Deneddylase Activity Research Articles

The COP9 signalosome reduces neuroinflammation and attenuates ischemic neuronal stress in organotypic brain slice culture model

The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) is a deNEDDylase controlling ubiquitination activity of cullin-RING-E3 ligases (CRLs) and thus the levels of key cellular proteins. While the CSN and its catalytic subunit CSN5 have been extensively studied in cancer, its role in inflammatory and neurological diseases is less understood. Following verification that CSN5 is expressed in mouse and human brain, here we studied the role of the CSN in neuroinflammation and ischemic neuronal damage employing models of relevant brain-resident cell types, an ex vivo organotypic brain slice culture model, and the CRL NEDDylation state-modifying drugs MLN4924 and CSN5i-3, which mimic and inhibit, respectively, CSN5 deNEDDylase activity. Untargeted mass spectrometry-based proteomics revealed that MLN4924 and CSN5i-3 substantially alter the microglial proteome, including inflammation-related proteins. Applying these drugs and mimicking microglial and endothelial inflammation as well as ischemic neuronal stress by TNF and oxygen-glucose-deprivation/reoxygenation (OGD/RO) treatment, respectively, we could link CSN5/CSN-mediated cullin deNEDDylation to reduction of microglial inflammation, attenuated cerebral endothelial inflammation, improved barrier integrity, as well as protection from ischemic stress-induced neuronal cell death. Specifically, MLN4924 reduced phagocytic activity, motility, and inflammatory cytokine expression of microglial cells, and this was linked to inhibition of inflammation-induced NF-κB and Akt signaling. Inversely, Csn5 knockdown and CSN5i-3 increased NF-κB signaling. Moreover, MLN4924 abrogated TNF-induced NF-κB signaling in cerebral microvascular endothelial cells (hCMECs) and rescued hCMEC monolayers from OGD/RO-triggered barrier leakage, while CSN5i-3 exacerbated permeability. In an ex vivo organotypic brain slice model of ischemia/reperfusion stress, MLN4924 protected from neuronal death, while CSN5i-3 impaired neuronal survival. Neuronal damage was attributable to microglial activation and inflammatory cytokines, as indicated by microglial shape tracking and TNF-blocking experiments. Our results indicate a protective role of the CSN in neuroinflammation via brain-resident cell types involved in ischemic brain disease and implicate CSN activity-mimicking deNEDDylating drugs as potential therapeutics.

Abstract 191: Cops5-mediated Nuclear Exclusion Of P27 Is Responsible For The Promotion Of Vascular Smooth Muscle Cells Proliferation By Cops8 Hypomorphism

Background: The COP9 signalosome (CSN) holocomplex is formed by 8 stoichiometric protein subunits: COPS1 through COPS8. Despite the strong evidence linking CSN to cell cycle regulation, direct study of CSN in VSMC proliferation is lacking. Our prior data show that vascular injury significantly increases Cops8 proteins in wild-type mice and induces more severe neointimal thickening and increased VSMC proliferation in Cops8 hypomorphic (Cops8-hypo) mice compared to non-hypomorphic littermates (CTL), suggesting an important role of the CSN in vascular pathology. The present study was carried out to further elucidate the mechanism underlying this phenomenon. We hypothesize that increased COPS5-mediated nuclear exclusion of p27 contributes to the promotion of VSMC proliferation by COPS8 hypomorphism. Methods and Results: We isolated and cultured VSMCs from the COPS8-hypo and CTL mice and used the treatment with platelet-derived growth factor (PDGF-BB) to mimic vascular injury. In response to PDGF-BB, COPS8-hypo VSMCs displayed increased hyperproliferation compared to CTL VSMCs. We detected markedly smaller nuclear to cytoplasmic ratio for p27 and COPS5; and native gel electrophoresis revealed increases in the cytoplasmic COPS5 mini-complex in hypomorphic VSMCs. Nuclear export inhibition with leptomycin attenuated the increases in PCNA and cytoplasmic p27 by COPS8 hypomorphism. Interestingly, CSN deneddylase inhibitor (COPS5i-3) did not have an effect on the proliferation of COPS8-hypo VSMCs but suppressed the PDGF-BB induced PCNA increases in CTL VSMCs. We then, constructed plasmids expressing full-length COPS5 harboring a mutation either in its nuclear export sequence (NES) or in its MPN domain. We found that genetically disabling the nuclear-export but not the deneddylase activity of COPS5 suppressed the hyperproliferation and restored p27 nuclear localization of hypomorphic VSMCs. These data suggest that promoting the nuclear export of p27 by COPS5 rather than the deneddylation activity of COPS5 is responsible for the greater PDGF-BB induced proliferation of COPS8-hypo VSMCs. Conclusion: COPS8 hypomorphism promotes the proliferation of VSMCs in injured vessels through the increased nuclear exclusion of p27 mediated by COPS5.

Phenotypic Differences Among Mice with Induced Cardiomyocyte‐Restricted Ablation of Cops5, Cops8, or Both

Background- Within a large subset of heart failure, the ubiquitin proteasome system has shown to be inadequate or even causative. The COP9 signalosome (CSN) holocomplex, a vital ubiquitination regulator, directly regulates Cullin-RING ligases (CRLs) via Cullin deneddylation. This holocomplex is formed by 8 unique subunits (COPS1~COPS8), where the deneddylase activity resides in COPS5. Our lab has shown in the past that cardiomyocyte-restricted knockout (CKO) of the Cops8 gene causes cardiomyocyte necroptosis, dilated cardiomyopathy, and shortened lifespan in mice. This study tests whether the protection against necroptosis by Cops8 is deneddylation-dependent or Cops8-specific, possibly alluding to subunit-specific functions independent of the holocomplex. Methods and Results- CKO of Cops8, Cops5, or both in adult mice was achieved using a tamoxifen-inducible Cre-LoxP system. Echocardiography performed 21 days after tamoxifen withdrawal showed no significant difference between MerCreMer (MCM) transgenic and Cops8-floxed/Cops5-floxed control groups. Compared with either control group, all CKO groups developed dilated cardiomyopathy, but the severity in the Cops5-CKO and the Cops8+Cops5 double CKO (dCKO) groups was greater than that in the Cops8-CKO group. Kaplan-Meier survival analyses revealed shortened lifespans for all CKO groups, compared with the MCM group. The post-tamoxifen lifespans of Cops5-CKO and dCKO mice were comparable (median 42 days vs. 42 days; p=0.66) but significantly shorter than that of the Cops8-CKO (81 days, p<0.0001). In Cops5-CKO and dCKO mice, we observed an increased number of cardiomyocytes positive for Evan's blue dye uptake and increased myocardial CD45 proteins compared to Cops8-CKO. All CKO myocardium showed increases in full-length caspase 8 but decreased cleaved caspase 8, indicating suppression of caspase 8 activation by defective CSN. Conclusions (1)Cops5-CKO and dCKO are similarly detrimental but are more so than Cops8-CKO; (2) the primary defect caused by Cops8-CKO results from impaired CSN holocomplex formation and thereby loss of Cullin deneddylation.

The Antioxidant Enzyme Methionine Sulfoxide Reductase A (MsrA) Interacts with Jab1/CSN5 and Regulates Its Function.

Methionine sulfoxide (MetO) is an oxidative posttranslational modification that primarily occurs under oxidative stress conditions, leading to alteration of protein structure and function. This modification is regulated by MetO reduction through the evolutionarily conserved methionine sulfoxide reductase (Msr) system. The Msr type A enzyme (MsrA) plays an important role as a cellular antioxidant and promotes cell survival. The ubiquitin- (Ub) like neddylation pathway, which is controlled by the c-Jun activation domain-binding protein-1 (Jab1), also affects cell survival. Jab1 negatively regulates expression of the cell cycle inhibitor cyclin-dependent kinase inhibitor 1B (P27) through binding and targeting P27 for ubiquitination and degradation. Here we report the finding that MsrA interacts with Jab1 and enhances Jab1′s deneddylase activity (removal of Nedd8). In turn, an increase is observed in the level of deneddylated Cullin-1 (Cul-1, a component of E3 Ub ligase complexes). Furthermore, the action of MsrA increases the binding affinity of Jab1 to P27, while MsrA ablation causes a dramatic increase in P27 expression. Thus, an interaction between MsrA and Jab1 is proposed to have a positive effect on the function of Jab1 and to serve as a means to regulate cellular resistance to oxidative stress and to enhance cell survival.

Structural dynamics of the human COP9 signalosome revealed by cross-linking mass spectrometry and integrative modeling

The COP9 signalosome (CSN) is an evolutionarily conserved eight-subunit (CSN1-8) protein complex that controls protein ubiquitination by deneddylating Cullin-RING E3 ligases (CRLs). The activation and function of CSN hinges on its structural dynamics, which has been challenging to decipher by conventional tools. Here, we have developed a multichemistry cross-linking mass spectrometry approach enabled by three mass spectometry-cleavable cross-linkers to generate highly reliable cross-link data. We applied this approach with integrative structure modeling to determine the interaction and structural dynamics of CSN with the recently discovered ninth subunit, CSN9, in solution. Our results determined the localization of CSN9 binding sites and revealed CSN9-dependent structural changes of CSN. Together with biochemical analysis, we propose a structural model in which CSN9 binding triggers CSN to adopt a configuration that facilitates CSN-CRL interactions, thereby augmenting CSN deneddylase activity. Our integrative structure analysis workflow can be generalized to define in-solution architectures of dynamic protein complexes that remain inaccessible to other approaches.

IKK-Mediated Regulation of the COP9 Signalosome via Phosphorylation of CSN5.

The COP9 signalosome (CSN) is an evolutionarily conserved multisubunit protein complex, which controls protein degradation through deneddylation and inactivation of cullin-RING ubiquitin E3 ligases (CRLs). Recently, the CSN complex has been linked to the NF-κB signaling pathway due to its association with the IKK complex. However, how the CSN complex is regulated in this signaling pathway remains unclear. Here, we have carried out biochemical experiments and confirmed the interaction between the CSN and IKK complexes. In addition, we have determined that overexpression of IKKα or IKKβ leads to enhanced phosphorylation of CSN5, the catalytic subunit for CSN deneddylase activity. Mutational analyses have revealed that phosphorylation at serine 201 and threonine 205 of CSN5 impairs CSN-mediated deneddylation activity in vitro. Interestingly, TNF-α treatment not only enhances the interaction between CSN and IKK but also induces an IKK-dependent phosphorylation of CSN5 at serine 201, linking CSN to TNF-α signaling through IKK. Moreover, TNF-α treatment affects the CSN interaction network globally, especially the associations of CSN with the proteasome complex, eukaryotic translation initiation factor complex, and CRL components. Collectively, our results provide new insights into IKK-mediated regulation of CSN associated with the NF-κB signaling pathway.

Role of the COP9 Signalosome (CSN) in Cardiovascular Diseases.

The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) is an evolutionarily conserved multi-protein complex, consisting of eight subunits termed CSN1-CSN8. The main biochemical function of the CSN is the control of protein degradation via the ubiquitin-proteasome-system through regulation of cullin-RING E3-ligase (CRL) activity by deNEDDylation of cullins, but the CSN also serves as a docking platform for signaling proteins. The catalytic deNEDDylase (isopeptidase) activity of the complex is executed by CSN5, but only efficiently occurs in the three-dimensional architectural context of the complex. Due to its positioning in a central cellular pathway connected to cell responses such as cell-cycle, proliferation, and signaling, the CSN has been implicated in several human diseases, with most evidence available for a role in cancer. However, emerging evidence also suggests that the CSN is involved in inflammation and cardiovascular diseases. This is both due to its role in controlling CRLs, regulating components of key inflammatory pathways such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and complex-independent interactions of subunits such as CSN5 with inflammatory proteins. In this case, we summarize and discuss studies suggesting that the CSN may have a key role in cardiovascular diseases such as atherosclerosis and heart failure. We discuss the implicated molecular mechanisms ranging from inflammatory NF-κB signaling to proteotoxicity and necrosis, covering disease-relevant cell types such as myeloid and endothelial cells or cardiomyocytes. While the CSN is considered to be disease-exacerbating in most cancer entities, the cardiovascular studies suggest potent protective activities in the vasculature and heart. The underlying mechanisms and potential therapeutic avenues will be critically discussed.

Analysis of RNA expression of normal and cancer tissues reveals high correlation of COP9 gene expression with respiratory chain complex components.

BackgroundThe COP9 signalosome, composed of eight subunits, is implicated in cancer genetics with its deneddylase activity to modulate cellular concentration of oncogenic proteins such as IkB and TGFβ. However, its function in the normal cell physiology remains elusive. Primarily focusing on gene expression data of the normal tissues of the head and neck, the cancer genome atlas (TCGA) database was used to identify groups of genes that were expressed synergistically with the COP9 genes, particularly with the COPS5 (CSN5), which possesses the catalytic activity of COP9.ResultsExpressions of seven of the COP9 genes (COPS2, COPS3, COPS4, COPS5, COPS6, COPS7A, and COPS8) were found to be highly synergistic in the normal tissues. In contrast, the tumor tissues decreased the coordinated expression pattern of COP9 genes. Pathway analysis revealed a high coordination of the expression of the COPS5 and the other COP9 genes with mitochondria-related functional pathways, including genes encoding the respiratory chain complex.ConclusionsThe results indicate that mRNA expression data for the matched normal tissues available in TCGA are statistically reliable, and are highly useful to assess novel associations of genes with functional pathways in normal physiology as well as in the cancer tissues. This study revealed the significant correlation between the expressions of the COP9 genes and those related to the mitochondrial activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3313-y) contains supplementary material, which is available to authorized users.

The emerging roles of Jab1/CSN5 in cancer.

C-Jun activation domain-binding protein-1 (Jab1) not only is full but also a subunit (CSN5) of the constitutive photomorphogenesis 9 signalosome (CSN), which is an evolutionarily conserved and multifunctional protein that involves in controlling cellular proliferation and apoptosis, affecting a series of pathways, as well as regulating genomic instability and DNA damage and repair. The CSN is a highly conservative protein from yeast to human and interacts with the cullin-RING family of ubiquitin ligases so that it could be execute a process of removing NEDD8, a ubiquitin-like polypeptide (deneddylase activity). The role of Jab1/CSN5's multi-function has been proved as being oncogenic in nature, what is more, Jab1/CSN5 has been confirmed by much evidence that it participates in the carcinogenesis progression and is tightly associated with poor prognosis. However, the biologic implication of Jab1/CSN5 activity during the cancer's development is unclear. We performed a systematic literature review and assessment from PubMed and Medline databases in this article. Jab1/CSN5 is participate in a lot of biologic responses, including cell proliferation, apoptosis, cell cycle regulation, DNA metabolism, invasion, DNA damage and repair, and recurrence. It also promotes cell transformation and tumorigenesis. In this review, we mainly expound the progress in the function and research advances of Jab1/CSN5 and in untangling the Jab1/CSN5 signaling pathway. Based on these bases, its potential as a therapeutic target for cancer can play a greater role in future cancer treatment.

Deneddylase 1 regulates deneddylase activity of the Cop9 signalosome in Drosophila melanogaster.

NEDD8 conjugation of Cullin has an important role in ubiquitin-mediated protein degradation. The COP9 signalosome, of which CSN5 is the major catalytic subunit, is a major Cullin deneddylase. Another deneddylase, Deneddylase 1, has also been shown to process the Nedd8 precursor. In Drosophila, the DEN1 mutants do not have increased levels of Cullin neddylation, but instead show a significant decrease in neddylated Cullin. This characteristic decrease in neddylated Cullins in the DEN1null background can be rescued by UAS-dDEN1WT overexpression but not by overexpression of mature NEDD8, indicating that this phenotype is distinct from the NEDD8-processing function of DEN1. We examined the role of DEN1-CSN interaction in regulating Cullin neddylation. Overexpression of DEN1 in a CSN5hypo background slightly reduced unneddylated Cullin levels. The CSN5, DEN1 double mutation partially rescues the premature lethality associated with the CSN5 single mutation. These results suggest that DEN1 regulates Cullin neddylation by suppressing CSN deneddylase activity.

COP9-Signalosome deneddylase activity is enhanced by simultaneous neddylation: insights into the regulation of an enzymatic protein complex.

BackgroundCullin-RING ubiquitin ligases (CRLs) are regulated by neddylation, which is a post translation modification of the Cullin family proteins. Neddylation of Cul1 activates the ligase through some means of biochemical mechanisms. The rate of neddylation and its extent are regulated by 2 opposing enzymatic processes: neddylation by an enzymatic cascade, and deneddylation by COP9-Signalosome (CSN) complex protein. The mechanism by which COP9-Signalosome catalytic activity is regulated is not well understood.MethodsWe set an in vitro neddylation and deneddylation reaction using as a source for specific COP9/Signalosome deneddylase activity either Hela cells extract or purified Signalosome. Neddylation reaction of either endogenic Cul1 from Hela cells extract or recombinant Cul1 was catalyzed by recombinant neddylation enzymes. Deneddylation rate was tested either simultaneous to neddylation or after termination of neddylation by using an ATP depleting reaction or by directly inhibiting the neddylation activation enzyme named APP-BP1/UBA3 by its specific inhibitor MLN-4924.ResultsWe demonstrated that neddylation and deneddylation are catalytically engaged and that inhibition of Cul1 neddylation significantly causes a decline in the rate of COP9-Signalosome deneddylase activity. Since neddylation is an ATP consuming reaction we managed to isolate the 2 opposing processes which surprisingly caused a decline in COP9 activity. Using MLN-4924 we demonstrated that direct inhibition of neddylation negatively influences the rate of deneddylation. The hypothesis that phosphorylation controls deneddylation was ruled out by the fact that no change in the rate of deneddylation was exemplified while converting the use of ATP with AMP-PNP.ConclusionsWe demonstrated that deneddylation of Cul1 is positively regulated through direct simultaneous neddylation and is not dependent upon autophosphorylation. Defining the mechanism that regulates neddylation and deneddylation of Cullin proteins is important due to their effect on highly conserved cellular processes. We showed that minor changes in the degree of Cul1 neddylation linearly control the degree of p27 conjugation to ubiquitin, which emphasizes the hypothetic physiologic significance of our findings.Electronic supplementary materialThe online version of this article (doi:10.1186/s13008-015-0011-0) contains supplementary material, which is available to authorized users.

Integration of the catalytic subunit activates deneddylase activity in vivo as final step in fungal COP9 signalosome assembly.

The eight-subunit COP9 signalosome (CSN) is conserved from filamentous fungi to humans and functions at the interface between cellular signalling and protein half-life control. CSN consists of six PCI and two MPN domain proteins and forms a scaffold for additional interacting proteins. CSN controls protein stability in the ubiquitin-proteasome system where the MPN domain CSN5/CsnE subunit inactivates cullin-RING ligases. The CSN5/CsnE isopeptidase functions as deneddylase and removes the ubiquitin-like protein Nedd8. The six PCI domain proteins of human CSN form a horseshoe-like ring and all eight subunits are connected by a bundle of C-terminal α-helices. We show that single deletions of any csn subunit of Aspergillus nidulans resulted in the lack of deneddylase activity and identical defects in the coordination of development and secondary metabolism. The CSN1/CsnA N-terminus is dispensable for deneddylase activity but required for asexual spore formation. Complex analyses in mutant strains revealed the presence of a seven-subunit pre-CSN without catalytic activity. Reconstitution experiments with crude extracts of deletion strains and recombinant proteins allowed the integration of CSN5/CsnE into pre-CSN resulting in an active deneddylase. This supports a stable seven subunit pre-CSN intermediate where deneddylase activation in vivo can be controlled by CSN5/CsnE integration as final assembly step.

The β-catenin E3 ubiquitin ligase SIAH-1 is regulated by CSN5/JAB1 in CRC cells

COP9 signalosome subunit 5 (CSN5) plays a decisive role in cellular processes such as cell cycle regulation and apoptosis via promoting protein degradation, gene transcription, and nuclear export. CSN5 regulates cullin-RING-E3 ligase (CRL) activity through its deNEDDylase function. It is overexpressed in several tumor entities, but its role in colorectal cancer (CRC) is poorly understood. Wnt/β-catenin signaling is aberrant in most CRC cells, resulting in increased levels of oncogenic β-catenin and thus tumor progression. Under physiological conditions, β-catenin levels are tightly regulated by continuous proteasomal degradation. We recently showed that knockdown of CSN5 in model and CRC cells results in decreased (phospho)-β-catenin levels. Reduced β-catenin levels were associated with an attenuated proliferation rate of different CRC cell types after CSN5 knockdown. The canonical Wnt pathway involves degradation of β-catenin by a β-TrCP1-containing E3 ligase, but is mostly non-functional in CRC cells. We thus hypothesized that alternative β-catenin degradation mediated by SIAH-1 (seven in absentia homolog-1), is responsible for the effect of CSN5 on β-catenin signaling in CRC cells. We found that SIAH-1 plays an essential role in β-catenin degradation in HCT116 CRC cells and that CSN5 affects β-catenin target gene expression in these cells. Of note, CSN5 affected SIAH-1 mRNA and SIAH-1 protein levels. Moreover, β-catenin and SIAH-1 form protein complexes with CSN5 in HCT116 cells. Lastly, we demonstrate that CSN5 promotes SIAH-1 degradation in HCT116 and SW480 cells and that this is associated with its deNEDDylase activity. In conclusion, we have identified a CSN5/β-catenin/SIAH-1 interaction network that might control β-catenin degradation in CRC cells.

The COP9 signalosome is involved in the regulation of lipid metabolism and of transition metals uptake in Saccharomyces cerevisiae

The COP9 signalosome (CSN) is a highly conserved eukaryotic protein complex which regulates the cullin RING family of ubiquitin ligases and carries out a deneddylase activity that resides in subunit 5 (CSN5). Whereas CSN activity is essential for the development of higher eukaryotes, several unicellular fungi including the budding yeast Saccharomyces cerevisiae can survive without a functional CSN. Nevertheless, the budding yeast CSN is biochemically active and deletion mutants of each of its subunits exhibit deficiency in cullins deneddylation, although the biological context of this activity is still unknown in this organism. To further characterize CSN function in budding yeast, we present here a transcriptomic and proteomic analysis of a S.cerevisiae strain deleted in the CSN5/RRI1 gene (hereafter referred to as CSN5), coding for the only canonical subunit of the complex. We show that Csn5 is involved in modulation of the genes controlling amino acid and lipid metabolism and especially ergosterol biosynthesis. These alterations in gene expression correlate with the lower ergosterol levels and increased intracellular zinc content which we observed in csn5 null mutant cells. We show that some of these regulatory effects of Csn5, in particular the control of isoprenoid biosynthesis, are conserved through evolution, since similar transcriptomic and/or proteomic effects of csn5 mutation were previously observed in other eukaryotic organisms such as Aspergillusnidulans, Arabidopsisthaliana and Drosophilamelanogaster. Our results suggest that the diverged budding yeast CSN is more conserved than was previously thought.

Caspase-1 Promotes Epstein-Barr Virus Replication by Targeting the Large Tegument Protein Deneddylase to the Nucleus of Productively Infected Cells

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.

Control of Multicellular Development by the Physically Interacting Deneddylases DEN1/DenA and COP9 Signalosome

Deneddylases remove the ubiquitin-like protein Nedd8 from modified proteins. An increased deneddylase activity has been associated with various human cancers. In contrast, we show here that a mutant strain of the model fungus Aspergillus nidulans deficient in two deneddylases is viable but can only grow as a filament and is highly impaired for multicellular development. The DEN1/DenA and the COP9 signalosome (CSN) deneddylases physically interact in A. nidulans as well as in human cells, and CSN targets DEN1/DenA for protein degradation. Fungal development responds to light and requires both deneddylases for an appropriate light reaction. In contrast to CSN, which is necessary for sexual development, DEN1/DenA is required for asexual development. The CSN-DEN1/DenA interaction that affects DEN1/DenA protein levels presumably balances cellular deneddylase activity. A deneddylase disequilibrium impairs multicellular development and suggests that control of deneddylase activity is important for multicellular development.

Insights into the regulation of the human COP9 signalosome catalytic subunit, CSN5/Jab1

The COP9 (Constitutive photomorphogenesis 9) signalosome (CSN), a large multiprotein complex that resembles the 19S lid of the 26S proteasome, plays a central role in the regulation of the E3-cullin RING ubiquitin ligases (CRLs). The catalytic activity of the CSN complex, carried by subunit 5 (CSN5/Jab1), resides in the deneddylation of the CRLs that is the hydrolysis of the cullin-neural precursor cell expressed developmentally downregulated gene 8 (Nedd8)isopeptide bond. Whereas CSN-dependent CSN5 displays isopeptidase activity, it is intrinsically inactive in other physiologically relevant forms. Here we analyze the crystal structure of CSN5 in its catalytically inactive form to illuminate the molecular basis for its activation state. We show that CSN5 presents a catalytic domain that brings essential elements to understand its activity control. Although the CSN5 active site is catalytically competent and compatible with di-isopeptide binding, the Ins-1 segment obstructs access to its substrate-binding site, and structural rearrangements are necessary for the Nedd8-binding pocket formation. Detailed study of CSN5 by molecular dynamics unveils signs of flexibility and plasticity of the Ins-1 segment. These analyses led to the identification of a molecular trigger implicated in the active/inactive switch that is sufficient to impose on CSN5 an active isopeptidase state. We show that a single mutation in the Ins-1 segment restores biologically relevant deneddylase activity. This study presents detailed insights into CSN5 regulation. Additionally, a dynamic monomer-dimer equilibrium exists both in vitro and in vivo and may be functionally relevant.

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.

A deneddylase encoded by Epstein–Barr virus promotes viral DNA replication by regulating the activity of cullin-RING ligases

The large tegument proteins of herpesviruses encode conserved cysteine proteases of unknown function. Here we show that BPLF1, the Epstein-Barr-virus-encoded member of this protease family, is a deneddylase that regulates virus production by modulating the activity of cullin-RING ligases (CRLs). BPLF1 hydrolyses NEDD8 conjugates in vitro, acts as a deneddylase in vivo, binds to cullins and stabilizes CRL substrates. Expression of BPLF1 alone or in the context of the productive virus cycle induces accumulation of the licensing factor CDT1 and deregulates S-phase DNA synthesis. Inhibition of BPLF1 during the productive virus cycle prevents cellular DNA re-replication and inhibits virus replication. Viral DNA synthesis is restored by overexpression of CDT1. Homologues encoded by other herpesviruses share the deneddylase activity. Thus, these enzymes are likely to have a key function in the virus life cycle by inducing a replication-permissive S-phase-like cellular environment.

NEDD8: A new ataxin-3 interactor

Machado–Joseph disease (MJD/SCA3) is an autosomal dominant neurodegenerative disease caused by the expansion of a CAG tract in the coding portion of the ATXN3 gene. The presence of ubiquitin-positive aggregates of the defective protein in affected neurons is characteristic of this and most of the polyglutamine disorders. Recently, the accumulation of the neural precursor cell expressed developmentally downregulated 8 (NEDD8), a ubiquitin-like protein, in the inclusions of MJD brains was reported. Here, we report a new molecular interaction between wild-type ataxin-3 and NEDD8, using in vitro and in situ approaches. Furthermore, we show that this interaction is not dependent on the ubiquitin-interacting motifs in ataxin-3, since the presence of the Josephin domain is sufficient for the interaction to occur. The conservation of the interaction between the Caenorhabditis elegans ataxin-3 homologue (atx-3) and NEDD8 suggests its biological and functional relevance. Molecular docking studies of the NEDD8 molecule to the Josephin domain of ataxin-3 suggest that NEDD8 interacts with ataxin-3 in a substrate-like mode. In agreement, ataxin-3 displays deneddylase activity against a fluorogenic NEDD8 substrate.