COP9 Signalosome Research Articles

DNA polymerase η is regulated by mutually exclusive mono-ubiquitination and mono-NEDDylation.

DNA polymerase eta (Pol η) is a Y-family translesion polymerase responsible for synthesizing new DNA across UV-damaged templates. It is recruited to replication forks following mono-ubiquitination of the PCNA DNA clamp. This interaction is mediated by PCNA-interacting protein (PIP) motifs within Pol η, as well as by its C-terminal ubiquitin-binding zinc finger (UBZ) domain. Previous work has suggested that Pol η itself is mono-ubiquitinated at four C-terminal lysine residues, which is dependent on prior ubiquitin-binding by its UBZ domain. Here, we show that Pol η can be modified at the same lysine residues by the ubiquitin-like protein, NEDD8. Like ubiquitination, this modification is driven by non-covalent interactions between NEDD8 and the UBZ domain. While only a small proportion of Pol η is mono-NEDDylated under normal conditions, these levels rapidly increase by inhibiting the COP9 signalosome, suggesting that mono-NEDDylation is maintained under strong negative regulation. Finally, we provide data to support that mono-ubiquitination is important for Pol η foci formation and suggest that NEDDylation disrupts this process. These results reveal a new mechanism of Pol η regulation by ubiquitin-like proteins.

Distal convoluted tubule-specific disruption of the COP9 signalosome but not its regulatory target cullin 3 causes tubular injury.

The disease familial hyperkalemic hypertension (FHHt; also known as Gordon syndrome) is caused by aberrant accumulation of with-no-lysine kinase (WNK4) activating the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT) of the kidney. Mutations in cullin 3 (CUL3) cause FHHt by disrupting interaction with the deneddylase COP9 signalosome (CSN). Deletion of Cul3 or Jab1 (the catalytically active CSN subunit) along the entire nephron causes a partial FHHt phenotype with activation of the WNK4-STE20/SPS1-related proline/alanine-rich kinase (SPAK)-NCC pathway. However, progressive kidney injury likely prevents hypertension, hyperkalemia, and hyperchloremic metabolic acidosis associated with FHHt. We hypothesized that DCT-specific deletion would more closely model the disease. We used Slc12a3-Cre-ERT2 mice to delete Cul3 (DCT-Cul3-/-) or Jab1 (DCT-Jab1-/-) only in the DCT and examined the mice after short- and long-term deletion. Short-term DCT-specific knockout of both Cul3 and Jab1 mice caused elevated WNK4, pSPAKS373, and pNCCT53 abundance. However, neither model demonstrated changes in plasma K+, Cl-, or total CO2, even though no injury was present. Long-term DCT-Jab1-/- mice showed significantly lower NCC and parvalbumin abundance and a higher abundance of kidney injury molecule-1, a marker of proximal tubule injury. No injury or reduction in NCC or parvalbumin was observed in long-term DCT-Cul3-/- mice. In summary, the prevention of injury outside the DCT did not lead to a complete FHHt phenotype despite activation of the WNK4-SPAK-NCC pathway, possibly due to insufficient NCC activation. Chronically, only DCT-Jab1-/- mice developed tubule injury and atrophy of the DCT, suggesting a direct JAB1 effect or dysregulation of other cullins as mechanisms for injury.NEW & NOTEWORTHY CUL3 degrades WNK4, which prevents activation of NCC in the DCT. CSN regulation of CUL3 is impaired in the disease FHHt, causing accumulation of WNK4. Short-term DCT-specific disruption of CUL3 or the CSN in mice resulted in activation of the WNK4-SPAK-NCC pathway but not hyperkalemic metabolic acidosis found in FHHt. Tubule injury was observed only after long-term CSN disruption. The data suggest that disruption of other cullins may be the cause for the injury.

COP9 Signalosome CSN4 and CSN5 Subunits Protect NKCC2 against Endoplasmic Reticulum Stress and Cellular Hypoxia

COP9 Signalosome CSN4 and CSN5 Subunits Protect NKCC2 against Endoplasmic Reticulum Stress and Cellular Hypoxia

Distal Convoluted Tubule (DCT)-Specific Disruption of the COP9 Signalosome Induces Autophagy Leading to Kidney Injury and DCT Atrophy

Distal Convoluted Tubule (DCT)-Specific Disruption of the COP9 Signalosome Induces Autophagy Leading to Kidney Injury and DCT Atrophy

Translocon Subunits of the COP9 Signalosome Complex Are a Central Hub for Regulating Multiple Photoresponsive Processes and Autophagic Flux in Magnaporthe oryzae.

Photodependent processes, including circadian rhythm, autophagy, ubiquitination, neddylation/deneddylation, and metabolite biosynthesis, profoundly influence microbial pathogenesis. Although a photomorphogenesis signalosome (COP9/CSN) has been identified, the mechanism by which this large complex contributes to the pathophysiological processes in filamentous fungi remains unclear. Here, we identified eight CSN complex subunits in the rice blast fungus Magnaporthe oryzae and functionally characterized the translocon subunits containing a nuclear export or localization signal (NES/NLS). Targeted gene replacement of these CSN subunits, including MoCSN3, MoCSN5, MoCSN6, MoCSN7, and MoCSN12, attenuated vegetative growth and conidiation and rendered the deletion strains nonpathogenic. MoCSN7 deletion significantly suppressed arachidonic acid catabolism, and compromised cell wall integrity in M. oryzae. Surprisingly, we also discovered that MoCSN subunits, particularly MoCsn7, are required for the cAMP-dependent regulation of autophagic flux. Therefore, MoCSN significantly contributes to morphological, physiological, and pathogenic differentiation in M. oryzae by fostering cross-talk between multiple pathways.

Circ_0084653 promotes the tumor progression and immune escape in triple-negative breast cancer via the deubiquitination of MYC and upregulation of SOX5

The role of circular RNAs (circRNAs) in cancers is gaining more and more attention, yet related reporters are limited. In triple-negative breast cancer (TNBC), circRNA circ_0084653 originated from COP9 signalosome subunit 5 (COPS5), and COPS5 has been validated to be upregulated in breast cancer before. In our research, COPS5 was also upregulated in TNBC cells, and knockdown of it repressed cell proliferation, invasion, EMT, stemness and PDL-1 protein expression but increased T-cell percentage. Further, circ_0084653 was an aberrantly upregulated circRNA in TNBC cells, and similarly, circ_0084653 silence inhibited TNBC development. Besides, circ_0084653 expression was distributed in both cytoplasm and nucleus. COPS5 overexpression partially rescued the suppressing effects of circ_0084653 depletion in TNBC. Subsequently, circ_0084653 triggered deubiquitination of MYC, the upstream transcription factor of COPS5, via recruiting ubiquitin specific peptidase 36 (USP36). Moreover, circ_0084653 served as the sponge of miR-1323 to release the expression the target gene SRY-box transcription factor 5 (SOX5). SOX5 upregulation completely remedied the inhibiting influence of circ_0084653 downregulation in TNBC. Meanwhile, transcription factor SOX5 activated transcriptionally circ_0084653. To sum up, SOX5-induced circ_0084653 promotes TNBC via the deubiquitination of USP36, which may provide some fresh ideas for TNBC-related molecular mechanisms.

Discovering New Metallo-Deubiquitinase CSN5 Inhibitors by a Non-Catalytic Activity Assay Platform.

COP9 signalosome catalytic subunit CSN5 plays a key role in tumorigenesis and tumor immunity, showing potential as an anticancer target. Currently, only a few CSN5 inhibitors have been reported, at least partially, due to the challenges in establishing assays for CSN5 deubiquitinase activity. Here, we present the establishment and validation of a simple and reliable non-catalytic activity assay platform for identifying CSN5 inhibitors utilizing a new fluorescent probe, CFP-1, that exhibits enhanced fluorescence and fluorescence polarization features upon binding to CSN5. By using this platform, we identified 2-aminothiazole-4-carboxylic acids as new CSN5 inhibitors, which inhibited CSN5 but slightly downregulated PD-L1 in cancer cells. Furthermore, through the integration of deep learning-enabled virtual screening, we discovered that shikonins are nanomolar CSN5 inhibitors, which can upregulate PD-L1 in HCT116 cells. The binding modes of these structurally distinct inhibitors with CSN5 were explored by using microsecond-scale molecular dynamics simulations and tryptophan quenching assays.

The COP9 signalosome stabilized MALT1 promotes Non-Small Cell Lung Cancer progression through activation of NF-κB pathway

MALT1 has been implicated as an upstream regulator of NF-κB signaling in immune cells and tumors. This study determined the regulatory mechanisms and biological functions of MALT1 in non-small cell lung cancer (NSCLC). In cell culture and orthotopic xenograft models, MALT1 suppression via gene expression interference or protein activity inhibition significantly impaired malignant phenotypes and enhanced radiation sensitivity of NSCLC cells. CSN5, the core subunit of COP9 signalosome, was firstly verified to stabilize MALT1 via disturbing the interaction with E3 ligase FBXO3. Loss of FBXO3 in NSCLC cells reduced MALT1 ubiquitination and promoted its accumulation, which was reversed by CSN5 interference. An association between CSN5/FBXO3/MALT1 regulatory axis and poor prognosis in NSCLC patients was identified. Our findings revealed the detail mechanism of continuous MALT1 activation in NF-κB signaling, highlighting its significance as predictor and potential therapeutic target in NSCLC.Graphical

The Toxoplasma gondii F-Box Protein L2 Functions as a Repressor of Stage Specific Gene Expression.

Toxoplasma gondii is a foodborne pathogen that can cause severe and life-threatening infections in fetuses and immunocompromised patients. Felids are its only definitive hosts, and a wide range of animals, including humans, serve as intermediate hosts. When the transmissible bradyzoite stage is orally ingested by felids, they transform into merozoites that expand asexually, ultimately generating millions of gametes for the parasite sexual cycle. However, bradyzoites in intermediate hosts differentiate exclusively to disease-causing tachyzoites, which rapidly disseminate throughout the host. Though tachyzoites are well-studied, the molecular mechanisms governing transitioning between developmental stages are poorly understood. Each parasite stage can be distinguished by a characteristic transcriptional signature, with one signature being repressed during the other stages. Switching between stages require substantial changes in the proteome, which is achieved in part by ubiquitination. F-box proteins mediate protein poly-ubiquitination by recruiting substrates to SKP1, Cullin-1, F-Box protein E3 ubiquitin ligase (SCF-E3) complexes. We have identified an F-box protein named Toxoplasma gondii F-Box Protein L2 (TgFBXL2), which localizes to distinct perinucleolar sites. TgFBXL2 is stably engaged in an SCF-E3 complex that is surprisingly also associated with a COP9 signalosome complex that negatively regulates SCF-E3 function. At the cellular level, TgFBXL2-depleted parasites are severely defective in centrosome replication and daughter cell development. Most remarkable, RNAseq data show that TgFBXL2 conditional depletion induces the expression of stage-specific genes including a large cohort of genes necessary for sexual commitment. Together, these data suggest that TgFBXL2 is a latent guardian of stage specific gene expression in Toxoplasma and poised to remove conflicting proteins in response to an unknown trigger of development.

MEN1 deficiency stabilizes PD-L1 and promotes tumor immune evasion of lung cancer.

Multiple Endocrine Neoplasia 1 gene (MEN1), which is known to be a tumor suppressor gene in lung tissues, encodes a 610 amino acid protein menin. Previous research has proven that MEN1 deficiency promotes the malignant progression of lung cancer. However, the biological role of this gene in the immune microenvironment of lung cancer remains unclear. In this study, we found that programmed cell death-ligand 1 (PD-L1) is upregulated in lung-specific KrasG12D mutation-induced lung adenocarcinoma in mice, after Men1 deficiency. Simultaneously, CD8+ and CD3+ T cells are depleted, and their cytotoxic effects are suppressed. Invitro, PD-L1 is inhibited by the overexpression of menin. Mechanistically, we found that MEN1 inactivation promotes the deubiquitinating activity of COP9 signalosome subunit 5 (CSN5) and subsequently increases the level of PD-L1.

Nematicidal activity and action mode of a methyl-accepting chemotaxis protein from Pseudomonas syringae against Caenorhabditis elegans

The conventional phytopathogen Pseudomonas syringae reportedly possesses several virulence determinants against Caenorhabditis elegans; however, their action mechanisms remain elusive. This study reports the nematicidal activity and action receptor of a methyl-accepting chemotaxis protein (MCP03) of a wild-type P. syringae MB03 against C. elegans. Purified MCP03 exhibited nematicidal toxicity against C. elegans at a half-lethal concentration of 124.4 μg mL−1, alongside detrimental effects on the growth and brood size of C. elegans. Additionally, MCP03-treated worms exhibited severe pathological destruction of the intestine and depressed wrinkles of the cuticle. Yeast two-hybrid assays identified a subunit of COP9 signalosome, namely CSN-5, which functioned as an MCP03 action receptor. In vitro pull-down verified the binding interaction between MCP03 and CSN-5. RNA interference assays confirmed that MCP03 antagonizes CSN-5, thereby adversely affecting the brood size and cuticle integrity of C. elegans. Following MCP03 infection, the expression of genes related to reproduction, growth, and cuticle formation, such as kgb-1, unc-98, and col-117, was considerably downregulated, indicating pathological changes in MCP03-treated nematodes. Therefore, we proposed that MCP03 antagonizes CSN-5, causing lethality as well as detrimental effects on the fertility, growth, and morphogenesis of C. elegans, which can provide new insights into the signaling pathways and mechanisms underlying the nematicidal action of MCP03 toward C. elegans.

Csn5 inhibits autophagy by regulating the ubiquitination of Atg6 and Tor to mediate the pathogenicity of Magnaporthe oryzae

Csn5 is subunit 5 of the COP9 signalosome (CSN), but the mechanism by which it strictly controls the pathogenicity of pathogenic fungi through autophagy remains unclear. Here, we found that Csn5 deficiency attenuated pathogenicity and enhanced autophagy in Magnaporthe oryzae. MoCSN5 knockout led to overubiquitination and overdegradation of MoTor (the core protein of the TORC1 complex [target of rapamycin]) thereby promoted autophagy. In addition, we identified MoCsn5 as a new interactor of MoAtg6. Atg6 was found to be ubiquitinated through linkage with lysine 48 (K48) in cells, which is necessary for infection-associated autophagy in pathogenic fungi. K48-ubiquitination of Atg6 enhanced its degradation and thereby inhibited autophagic activity. Our experimental results indicated that MoCsn5 promoted K48-ubiquitination of MoAtg6, which reduced the MoAtg6 protein content and thus inhibited autophagy. Aberrant ubiquitination and autophagy in ΔMocsn5 led to pleiotropic defects in the growth, development, stress resistance, and pathogenicity of M. oryzae. In summary, our study revealed a novel mechanism by which Csn5 regulates autophagy and pathogenicity in rice blast fungus through ubiquitination.

A Characterization of the Functions of OsCSN1 in the Control of Sheath Elongation and Height in Rice Plants under Red Light

The COP9 signalosome (CSN) is a conserved protein complex, with CSN1 being one of the largest and most important subunits in the COP9 complex. To investigate the N-terminus function of OsCSN1, we edited the N-terminus of OsCSN1 and found that the mutant of OsCSN1 with 102 amino acids missing at the N-terminus showed insensitivity to red light in terms of the embryonic sheath, stem elongation, and main-root elongation. Moreover, the mutant was able to produce, develop, and bear fruit normally. The research results indicate that OsCSN1 is a negative regulator of stem elongation in rice seedlings regulated by red light. Under red-light treatment, OsCSN1 assembles into CSN, which degrades SLR1 through de NEDDylation, affecting PIL11 activity and ultimately inhibiting stem elongation. OsCSN1 also plays an important regulatory role in the inhibition of rice embryonic sheath elongation under red light. By regulating the degradation of SLR1 and PIL14 through the ubiquitin/26S protease pathway, the elongation of the embryonic sheath is ultimately inhibited. OsCSN1 forms a COP9 complex and is modified with RUB/NEDD8 of the E3 ligase of CUL1 to promote the degradation of SLR1 and PIL14, ultimately affecting the elongation of the embryonic sheath. The regulatory domain is located at the N-terminus of CSN1.

COP9 signalosome complex is a prognostic biomarker and corresponds with immune infiltration in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is among the most common deadly tumors but still lacks specific biomarkers for diagnosis, prognosis, and treatment guidance. The COP9 signalosome (COPS) is an essential regulator of the ubiquitin conjugation pathway upregulated in various cancers. We evaluated the contributions of COPS subunits to HCC tumorigenesis and their utility for prognosis. We comprehensively evaluated the tumor expression pattern and tumorigenic functions of COPS subunits using The Cancer Genome Atlas (TCGA), The Human Protein Atlas and immunohistochemistry. Kaplan-Meier, Cox regression, ROC curve, and nomogram analyses were used to assess the predictive values of COPS subunits for clinical outcome. Expression levels of COPS subunits were significantly upregulated in HCC tissues, which predicted shorter overall survival (OS). Further, Cox regression analysis identified COPS5, COPS7B, and COPS9 as independent prognostic biomarkers for OS. High mutation rates were also found in COPS subunits. Functional network analysis indicated that COPS and neighboring genes regulate 'protein neddylation', 'protein deneddylation', and 'protein ubiquitination'. The COPS PPI included strong interactions with p53, CUL1/2/3/4, and JUN. Moreover, the correlations between COPS subunit expression levels and tumor immune cell infiltration rates were examined using TIMER, TISIDB, ssGSEA, and ESTIMATE packages. COPS subunits expression levels were positively correlated with specific tumor immune cell infiltration rates, immunoregulator expression levels, and microsatellite instability in HCC. Finally, knockout of COPS6 and COPS9 in HCC cells reduced while overexpression enhanced proliferation rate and metastasis capacity. Our study revealed that COPS potential biomarker for unfavorable HCC prognosis and indicators of immune infiltration, tumorigenicity, and metastasis.

COP9 signalosome component CSN-5 stabilizes PUF proteins FBF-1 and FBF-2 in Caenorhabditis elegans germline stem and progenitor cells.

RNA-binding proteins FBF-1 and FBF-2 (FBFs) are required for germline stem cell maintenance and the sperm/oocyte switch in Caenorhabditis elegans, although the mechanisms controlling FBF protein levels remain unknown. We identified an interaction between both FBFs and CSN-5), a component of the constitutive photomorphogenesis 9 (COP9) signalosome best known for its role in regulating protein degradation. Here, we find that the Mpr1/Pad1 N-terminal metalloprotease domain of CSN-5 interacts with the Pumilio and FBF RNA-binding domain of FBFs and the interaction is conserved for human homologs CSN5 and PUM1. The interaction between FBF-2 and CSN-5 can be detected in vivo by proximity ligation. csn-5 mutation results in the destabilization of FBF proteins, which may explain previously observed decrease in the numbers of germline stem and progenitor cells, and disruption of oogenesis. The loss of csn-5 does not decrease the levels of a related PUF protein PUF-3, and csn-5(lf) phenotype is not enhanced by fbf-1/2 knockdown, suggesting that the effect is specific to FBFs. The effect of csn-5 on oogenesis is largely independent of the COP9 signalosome and is cell autonomous. Surprisingly, the regulation of FBF protein levels involves a combination of COP9-dependent and COP9-independent mechanisms differentially affecting FBF-1 and FBF-2. This work supports a previously unappreciated role for CSN-5 in the stabilization of germline stem cell regulatory proteins FBF-1 and FBF-2.

Weighted gene co-expression network analysis reveals immune evasion related genes in Echinococcus granulosus sensu stricto.

Cystic echinococcosis (CE) is a zoonotic disease caused by the tapeworm Echinococcus granulosus sensu lato (s.l). In the intermediate host, this disease is characterized by the growth of cysts in viscera such as liver and lungs, inside of which the parasite develops to the next infective stage known as protoscoleces. There are records that the infected viscera affect the development and morphology of E. granulosus s.l. protoscolex in hosts such as buffalo or humans. However, the molecular mechanisms that drive these differences remains unknown. Weighted gene co-expression network analysis (WGCNA) using a set of RNAseq data obtained from E. granulosus sensu stricto (s.s.) protoscoleces found in liver and lung cysts reveals 34 modules in protoscoleces of liver origin, of which 12 have differential co-expression from protoscoleces of lung origin. Three of these twelve modules contain hub genes related to immune evasion: tegument antigen, tegumental protein, ubiquitin hydrolase isozyme L3, COP9 signalosome complex subunit 3, tetraspanin CD9 antigen, and the methyl-CpG-binding protein Mbd2. Also, two of the twelve modules contain only hypothetical proteins with unknown orthology, which means that there are a group of unknown function proteins co-expressed inside the protoscolex of liver CE cyst origin. This is the first evidence of gene expression differences in protoscoleces from CE cysts found in different viscera, with co-expression networks that are exclusive to protoscoleces from liver CE cyst samples. This should be considered in the control strategies of CE, as intermediate hosts can harbor CE cysts in liver, lungs, or both organs simultaneously.

OsCSN2 orchestrates Oryza sativa L. growth and development through modulation of the GA and BR pathways.

The COP9 signalosome (CSN) is a conserved protein complex found in higher eukaryotes, consisting of eight subunits, and it plays a crucial role in regulating various processes of plant growth and development. Among these subunits, CSN2 is one of the most conserved components within the COP9 signalosome complex. Despite its prior identification in other species, its specific function in Oryza sativa L. (Rice) has remained poorly understood. In this study, we investigated the role of CSN2 in rice using gene editing CRISPR/Cas9 technology and overexpression techniques. We created two types of mutants: the oscsn2 mutant and the OsCSN2-OE mutant, both in the background of rice, and also generated point mutants of OsCSN2 (OsCSN2K64E, OsCSN2K67E, OsCSN2K71E and OsCSN2K104E) to further explore the regulatory function of OsCSN2. Phenotypic observation and gene expression analysis were conducted on plants from the generated mutants, tracking their growth from the seedling to the heading stages. The results showed that the loss and modification of OsCSN2 had limited effects on plant growth and development during the early stages of both the wild-type and mutant plants. However, as the plants grew to 60days, significant differences emerged. The OsCSN2 point mutants exhibited increased tillering compared to the OsCSN2-OE mutant plants, which were already at the tillering stage. On the other hand, the OsCSN2 point mutant had already progressed to the heading and flowering stages, with the shorter plants. These results, along with functional predictions of the OsCSN2 protein, indicated that changes in the 64th, 67th, 71st, and 104th amino acids of OsCSN2 affected its ubiquitination site, influencing the ubiquitination function of CSN and consequently impacting the degradation of the DELLA protein SLR1. Taken together, it can be speculated that OsCSN2 plays a key role in GA and BR pathways by influencing the functional regulation of the transcription factor SLR1 in CSN, thereby affecting the growth and development of rice and the number of tillers.

SMC5/6 Promotes Replication Fork Stability via Negative Regulation of the COP9 Signalosome.

It is widely accepted that DNA replication fork stalling is a common occurrence during cell proliferation, but there are robust mechanisms to alleviate this and ensure DNA replication is completed prior to chromosome segregation. The SMC5/6 complex has consistently been implicated in the maintenance of replication fork integrity. However, the essential role of the SMC5/6 complex during DNA replication in mammalian cells has not been elucidated. In this study, we investigate the molecular consequences of SMC5/6 loss at the replication fork in mouse embryonic stem cells (mESCs), employing the auxin-inducible degron (AID) system to deplete SMC5 acutely and reversibly in the defined cellular contexts of replication fork stall and restart. In SMC5-depleted cells, we identify a defect in the restart of stalled replication forks, underpinned by excess MRE11-mediated fork resection and a perturbed localization of fork protection factors to the stalled fork. Previously, we demonstrated a physical and functional interaction of SMC5/6 with the COP9 signalosome (CSN), a cullin deneddylase that enzymatically regulates cullin ring ligase (CRL) activity. Employing a combination of DNA fiber techniques, the AID system, small-molecule inhibition assays, and immunofluorescence microscopy analyses, we show that SMC5/6 promotes the localization of fork protection factors to stalled replication forks by negatively modulating the COP9 signalosome (CSN). We propose that the SMC5/6-mediated modulation of the CSN ensures that CRL activity and their roles in DNA replication fork stabilization are maintained to allow for efficient replication fork restart when a replication fork stall is alleviated.

Involvement of the Constitutive Photomorphogenesis 9 Signalosome Subunit 5 With Programmed Cell Death Protein 1 Ligand in Asthma.

The constitutive photomorphogenesis 9 signalosome (CSN) is a highly conserved protein complex comprised of eight subunits, each of which play crucial roles in diverse cellular processes, such as signal transduction, gene transcription, angiogenesis, and cell proliferation. In the context of asthma, a potential emerging target is the programmed death-ligand 1 (PD-L1)-mediated pathway, which serves as a significant immune checkpoint inhibitor in this condition. However, the precise involvement of CSN subunit 5 (CSN5) in bronchial asthma and the interplay between CSN5 and PD-L1 in asthma remain poorly understood. The potential association between CSN5 and bronchial asthma was explored in a mouse model of ovalbumin (OVA)-induced asthma. Samples were obtained from human lung microvascular endothelial cell (HMVEC-L) treated with Dermatophagoides pteronyssinus (Der p 1) and CSN5 small interfering RNA. The expression of nuclear factor (NF)-κB, IκBα, inhibitor of κB kinase β (IKKβ), PD-L1, and CSN5 was assessed. Additionally, plasma CSN5 levels in asthma patients, both in stable and exacerbated states, were examined. Plasma levels of CSN5 were elevated in patients with exacerbated asthma (n = 19) compared to both healthy controls (n = 10) and patients with stable asthma (n = 19). The CSN5 level demonstrated a correlation with lung function in individuals with asthma. Silencing CSN5 in HMVEC-L led to a reduction in NF-κB protein levels at 4 hours and PD-L1 levels at 4, 8, and 24 hours after Der p 1 treatment. In OVA-sensitized/challenged mice, goblet cell hyperplasia, lung fibrosis, and the levels of CSN5, PD-L1, interleukin-13, interferon-γ, phospho (p)-NF-κB, p-IκBα, and p-IKKβ proteins increased at 33 and 80 days compared to control mice. However, these changes were mitigated by treatment with a PD-L1 inhibitor. These findings suggest that CSN5, along with PD-L1, could serve as a promising target for the treatment of asthma.

COP9 signalosome-mediated deneddylation of CULLIN1 is necessary for SCFEBF1 assembly in Arabidopsis thaliana

Functions of the SKP1-CUL1-F box (SCF) ubiquitin E3 ligases are essential in plants. The F box proteins (FBPs) are substrate receptors that recruit substrates and assemble an active SCF complex, but the regulatory mechanism underlying the FBPs binding to CUL1 to activate the SCF cycle is not fully understood. We show that Arabidopsis csn1-10 is defective in SCFEBF1-mediated PIF3 degradation during de-etiolation, due to impaired association of EBF1 with CUL1 in csn1-10. EBF1 preferentially associates with un-neddylated CUL1 that is deficient in csn1-10 and the EBF1-CUL1 binding is rescued by the neddylation inhibitor MLN4924. Furthermore, we identify a subset of FBPs with impaired binding to CUL1 in csn1-10, indicating their assembly to form SCF complexes may depend on COP9 signalosome (CSN)-mediated deneddylation of CUL1. This study reports that a key role of CSN-mediated CULLIN deneddylation is to gate the binding of the FBP-substrate module to CUL1, thus initiating the SCF cycle of substrate ubiquitination.