated proteins in Jurkat T cells. Another functional consequence of CSN6 cleavage is the enhancement of CSN-mediated deneddylating activity causing deneddylation of cullin 1 in cells. The CSN-associated deubiquitinating as well as kinase activity remained unchanged in presence of active caspase 3. The cleavage of Rbx1 and increased deneddylation of cullins inactivate CRLs and presumably stabilize pro-apoptotic factors for final apoptotic steps. Bettina K. J. Hetfeld and Andreas Peth contributed equally.     

COP9 signalosome function in the DDR

The COP9 signalosome (CSN) is a platform for protein communication in eukaryotic cells. It has an intrinsic metalloprotease that removes the ubiquitin (Ub)-like protein Nedd8 from cullins. CSN-mediated deneddylation regulates culling-RING Ub ligases (CRLs) and controls ubiquitination of proteins involved in DNA damage response (DDR). CSN forms complexes with CRLs containing cullin 4 (CRL4s) which act on chromatin playing crucial roles in DNA repair, checkpoint control and chromatin remodeling. Furthermore, via associated kinases the CSN controls the stability of DDR effectors such as p53 and p27 and thereby the DDR outcome. DDR is a protection against cancer and deregulation of CSN function causes cancer making it an attractive pharmacological target. Here we review current knowledge on CSN function in DDR.     

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

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 k(cat) of ～1 s(-1) and K(m)for neddylated Cul1-Rbx1 of ～200 nm, yielding a k(cat)/K(m) 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 SCF(Fbw7) and SCF(Skp2-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.     

Characterization of the role of COP9 signalosome in regulating cullin E3 ubiquitin ligase activity

Cullin RING ligases (CRLs) are the largest family of cellular E3 ubiquitin ligases and mediate polyubiquitination of a number of cellular substrates. CRLs are activated via the covalent modification of the cullin protein with the ubiquitin-like protein Nedd8. This results in a conformational change in the cullin carboxy terminus that facilitates the ubiquitin transfer onto the substrate. COP9 signalosome (CSN)-mediated cullin deneddylation is essential for CRL activity in vivo. However, the mechanism through which CSN promotes CRL activity in vivo is currently unclear. In this paper, we provide evidence that cullin deneddylation is not intrinsically coupled to substrate polyubiquitination as part of the CRL activation cycle. Furthermore, inhibiting substrate-receptor autoubiquitination is unlikely to account for the major mechanism through which CSN regulates CRL activity. CSN also did not affect recruitment of the substrate-receptor SPOP to Cul3, suggesting it may not function to facilitate the exchange of Cul3 substrate receptors. Our results indicate that CSN binds preferentially to CRLs in the neddylation-induced, active conformation. Binding of the CSN complex to active CRLs may recruit CSN-associated proteins important for CRL regulation. The deneddylating activity of CSN would subsequently promote its own dissociation to allow progression through the CRL activation cycle.     

Multimodal activation of the ubiquitin ligase SCF by Nedd8 conjugation

Conjugation of ubiquitin-like protein Nedd8 to cullins (neddylation) is essential for the function of cullin-RING ubiquitin ligases (CRLs). Here, we show that neddylation stimulates CRL activity by multiple mechanisms. For the initiator ubiquitin, the major effect is to bridge the approximately 50 A gap between naked substrate and E2 approximately Ub bound to SCF. The gap between the acceptor lysine of ubiquitinated substrate and E2 approximately Ub is much smaller, and, consequentially, the impact of neddylation on transfer of subsequent ubiquitins by Cdc34 arises primarily from improved E2 recruitment and enhanced amide bond formation in the E2 active site. The combined effects of neddylation greatly enhance the probability that a substrate molecule acquires >or= 4 ubiquitins in a single encounter with a CRL. The surprisingly diverse effects of Nedd8 conjugation underscore the complexity of CRL regulation and suggest that modification of other ubiquitin ligases with ubiquitin or ubiquitin-like proteins may likewise have major functional consequences.     

Protection of cullin-RING E3 ligases by CSN-UBP12

Neddylation, a process that conjugates the ubiquitin-like polypeptide NEDD8 to cullin proteins, activates cullin-RING ubiquitin ligases (CRLs). Deneddylation, in which the COP9 signalosome (CSN) removes NEDD8 from cullins, inactivates CRLs. However, genetic studies of CSN function conclude that deneddylation also promotes CRL activity. It has been proposed that a cyclic transition through neddylation and deneddylation is required for the regulation of CRL activity in vivo. Recent discoveries suggest that an additional level of complexity exists, whereby CRL components are targets for degradation, mediated either by autocatalytic ubiquitination or by unknown mechanisms. Deneddylation by CSN and deubiquitylation by CSN-associated ubiquitin-specific protease 12 protect CRL components from cellular depletion, thus maintaining the physiological CRL activities.     

COMMD1 (copper metabolism MURR1 domain-containing protein 1) regulates Cullin RING ligases by preventing CAND1 (Cullin-associated Nedd8-dissociated protein 1) binding

Cullin RING ligases (CRLs), the most prolific class of ubiquitin ligase enzymes, are multimeric complexes that regulate a wide range of cellular processes. CRL activity is regulated by CAND1 (Cullin-associated Nedd8-dissociated protein 1), an inhibitor that promotes the dissociation of substrate receptor components from the CRL. We demonstrate here that COMMD1 (copper metabolism MURR1 domain-containing 1), a factor previously found to promote ubiquitination of various substrates, regulates CRL activation by antagonizing CAND1 binding. We show that COMMD1 interacts with multiple Cullins, that the COMMD1-Cul2 complex cannot bind CAND1, and that, conversely, COMMD1 can actively displace CAND1 from CRLs. These findings highlight a novel mechanism of CRL activation and suggest that CRL regulation may underlie the pleiotropic activities of COMMD1.     

CAND1 enhances deneddylation of CUL1 by COP9 signalosome

Cullin-RING ligases (CRLs) regulate diverse cellular functions such as cell cycle progression and cytokine signaling by ubiquitinating key regulatory proteins. The activity of CRLs is controlled by Nedd8 modification of the cullin subunits. Recent reports have suggested that CAND1, which specifically binds to unmodified CUL1 but not to neddylated one, is required for the in vivo function of SCFs, the CUL1-containing CRLs. We show here that CAND1 and COP9 signalosome (CSN), the major deneddylase of cullins, bind to unneddylated CUL1 in a mutually exclusive way. The suppression of CAND1 expression by small inhibitory RNA enhanced the interaction between CUL1 and CSN, suggesting that CAND1 inhibited the binding of CSN to CUL1. We found that the binding of CSN to CUL1 required the four helix bundle in CUL1 C-terminal domain, which was wrapped around by CAND1 in the CAND1-CUL1-Rbx1 complex. CAND1 greatly facilitated CSN-mediated deneddylation of CUL1 in vitro, which was dependent on its binding to CUL1. Our data suggest that enhancement of CSN-mediated deneddylation by CAND1 may contribute to its function as a positive regulator of SCFs in vivo.     

Neddylation and deneddylation regulate Cul1 and Cul3 protein accumulation

Cullin family proteins organize ubiquitin ligase (E3) complexes to target numerous cellular proteins for proteasomal degradation. Neddylation, the process that conjugates the ubiquitin-like polypeptide Nedd8 to the conserved lysines of cullins, is essential for in vivo cullin-organized E3 activities. Deneddylation, which removes the Nedd8 moiety, requires the isopeptidase activity of the COP9 signalosome (CSN). Here we show that in cells deficient for CSN activity, cullin1 (Cul1) and cullin3 (Cul3) proteins are unstable, and that to preserve their normal cellular levels, CSN isopeptidase activity is required. We further show that neddylated Cul1 and Cul3 are unstable - as suggested by the evidence that Nedd8 promotes the instability of both cullins - and that the unneddylatable forms of cullins are stable. The protein stability of Nedd8 is also subject to CSN regulation and this regulation depends on its cullin-conjugating ability, suggesting that Nedd8-conjugated cullins are degraded en bloc. We propose that while Nedd8 promotes cullin activation through neddylation, neddylation also renders cullins unstable. Thus, CSN deneddylation recycles the unstable, neddylated cullins into stable, unneddylated ones, and promotes cullin-organized E3 activity in vivo.     

VHL protein-interacting deubiquitinating enzyme 2 deubiquitinates and stabilizes HIF-1alpha

Hypoxia-inducible factor (HIF)-1alpha is a short-lived protein and is ubiquitinated and degraded through the von Hippel-Lindau protein (pVHL)-E3 ubiquitin ligase pathway at normoxia. Deubiquitination, by reversing ubiquitination, has been recognized as an important regulatory step in ubiquitination-related processes. Here, we show that pVHL-interacting deubiquitinating enzyme 2, VDU2, but not VDU1, interacts with HIF-1alpha. VDU2 can specifically deubiquitinate and stabilize HIF-1alpha and, therefore, increase expression of HIF-1alpha targeted genes, such as vascular endothelial growth factor (VEGF). These findings suggest that ubiquitination of HIF-1alpha is a dynamic process and that ubiquitinated HIF-1alpha might be rescued from degradation by VDU2 through deubiquitination. Although pVHL functions as a master control for HIF-1alpha stabilization, as pVHL-E3 ligase mediates the ubiquitination of both HIF-1alpha and VDU2, the balance between the pVHL-mediated ubiquitination and VDU2-mediated deubiquitination of HIF-1alpha provides another level of control for HIF-1alpha stabilization.     

Inhibition of cullin RING ligases by cycle inhibiting factor: evidence for interference with Nedd8-induced conformational control

Cycle inhibiting factor (Cif) is produced by pathogenic intracellular bacteria and injected into the host cells via a type III secretion system. Cif is known to interfere with the eukaryotic cell cycle by inhibiting the function of cullin RING E3 ubiquitin ligases (CRLs). Cullin proteins form the scaffold protein of CRLs and are modified with the ubiquitin-like protein Nedd8, which exerts important conformational control required for CRL activity. Cif has recently been shown to catalyze the deamidation of Gln40 in Nedd8 to Glu. Here, we addressed how Nedd8 deamidation inhibits CRL activity. Our results indicate that Burkholderia pseudomallei Cif (also known as CHBP) inhibits the deconjugation of Nedd8 in vivo by inhibiting binding of the deneddylating COP9 signalosome (CSN) complex. We provide evidence that the reduced binding of CSN and the inhibition of CRL activity by Cif are due to interference with Nedd8-induced conformational control, which is dependent on the interaction between the Nedd8 hydrophobic patch and the cullin winged-helix B subdomain. Of note, mutation of Gln40 to Glu in ubiquitin, an additional target of Cif, inhibits the interaction between the hydrophobic surface of ubiquitin and the ubiquitin-binding protein p62/SQSTM1, showing conceptually that Cif activity can impair ubiquitin/ubiquitin-like protein non-covalent interactions. Our results also suggest that Cif may exert additional cellular effects by interfering with the association between ubiquitin and ubiquitin-binding proteins.     

Structural and Biochemical Characterization of the Cop9 Signalosome CSN5/CSN6 Heterodimer

The Cop9 signalosome complex (CSN) regulates the functional cycle of the major E3 ubiquitin ligase family, the cullin RING E3 ubiquitin ligases (CRLs). Activated CRLs are covalently modified by the ubiquitin-like protein Nedd8 (neural precursor cell expressed developmentally down-regulated protein 8). CSN serves an essential role in myriad cellular processes by reversing this modification through the isopeptidase activity of its CSN5 subunit. CSN5 alone is inactive due to an auto-inhibited conformation of its catalytic domain. Here we report the molecular basis of CSN5 catalytic domain activation and unravel a molecular hierarchy in CSN deneddylation activity. The association of CSN5 and CSN6 MPN (for Mpr1/Pad1 N-terminal) domains activates its isopeptidase activity. The CSN5/CSN6 module, however, is inefficient in CRL deneddylation, indicating a requirement of further elements in this reaction such as other CSN subunits. A hybrid molecular model of CSN5/CSN6 provides a structural framework to explain these functional observations. Docking this model into a published CSN electron density map and using distance constraints obtained from cross-linking coupled to mass-spectrometry, we find that the C-termini of the CSN subunits could form a helical bundle in the centre of the structure. They likely play a key scaffolding role in the spatial organization of CSN and precise positioning of the dimeric MPN catalytic core.     

Identification of a deubiquitinating enzyme subfamily as substrates of the von Hippel-Lindau tumor suppressor

The VHL protein (pVHL) is a component of an E3 ubiquitin ligase complex which is involved in the ubiquitination and degradation of the alpha subunits of HIF (hypoxia-inducible factor) in the presence of oxygen. However, it is of considerable interest to identify pVHL substrates other than HIF. In our previous studies, we have shown that VDU1 (pVHL-interacting deubiquitinating enzyme-1) can be ubiquitinated for rapid degradation in a pVHL-dependent manner. In this report we show that another uncharacterized deubiquitinating enzyme, named VDU2 (pVHL-interacting deubiquitinating enzyme-2), is a substrate of pVHL. Based on human and mouse cDNA sequences, VDU1 and VDU2 are identical in approximately 59% of the amino acids with strong homology in the N-terminus and C-terminus and a weaker similarity in the middle region. VDU2 contains the signature motifs of the ubiquitin-specific processing protease family and possesses deubiquitinating activity. Like VDU1, VDU2 interacts with pVHL beta-domain and these two proteins can compete with each other to bind to pVHL. Finally, we demonstrate that VDU2 can also be ubiquitinated and degraded in a pVHL-dependent manner. Based on their amino acid sequence homology and functional interaction with pVHL, VDU1 and VDU2 define a subfamily of ubiquitin specific processing proteases. Since deubiquitination, by reversing ubiquitination, has been recognized as an important regulatory step in ubiquitination-related processes, VDU1 and VDU2 could be important substrates of pVHL E3 ligase complex.     

E2-EPF UCP targets pVHL for degradation and associates with tumor growth and metastasis

The von Hippel-Lindau tumor suppressor, pVHL, forms part of an E3 ubiquitin ligase complex that targets specific substrates for degradation, including hypoxia-inducible factor-1alpha (HIF-1alpha), which is involved in tumor progression and angiogenesis. It remains unclear, however, how pVHL is destabilized. Here we show that E2-EPF ubiquitin carrier protein (UCP) associates with and targets pVHL for ubiquitin-mediated proteolysis in cells, thereby stabilizing HIF-1alpha. UCP is detected coincidently with HIF-1alpha in human primary liver, colon and breast tumors, and metastatic cholangiocarcinoma and colon cancer cells. UCP level correlates inversely with pVHL level in most tumor cell lines. In vitro and in vivo, forced expression of UCP boosts tumor-cell proliferation, invasion and metastasis through effects on the pVHL-HIF pathway. Our results suggest that UCP helps stabilize HIF-1alpha and may be a new molecular target for therapeutic intervention in human cancers.     

Quantitative proteomic analysis of cellular protein modulation upon inhibition of the NEDD8-activating enzyme by MLN4924

Cullin-RING ubiquitin ligases (CRLs) are responsible for the ubiquitination of many cellular proteins, thereby targeting them for proteasomal degradation. In most cases the substrates of the CRLs have not been identified, although many of those that are known have cancer relevance. MLN4924, an investigational small molecule that is a potent and selective inhibitor of the Nedd8-activating enzyme (NAE), is currently being explored in Phase I clinical trials. Inhibition of Nedd8-activating enzyme by MLN4924 prevents the conjugation of cullin proteins with NEDD8, resulting in inactivation of the entire family of CRLs. We have performed stable isotope labeling with amino acids in cell culture analysis of A375 melanoma cells treated with MLN4924 to identify new CRL substrates, confidently identifying and quantitating 5122-6012 proteins per time point. Proteins such as MLX, EID1, KLF5, ORC6L, MAGEA6, MORF4L2, MRFAP1, MORF4L1, and TAX1BP1 are rapidly stabilized by MLN4924, suggesting that they are novel CRL substrates. Proteins up-regulated at later times were also identified and siRNA against their corresponding genes were used to evaluate their influence on MLN4924-induced cell death. Thirty-eight proteins were identified as being particularly important for the cytotoxicity of MLN4924. Strikingly, these proteins had roles in cell cycle, DNA damage repair, and ubiquitin transfer. Therefore, the combination of RNAi with stable isotope labeling with amino acids in cell culture provides a paradigm for understanding the mechanism of action of novel agents affecting the ubiquitin proteasome system and a path to identifying mechanistic biomarkers.