The Minimal Deneddylase Core of the COP9 Signalosome Excludes the Csn6 MPN(-) Domain

The COP9 signalosome (CSN) is a eukaryotic protein complex, which regulates a wide range of biological processes mainly through modulating the cullin ubiquitin E3 ligases in the ubiquitin-proteasome pathway. The CSN possesses a highly conserved deneddylase activity that centers at the JAMM motif of the Csn5 subunit but requires other subunits in a complex assembly. The classic CSN is composed of 8 subunits (Csn1-8), yet in several Ascomycota, the complex is smaller and lacks orthologs for a few CSN subunits, but nevertheless contains a conserved Csn5. This feature makes yeast a powerful model to determine the minimal assemblage required for deneddylation activity. Here we report, that Csi1, a diverged S. cerevisiae CSN subunit, displays significant homology with the carboxyl terminal domain of the canonical Csn6, but lacks the amino terminal MPN(-) domain. Through the comparative and experimental analyses of the budding yeast and the mammalian CSNs, we demonstrate that the MPN(-) domain of the canonical mouse Csn6 is not part of the CSN deneddylase core. We also show that the carboxyl domain of Csn6 has an indispensable role in maintaining the integrity of the CSN complex. The CSN complex assembled with the carboxyl fragment of Csn6, despite its lack of an MPN(-) domain, is fully active in deneddylation of cullins. We propose that the budding yeast Csi1 is a functional equivalent of the canonical Csn6, and thus the composition of the CSN across phyla is more conserved than hitherto appreciated.     

The COP9 signalosome and its role in plant development

The COP9 signalosome (CSN) is an evolutionarily conserved multiprotein complex with a role in the regulation of cullin-RING type E3 ubiquitin ligases (CRLs). CSN exerts its function on E3 ligases by deconjugating the ubiquitin-related protein NEDD8 from the CRL cullin subunit. Thereby, CSN has an impact on multiple CRL-dependent processes. In recent years, advances have been made in understanding the structural organisation and biochemical function of CSN: Crystal structure analysis and mass spectrometry-assisted studies have come up with first models of the pair-wise and complex interactions of the 8 CSN subunits. Based on the analysis of mutant phenotypes, it can now be taken as an accepted fact that – at least in plants –the major biochemical function of CSN resides in its deneddylation activity, which is mediated by CSN subunit 5 (CSN5). Furthermore, it could be demonstrated that CSN function and deneddylation are required but not essential for CRL-mediated processes, and models for the role of neddylation and deneddylation in controlling CRL activity are emerging. Significant advances have also been made in identifying pathways that are growth restricting in the Arabidopsis csn mutants. Recently it has been shown that a G2 phase arrest, possibly due to genomic instability, restricts growth in Arabidopsis csn mutants. This review provides an update on recent advances in understanding CSN structure and function and summarises the current knowledge on its role in plant development and cell cycle progression.     

COP9 Signalosome Subunit Csn8 Is Involved in Maintaining Proper Duration of the G1 Phase

The COP9 signalosome (CSN) is a conserved protein complex known to be involved in developmental processes of eukaryotic organisms. Genetic disruption of a CSN gene causes arrest during early embryonic development in mice. The Csn8 subunit is the smallest and the least conserved subunit, being absent from the CSN complex of several fungal species. Nevertheless, Csn8 is an integral component of the CSN complex in higher eukaryotes, where it is essential for life. By characterizing the mouse embryonic fibroblasts (MEFs) that express Csn8 at a low level, we found that Csn8 plays an important role in maintaining the proper duration of the G₁ phase of the cell cycle. A decreased level of Csn8, either in Csn8 hypomorphic MEFs or following siRNA-mediated knockdown in HeLa cells, accelerated cell growth rate. Csn8 hypomorphic MEFs exhibited a shortened G₁ duration and affected expression of G₁ regulators. In contrast to Csn8, down-regulation of Csn5 impaired cell proliferation. Csn5 proteins were found both as a component of the CSN complex and outside of CSN (Csn5-f), and the amount of Csn5-f relative to CSN was increased in the Csn8 hypomorphic cells. We conclude that CSN harbors both positive and negative regulators of the cell cycle and therefore is poised to influence the fate of a cell at the crossroad of cell division, differentiation, and senescence.     

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.     

The COP9 signalosome-like complex in S. cerevisiae and links to other PCI complexes

The COP9 signalosome (CSN), the lid subcomplex of the proteasome and translational initiation factor 3 (eIF3) share structural similarities and are often referred to as the PCI family of complexes. In multicellular eukaryotes, the CSN is highly conserved as an 8-subunit complex but in Saccharomyces cerevisiae the complex is rather divergent. We further characterize the composition and properties of the CSN in budding yeast and its interactions with these related complexes. Using the generalized profile method we identified CSN candidates, four with PCI domains: Csn9, Csn10, Pci8/Csn11, and Csn12, and one with an MPN domain, Csn5/Rri1. These proteins and an additional interactor, Csi1, were tested for pairwise interactions by yeast two-hybrid and were found to form a cluster surrounding Csn12. Csn5 and Csn12 cofractionate in a complexed form with an apparent molecular weight of roughly 250kDa. However, Csn5 migrates as a monomer in Deltacsn12 supporting the pivotal role of Csn12 in stabilizing the complex. Confocal fluorescence microscopy detects GFP-tagged Csn5 preferentially in the nucleus, whereas in absence of Csn12, Csn10, Pci8/Csn11, or Csi1, Csn5 is delocalized throughout the cell, indicating that multiple subunits are required for nuclear localization of Csn5. Two CSN subunits, Csn9 and Csi1, interact with the proteasome lid subunit Rpn5. Pci8/Csn11 has previously been shown to interact with eIF3. Together, these results point to a network of interactions between these three structurally similar, yet functionally diverse, complexes.     

CIF-1, a shared subunit of the COP9/signalosome and eukaryotic initiation factor 3 complexes, regulates MEL-26 levels in the Caenorhabditis elegans embryo

The COP9/signalosome (CSN) is an evolutionarily conserved macromolecular complex that regulates the cullin-RING ligase (CRL) class of E3 ubiquitin ligases, primarily by removing the ubiquitin-like protein Nedd8 from the cullin subunit. In the Caenorhabditis elegans embryo, the CSN controls the degradation of the microtubule-severing protein MEI-1 through CUL-3 deneddylation. However, the molecular mechanisms of CSN function and its subunit composition remain to be elucidated. Here, using a proteomic approach, we have characterized the CSN and CUL-3 complexes from C. elegans embryos. We show that the CSN physically interacts with the CUL-3-based CRL and regulates its activity by counteracting the autocatalytic instability of the substrate-specific adaptor MEL-26. Importantly, we identified the uncharacterized protein K08F11.3/CIF-1 (for CSN-eukaryotic initiation factor 3 [eIF3]) as a stoichiometric and functionally important subunit of the CSN complex. CIF-1 appears to be the only ortholog of Csn7 encoded by the C. elegans genome, but it also exhibits extensive sequence similarity to eIF3m family members, which are required for the initiation of protein translation. Indeed, CIF-1 binds eIF-3.F and inactivation of cif-1 impairs translation in vivo. Taken together, our results indicate that CIF-1 is a shared subunit of the CSN and eIF3 complexes and may therefore link protein translation and degradation.     

CSN5/Jab1 controls multiple events in the mammalian cell cycle

The COP9 signalosome (CSN) complex is critical for mammalian cell proliferation and survival, but it is not known how the CSN affects the cell cycle. In this study, MEFs lacking CSN5/Jab1 were generated using a CRE-flox system. MEFs ceased to proliferate upon elimination of CSN5/Jab1. Rescue experiments indicated that the JAMM domain of CSN5/Jab1 was essential. CSN5/Jab1-elimination enhanced the neddylation of cullins 1 and 4 and altered the expression of many factors including cyclin E and p53. CSN5/Jab1-elimination inhibited progression of the cell cycle at multiple points, seemed to initiate p53-independent senescence and increased the ploidy of cells. Thus, CSN5/Jab1 controls different events of the cell cycle, preventing senescence and endocycle as well as the proper progression of the somatic cell cycle.

Structured summary

MINT-8046253: Csn1 (uniprotkb:Q99LD4) physically interacts (MI:0914) with Csn5 (uniprotkb:O35864), Csn8 (uniprotkb:Q8VBV7), Csn3 (uniprotkb:O88543), Csn7b (uniprotkb:Q8BV13) and Csn6 (uniprotkb:O88545) by anti bait coimmunoprecipitation (MI:0006)     

CSN facilitates Cullin-RING ubiquitin ligase function by counteracting autocatalytic adapter instability

The COP9 signalosome (CSN) is known to bind cullin-RING ubiquitin ligases (CRLs) and to promote their activity in vivo. The mechanism of this stimulation has remained enigmatic because CSN's intrinsic and associated enzymatic activities paradoxically inhibit CRL activity in vitro. Reconciling this paradox, we show here that Csn5-catalysed cullin (Cul) deneddylation and Ubp12-mediated deubiquitination cooperate in maintaining the stability of labile substrate adapters, thus facilitating CRL function. Various fission-yeast csn and ubp12 deletion mutants have lower levels of the Cul3p adapter Btb3p. This decrease is due to increased autocatalytic, Cul3p-dependent, ubiquitination and the subsequent degradation of Btb3p. The CSN-Ubp12p pathway also maintains the stability of the Cul1p adapter Pop1p, a mechanism required for the efficient destruction of its cognate substrate Rum1p. Emphasizing the physiological importance of this mechanism, we found that the dispensable csn5 and ubp12 genes become essential for viability when adapter recruitment to Cul1p is compromised. Our data suggest that maintenance of adapter stability is a general mechanism of CRL control by the CSN.     

COP9 signalosome subunit 8 is required for postnatal hepatocyte survival and effective proliferation

Studies using lower organisms and cultured mammalian cells have revealed that the COP9 signalosome (CSN) has important roles in multiple cellular processes. Conditional gene targeting was recently used to study CSN function in murine T-cell development and activation. Using the Cre-loxP system, here we have achieved postnatal hepatocyte-restricted knockout of the csn8 gene (HR-Csn8KO) in mice. The protein abundance of other seven CSN subunits was differentially downregulated by HR-Csn8KO and the deneddylation of all cullins examined was significantly impaired. Moreover, HR-Csn8KO-induced massive hepatocyte apoptosis and evoked extensive reparative responses in the liver, including marked intralobular proliferation of biliary lineage cells and trans-differentiation and proliferation of the oval cells. However, division of pre-existing hepatocytes was significantly diminished in HR-Csn8KO livers. These findings indicate that Csn8 is essential to the ability of mature hepatocytes to proliferate effectively in response to hepatic injury. The histopathological examinations revealed striking hepatocytomegaly in Csn8-deficient livers. The hepatocyte nuclei were dramatically enlarged and pleomorphic with hyperchromasia and prominent nucleoli, consistent with dysplasia or preneoplastic cellular alteration in HR-Csn8KO mice at 6 weeks. Pericellular and perisinusoid fibrosis with distorted architecture was also evident at 6 weeks. It is concluded that CSN8/CSN is essential to postnatal hepatocyte survival and effective proliferation.     

Neurospora COP9 signalosome integrity plays major roles for hyphal growth, conidial development, and circadian function

The COP9 signalosome (CSN) is a highly conserved multifunctional complex that has two major biochemical roles: cleaving NEDD8 from cullin proteins and maintaining the stability of CRL components. We used mutation analysis to confirm that the JAMM domain of the CSN-5 subunit is responsible for NEDD8 cleavage from cullin proteins in Neurospora crassa. Point mutations of key residues in the metal-binding motif (EX(n)HXHX(10)D) of the CSN-5 JAMM domain disrupted CSN deneddylation activity without interfering with assembly of the CSN complex or interactions between CSN and cullin proteins. Surprisingly, CSN-5 with a mutated JAMM domain partially rescued the phenotypic defects observed in a csn-5 mutant. We found that, even without its deneddylation activity, the CSN can partially maintain the stability of the SCF(FWD-1) complex and partially restore the degradation of the circadian clock protein FREQUENCY (FRQ) in vivo. Furthermore, we showed that CSN containing mutant CSN-5 efficiently prevents degradation of the substrate receptors of CRLs. Finally, we found that deletion of the CAND1 ortholog in N. crassa had little effect on the conidiation circadian rhythm. Our results suggest that CSN integrity plays major roles in hyphal growth, conidial development, and circadian function in N. crassa.     

Loss of the CONSTITUTIVE PHOTOMORPHOGENIC9 signalosome subunit 5 is sufficient to cause the cop/det/fus mutant phenotype in Arabidopsis

The COP9 signalosome (CSN) was originally identified based on the constitutively photomorphogenic/de-etiolated/fusca (cop/det/fus) mutants from Arabidopsis thaliana. CSN is evolutionary conserved, and its subunit 5 (CSN5) mediates the deconjugation of NEDD8 from the cullin subunit of E3 ubiquitin ligases (deneddylation). Here, we report on Arabidopsis mutants deficient in CSN5 function. We show that these mutants are phenotypically indistinguishable from the previously described cop/det/fus mutants of other CSN subunits. However, we also show that these mutants retain the CSN complex (lacking CSN5), and this finding is in contrast with the previously described CSN subunit mutants, which lack the CSN complex. We therefore conclude that loss of CSN5 as part of CSN is sufficient to cause the cop/det/fus mutant phenotype. Furthermore, we show that mutants defective in CSN5 as well as mutants defective in CSN are unable to deneddylate the Arabidopsis cullins AtCUL1, AtCUL3A, and AtCUL4. Because these are representative cullin subunits of the three cullin-containing E3 families present in Arabidopsis, we postulate that the cop/det/fus mutant phenotype may be the result of the defects caused by impaired CSN5-dependent deneddylation of cullin-containing E3s.     

Hepatic Deficiency of COP9 Signalosome Subunit 8 Induces Ubiquitin-Proteasome System Impairment and Bim-Mediated Apoptosis in Murine Livers

The COP9 signalosome (CSN), an evolutionally highly conserved protein complex composed of 8 unique subunits (CSN1 through CSN8) in higher eukaryotes, is purported to modulate protein degradation mediated by the ubiquitin-proteasome system (UPS) but this has not been demonstrated in a critical mitotic parenchymal organ of vertebrates. Hepatocyte-specific knockout of the Cops8 gene (HS-Csn8KO) was shown to cause massive hepatocyte apoptosis and liver malfunction but the underlying mechanism remains unclear. Here, we report that Csn8/CSN exerts profound impacts on hepatic UPS function and is critical to the stability of the pro-apoptotic protein Bim. Significant decreases in CIS (cytokine-inducible Src homology 2 domain-containing protein), a Bim receptor of a cullin2-based ubiquitin ligase, were found to co-exist with a marked increase of Bim proteins. Csn8 deficiency also significantly decreased 19S proteasome subunit Rpt5 and markedly increased high molecular weight neddylated and ubiquitinated proteins. The use of a surrogate UPS substrate further reveals severe impairment of UPS-mediated proteolysis in HS-Csn8KO livers. Inclusion body-like materials were accumulated in Csn8 deficient hepatocytes. In addition to Bim, massive hepatocyte apoptosis in HS-Csn8KO livers is also associated with elevated expression of other members of the Bcl2 family, including pro-apoptotic Bax as well as anti-apoptotic Bcl2 and Bcl-XL. Increased interaction between Bcl2 and Bim, but not between Bcl2 and Bax, was detected. Hence, it is concluded that hepatic CSN8 deficiency impairs the UPS in the liver and the resultant Bim upregulation likely plays an important role in triggering hepatocyte apoptosis via sequestering Bcl2 away from Bax.     

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.     

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.     

The COP9 signalosome inhibits p27(kip1) degradation and impedes G1-S phase progression via deneddylation of SCF Cul1

The COP9 signalosome (CSN) is a conserved protein complex with homologies to the lid subcomplex of the 26S proteasome. It promotes cleavage of the Nedd8 conjugate (deneddylation) from the cullin component of SCF ubiquitin ligases. We provide evidence that cullin neddylation and deneddylation is highly dynamic, that its equilibrium can be effectively modulated by CSN, and that neddylation allows Cul1 to form larger protein complexes. CSN2 integrates into the CSN complex via its C-terminal region and its N-terminal half region is necessary for direct interaction with Cul1. The polyclonal antibodies against CSN2 but not other CSN subunits cause accumulation of neddylated Cul1/Cul2 in HeLa cell extract, indicating that CSN2 is essential in cullin deneddylation. Further, CSN inhibits ubiquitination and degradation of the cyclin-dependent kinase inhibitor p27(kip1) in vitro. Microinjection of the CSN complex impeded the G1 cells from entering the S phase. Moreover, anti-CSN2 antibodies negate the CSN-dependent p27 stabilization and the G1/S blockage, suggesting that these functions require the deneddylation activity. We conclude that CSN inhibits SCF ubiquitin ligase activity in targeting p27 proteolysis and negatively regulates cell cycle at the G1 phase by promoting deneddylation of Cul1.     

The eta7/csn3-3 Auxin Response Mutant of Arabidopsis Defines a Novel Function for the CSN3 Subunit of the COP9 Signalosome

The COP9 signalosome (CSN) is an eight subunit protein complex conserved in all higher eukaryotes. In Arabidopsis thaliana, the CSN regulates auxin response by removing the ubiquitin-like protein NEDD8/RUB1 from the CUL1 subunit of the SCF^TIR1/AFB ubiquitin-ligase (deneddylation). Previously described null mutations in any CSN subunit result in the pleiotropic cop/det/fus phenotype and cause seedling lethality, hampering the study of CSN functions in plant development. In a genetic screen to identify enhancers of the auxin response defects conferred by the tir1-1 mutation, we identified a viable csn mutant of subunit 3 (CSN3), designated eta7/csn3-3. In addition to enhancing tir1-1 mutant phenotypes, the csn3-3 mutation alone confers several phenotypes indicative of impaired auxin signaling including auxin resistant root growth and diminished auxin responsive gene expression. Unexpectedly however, csn3-3 plants are not defective in either the CSN-mediated deneddylation of CUL1 or in SCF^TIR1-mediated degradation of Aux/IAA proteins. These findings suggest that csn3-3 is an atypical csn mutant that defines a novel CSN or CSN3-specific function. Consistent with this possibility, we observe dramatic differences in double mutant interactions between csn3-3 and other auxin signaling mutants compared to another weak csn mutant, csn1-10. Lastly, unlike other csn mutants, assembly of the CSN holocomplex is unaffected in csn3-3 plants. However, we detected a small CSN3-containing protein complex that is altered in csn3-3 plants. We hypothesize that in addition to its role in the CSN as a cullin deneddylase, CSN3 functions in a distinct protein complex that is required for proper auxin signaling.     

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.