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

PURPOSE: 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. METHODS: 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 ß (IKKß), PD-L1, and CSN5 was assessed. Additionally, plasma CSN5 levels in asthma patients, both in stable and exacerbated states, were examined. RESULTS: 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-IKKß proteins increased at 33 and 80 days compared to control mice. However, these changes were mitigated by treatment with a PD-L1 inhibitor. CONCLUSIONS: These findings suggest that CSN5, along with PD-L1, could serve as a promising target for the treatment of asthma.

Hormone-mediated disassembly and inactivation of a plant E3 ubiquitin ligase complex.

Phytohormone abscisic acid (ABA) regulates key plant development and environmental stress responses. The ubiquitin-proteasome system tightly controls ABA signaling. CULLIN4-RING (CRL4) E3 ubiquitin ligases use the substrate receptor module CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP10)-DDB1-DET1-DDA1 (CDDD) to target Arabidopsis ABA receptor PYL8, acting as negative regulators of ABA responses. Conversely, ABA treatment attenuates PYL8 receptor degradation, although the molecular mechanism remained elusive. Here, we show that ABA promotes the disruption of CRL4-CDDD complexes, leading to PYL8 stabilization. ABA-mediated CRL4-CDDD dissociation likely involves an altered association between DDA1-containing complexes and the COP9 signalosome (CSN), a master regulator of the assembly of cullin-based E3 ligases, including CRL4-CDDD. Indeed, treatment with CSN inhibitor CSN5i-3 suppresses the ABA effect on CRL4-CDDD assembly. Our findings indicate that ABA stabilizes PYL8 by altering the dynamics of the CRL4-CDDD-CSN complex association, showing a regulatory mechanism by which a plant hormone inhibits an E3 ubiquitin ligase to protect its own receptors from degradation.

Ubiquitin-mediated degradation of SlPsbS regulates low night temperature tolerance in tomatoes.

PsbS protein is essential for the rapid induction of non-photochemical quenching (NPQ) under low night temperatures (LNTs), but its stability is often affected by adverse environmental conditions. However, the regulatory mechanism for the stability of PsbS or chloroplast proteins remains to be fully characterized. We show that LNT decreases NPQ levels and SlPsbS protein abundance in tomato leaves. LNT-activated chloroplast vesicles (SlCVs) targeting the chloroplasts induce the formation of CV-containing vesicles (CCVs) containing SlPsbS, exported from the chloroplasts. Subsequently, SlCV and SlPsbS contact COP9 signalosome subunit 5A (SlCSN5A) in the cytosol and are ubiquitinated and degraded. Genetic evidence demonstrates that the overexpression of SlCV aggravates SlPsbS protein degradation, whereas silencing of SlCSN5 and SlCV delays LNT-induced NPQ reduction and SlPsbS protein turnover. This study reveals a ubiquitin-dependent degradation pathway of chloroplast proteins co-mediated by CV and CSN5A, thereby providing a basic reference for the regulation of chloroplast protein stability under stress conditions.

Identification of COP9 signalosome (CSN) subunits and antiviral function analysis of CSN5 in shrimp.

The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) typically composing of eight subunits (CSN1-8) mediates the process of deneddylation and deubiquitination. The fifth subunit of COP9 signalosome, CSN5, has special characteristics compared with the other seven subunits, and plays vital roles in the deneddylation activity and diverse cellular processes. However, the role of CSN5 in antiviral immunity is not clear. In this study, we identified 8 subunits (CSN1-8) of COP9 signalosome in shrimp Marsupenaeus japonicus. CSN1-6 were existed in all tested tissues, but CSN7-CSN8 were not detected in hepatopancreas. After WSSV challenged, the expression level of Csn1 to Csn4, and Csn6 to Csn8 were highly decreased, but the expression level of Csn5 was conspicuously increased in shrimp challenged by white spot syndrome virus (WSSV). The CSN5 was recombinantly expressed in Escherichia coli and its polyclonal antibody was prepared. The expression level of CSN5 was conspicuously increased at RNA and protein levels in the shrimp challenged by WSSV. After knockdown of Csn5 by RNA interference, the WSSV replication was obviously increased in shrimp. When injected the recombinant protein of CSN5 with the membrane penetrating peptide into shrimp, WSSV replication was inhibited and the survival rate of shrimp was significantly improved compared with control. We further analyzed the expression of antimicrobial peptides (AMPs) in Csn5-RNAi shrimp, and the results showed that the expression of several AMPs was declined significantly. These results indicate that CSN5 inhibits replication of WSSV via regulating expression of AMPs in shrimp, and the recombinant CSN5 might be used in shrimp aquaculture for the white spot syndrome disease control.

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. In vitro, 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.

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.

EARLY FLOWERING is a dominant gain-of-function allele of FANTASTIC FOUR 1/2c that promotes early flowering in tomato.

Flowering time, an important factor in plant adaptability and genetic improvement, is regulated by various genes in tomato (Solanum lycopersicum). In this study, we characterized a tomato mutant, EARLY FLOWERING (EF), that developed flowers much earlier than its parental control. EF is a dominant gain-of-function allele with a T-DNA inserted 139 bp downstream of the stop codon of FANTASTIC FOUR 1/2c (FAF1/2c). The transcript of SlFAF1/2c was at elevated levels in the EF mutant. Overexpressing SlFAF1/2c in tomato plants phenocopied the early flowering trait of the EF mutant. Knocking out SlFAF1/2c in the EF mutant reverted the early flowering phenotype of the mutant to the normal flowering time of the wild-type tomato plants. SlFAF1/2c promoted the floral transition by shortening the vegetative phase rather than by reducing the number of leaves produced before the emergence of the first inflorescence. The COP9 signalosome subunit 5B (CSN5B) was shown to interact with FAF1/2c, and knocking out CSN5B led to an early flowering phenotype in tomato. Interestingly, FAF1/2c was found to reduce the accumulation of the CSN5B protein by reducing its protein stability. These findings imply that FAF1/2c regulates flowering time in tomato by reducing the accumulation and stability of CSN5B, which influences the expression of SINGLE FLOWER TRUSS (SFT), JOINTLESS (J) and UNIFLORA (UF). Thus, a new allele of SlFAF1/2c was discovered and found to regulate flowering time in tomato.

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.

Adaptive exchange sustains cullin-RING ubiquitin ligase networks and proper licensing of DNA replication.

Cop9 signalosome (CSN) regulates the function of cullin-RING E3 ubiquitin ligases (CRLs) by deconjugating the ubiquitin-like protein NEDD8 from the cullin subunit. To understand the physiological impact of CSN function on the CRL network and cell proliferation, we combined quantitative mass spectrometry and genome-wide CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) screens to identify factors that modulate cell viability upon inhibition of CSN by the small molecule CSN5i-3. CRL components and regulators strongly modulated the antiproliferative effects of CSN5i-3, and in addition we found two pathways involved in genome integrity, SCFFBXO5-APC/C-GMNN and CUL4DTL-SETD8, that contribute substantially to the toxicity of CSN inhibition. Our data highlight the importance of CSN-mediated NEDD8 deconjugation and adaptive exchange of CRL substrate receptors in sustaining CRL function and suggest approaches for leveraging CSN inhibition for the treatment of cancer.

Prognostic value of CSN5 in patients with digestive system cancers: a systematic review and meta-analysis.

BACKGROUND: Despite the understanding of the COP9 signalosome subunit 5 (CSN5) in tumor genesis, there is no conclusive evidence on its value to predict the survival and prognosis of digestive system tumor patients. Hence this study aimed to evaluate the impact of CSN5 levels on the survival and clinicopathological parameters of digestive system neoplasm patients. METHODS: First, a comprehensive search was conducted in four databases. We utilized the Hazard Ratio (HR) with a 95% confidence interval (CI) to evaluate the prognostic value of CSN5 for the overall survival (OS) and recurrence-free survival (RFS) of patients. Then, we estimated the connection between CSN5 and the clinicopathological parameters based on the Odds Ratio (OR) with the corresponding 95% CI. RESULTS: This meta-analysis included 22 studies and 2193 patients diagnosed with digestive system tumors. High expression of CSN5 was correlated to poorer OS (HR = 2.28, 95% CI: 1.71-3.03; p < 0.00001). Additionally, high CSN5 levels were correlated with worse invasion depth (OR = 0.49, 95% CI: 0.25-0.96, p = 0.04), positive lymphatic metastasis (OR = 0.28, 95% CI: 0.16-0.47, p = 0.00001), positive distant metastasis (OR = 0.32, 95% CI: 0.13-0.76, p = 0.01) and poorer differentiation degree (OR = 0.34, 95% CI: 0.19-0.60, p = 0.0003). However, we did not detect a correlation between CSN5 expression and age, gender, tumor stage, tumor size or vascular invasion. Furthermore, no significant publication bias was detected. CONCLUSION: This meta-analysis demonstrated that the overexpression of CSN5 level might foresee poorer OS in digestive system cancer patients. Additionally, CSN5 levels might be related to the prognosis of digestive system tumors.

The generation of PD-L1 and PD-L2 in cancer cells: From nuclear chromatin reorganization to extracellular presentation.

The immune checkpoint blockade (ICB) targeting on PD-1/PD-L1 has shown remarkable promise in treating cancers. However, the low response rate and frequently observed severe side effects limit its broad benefits. It is partially due to less understanding of the biological regulation of PD-L1. Here, we systematically and comprehensively summarized the regulation of PD-L1 from nuclear chromatin reorganization to extracellular presentation. In PD-L1 and PD-L2 highly expressed cancer cells, a new TAD (topologically associating domain) (chr9: 5,400,000-5,600,000) around CD274 and CD273 was discovered, which includes a reported super-enhancer to drive synchronous transcription of PD-L1 and PD-L2. The re-shaped TAD allows transcription factors such as STAT3 and IRF1 recruit to PD-L1 locus in order to guide the expression of PD-L1. After transcription, the PD-L1 is tightly regulated by miRNAs and RNA-binding proteins via the long 3'UTR. At translational level, PD-L1 protein and its membrane presentation are tightly regulated by post-translational modification such as glycosylation and ubiquitination. In addition, PD-L1 can be secreted via exosome to systematically inhibit immune response. Therefore, fully dissecting the regulation of PD-L1/PD-L2 and thoroughly detecting PD-L1/PD-L2 as well as their regulatory networks will bring more insights in ICB and ICB-based combinational therapy.

Suppressed COP9 signalosome 5 promotes hemocyte proliferation through Cyclin E in the early G1 phase to defend against bacterial infection in crab.

Hemocytes are invertebrate immune cells that are similar to blood cells in vertebrates and play a crucial role in innate immunity. Previous work has found that mature circulating hemocytes lack the ability to proliferate. However, recent single-cell RNA sequencing and functional studies in invertebrate have challenged this view. Here, we report that bacteria induced hemocytes proliferation in the Chinese mitten crab, Eriocheir sinensis. Flow cytometry was used to collect non-proliferating and proliferating hemocytes populations, while the expression of EsCyclin E was highly expressed in proliferating hemocytes, but the expression of EsCsn5 was significantly suppressed in proliferating hemocytes. Subsequent studies have found EsCsn5 distributed in two fractions include holo-complex and monomeric form, whereas knockdown of EsCsn5 has little impact on the amount of the holo-complex. EsCsn5 was widely expressed in different crab tissues, while its expression was significantly reduced upon bacterial infection. Crab hemocytes showed significantly enhanced proliferation when EsCsn5 was genetically knocked down, suggesting a critical role for CSN5 in the negative regulation of crab hemocyte proliferation. Moreover, EsCSN5 but not the EsCSN8 was demonstrated to negatively regulate the early G1 phase of the cell cycle by controlling the degradation of EsCyclin E through ubiquitination steps, rather than affecting its transcription. Furthermore, in the EsCyclin E-suppressed crab there was a significantly reduced survival rate and an up-regulated hemolymph bacterial concentration. Taken together, this study provides evidence demonstrating that invertebrate hemocytes down-regulate the expression of EsCsn5 upon bacterial challenge, thus promoting proliferation in an EsCyclin E-dependent manner in order to protect the crab from infection.

SENP1 promotes triple-negative breast cancer invasion and metastasis via enhancing CSN5 transcription mediated by GATA1 deSUMOylation.

TNBC is characterized by high incidence of visceral metastasis and lacks effective clinical targets. This study aims to delineate the molecular mechanisms of SENP1 in TNBC invasion and metastasis. By using IHC to test the SENP1 expression in TNBC tissues, we analyzed the relationship between SENP1 expression and TNBC prognosis. We showed that SENP1 expression was higher in TNBC tumor tissues and related to TNBC prognosis, supporting SENP1 as an independent risk factor. High expression of SENP1 was significantly associated with histologic grade and tumor lymph node invasion. Intriguingly, the expression levels of SENP1 in TNBC tumors were significantly correlated with that of CSN5, GATA1 and ZEB1. Importantly, SENP1 promoted TNBC cell migration and invasion by regulating ZEB1 deubiquitination and expression through CSN5. Further studies showed that deSUMOylation at lysine residue K137 of GATA1 enhanced the binding of GATA1 to the CSN5 promoter and transactivated CSN5 expression. In addition, we showed that ZEB1 is deubiquitinated at lysine residue K1108. Our in vivo studies also indicated that reduction in SENP1 expression upregulated GATA1 SUMOylation, and thus resulted in decreased expression of CSN5 and ZEB1 in the tumor microenvironment, which decelerated TNBC progression and metastasis. SENP1 promoted CSN5-mediated ZEB1 protein degradation via deSUMOylation of GATA1, and thus influenced TNBC progression. These findings suggest that SENP1 could be utilized as a potential target for blockade of TNBC development and thus provide a totally new approach for TNBC treatment.

Overexpression of DAPK1-mediated inhibition of IKKß/CSN5/PD-L1 axis enhances natural killer cell killing ability and inhibits tumor immune evasion in gastric cancer.

Gastric cancer (GC) originates from the stomach and is a prevalent human malignancy. Dysfunction of death associated protein kinase 1 (DAPK1) has been identified as a major regulator involved in the development and progression of GC. However, there's limited data regarding the regulatory mechanism of GC. Herein, we investigated role of DAPK1 in natural killer (NK) cell killing ability and immune evasion of GC cells and mediated pathway. Samples from GC-related gene expression profile and clinical samples from 67 patients with GC were collected to determine the expression of DAPK1, IκB kinase ß (IKKß), programmed death receptor-ligand 1 (PD-L1), and photomorphogenesis 9 (COP9) signalosome 5 (CSN5). The binding affinity among DAPK1, IKKß, CSN5, and PD-L1 was characterized to verify the underlying mechanism. GC lines were transfected with overexpressed plasmid or siRNA to determine the effect of DAPK1/IKKß/CSN5/PD-L1 axis on NK cell killing ability and immune evasion of GC cells. GC cells and tissues presented low expression of DAPK1 and high expression of IKKß, CSN5 and PD-L1. IKKß, negatively regulated by DAPK1, was capable of activating CSN5 and upregulating PD-L1 expression. Overexpression of DAPK1 promoted NK cell killing ability and reduced immune evasion, coupled with reduction of NK cell apoptosis and increases in levels of TNF-α, IFN-γ, CD107a, and Granzyme B cytokines. The tumor-suppressing properties of DAPK1 through downregulation of IKKß/CSN5/PD-L1 axis in GC were further confirmed in vivo. In summary, overexpression of DAPK1 promoted the NK cell killing ability and restrained immune evasion of GC cells, providing a potential therapeutic strategy for GC treatment by modulating immune evasion.

The potential oncogenic and MLN4924-resistant effects of CSN5 on cervical cancer cells.

BACKGROUND: CSN5, a member of Cop9 signalosome, is essential for protein neddylation. It has been supposed to serve as an oncogene in some cancers. However, the role of CSN5 has not been investigated in cervical cancer yet. METHODS: Data from TCGA cohorts and GEO dataset was analyzed to examine the expression profile of CSN5 and clinical relevance in cervical cancers. The role of CSN5 on cervical cancer cell proliferation was investigated in cervical cancer cell lines, Siha and Hela, through CSN5 knockdown via CRISPR-CAS9. Western blot was used to detect the effect of CSN5 knockdown and overexpression. The biological behaviors were analyzed by CCK8, clone formation assay, 3-D spheroid generation assay and cell cycle assay. Besides, the role CSN5 knockdown in vivo was evaluated by xenograft tumor model. MLN4924 was given in Siha and Hela with CSN5 overexpression. RESULTS: We found that downregulation of CSN5 in Siha and Hela cells inhibited cell proliferation in vitro and in vivo, and the inhibitory effects were largely rescued by CSN5 overexpression. Moreover, deletion of CSN5 caused cell cycle arrest rather than inducing apoptosis. Importantly, CSN5 overexpression confers resistance to the anti-cancer effects of MLN4924 (pevonedistat) in cervical cancer cells. CONCLUSIONS: Our findings demonstrated that CSN5 functions as an oncogene in cervical cancers and may serve as a potential indicator for predicting the effects of MLN4924 treatment in the future.

PDIA6 promotes pancreatic cancer progression and immune escape through CSN5-mediated deubiquitination of ß-catenin and PD-L1.

Protein Disulfide Isomerase Family A Member 6 (PDIA6) is an endoplasmic reticulum protein that is capable of catalyzing protein folding and disulfide bond formation. Abnormally elevated expression of PDIA6 has been reported to predict poor outcomes in various cancers. Herein, gain-of- and loss-of-function experiments were performed to investigate how PDIA6 participated in the carcinogenesis of pancreatic cancer (PC). By analyzing the protein expression of PDIA6 in 28 paired PC and para carcinoma specimens, we first found that PDIA6 expression was higher in PC samples. Both the overall survival and disease-free survival rates of PC patients with higher PDIA6 expression were poorer than those with lower PDIA6 (n = 178). Furthermore, knockdown of PDIA6 impaired the malignancies of PC cells - suppressed cell proliferation, invasion, migration, cisplatin resistance, and xenografted tumor growth. PDIA6-silenced PC cells were more sensitive to cytotoxic natural killer (NK) cells. Overexpression of PDIA6 had opposite effects on PC cells. Interestingly, COP9 signalosome subunit 5 (CSN5), a regulator of E3 ubiquitin ligases known to promote deubiquitination of its downstream targets, was demonstrated to interact with PDIA6, and its expression was increased in PC cells overexpressing PDIA6. Additionally, PDIA6 overexpression promoted deubiquitination of ß-catenin and PD-L1 and subsequently upregulated their expression in PC cells. These alterations were partly reversed by CSN5 shRNA. Collectively, the above results demonstrate that PDIA6 contributes to PC progression, which may be associated with CSN5-regulated deubiquitination of ß-catenin and PD-L1. Our findings suggest PDIA6 as a potential target for the treatment of PC.

Downregulation of Cullin 3 Ligase Signaling Pathways Contributes to Hypertension in Preeclampsia.

Background: Preeclampsia (PE) is a leading cause of maternal and perinatal morbidity and mortality; however, its etiology and pathophysiology remain obscure. PE is initiated by inadequate spiral artery remodeling and subsequent placental ischemia/hypoxia, which stimulates release of bioactive factors into maternal circulation, leading to hypertension and renal damage. Methods and Results: Abundance of key components of cullin 3-ring ubiquitin ligase (CRL3), including cullin 3 (CUL3) and its neddylated modification, and adaptors including Kelch-like 2 (KLHL2) and Rho-related BTB domain containing protein 1 was all decreased in spiral arteries and placentas of PE patients. Similar changes were found in aortic tissues and renal distal tubules of pregnant mice treated with Nω-nitro-l-arginine methyl ester hydrochloride. The downregulation of CRL3 function led to accumulation of with-no-lysine kinases, phosphodiesterase 5, and RhoA in vessels and renal distal tubules, which promoted vasoconstriction and Na-Cl cotransporter activation in the distal convoluted tubule (DCT), as well as vascular and DCT structure remodeling. Proton pump inhibitor esomeprazole partially restored CRL3 function. In vitro studies have shown that increased abundance of JAB1, a component of the COP9 signalosome, inhibited CUL3 neddylation and promoted the expression of hypoxia-inducible factor 1α, which downregulated peroxisome proliferator-activated receptor γ and further promoted CUL3 inactivation. KLHL3/2 was degraded by increased autophagy. Conclusion: These findings support that the downregulation of CRL3 function disrupts the balance of vasoconstriction and vasodilation and aggravates excess reabsorption of sodium in PE.

Saccharomyces cerevisiae as a Toolkit for COP9 Signalosome Research.

The COP9 signalosome (CSN) is a highly conserved eukaryotic multi-subunit enzyme, regulating cullin RING ligase activities and accordingly, substrate ubiquitination and degradation. We showed that the CSN complex of Saccharomyces cerevisiae that is deviated in subunit composition and in sequence homology harbors a highly conserved cullin deneddylase enzymatic core complex. We took advantage of the non-essentiality of the S. cerevisiae CSN-NEDD8/Rub1 axis, together with the enzyme-substrate cross-species activity, to develop a sensitive fluorescence readout assay, suitable for biochemical assessment of cullin deneddylation by CSNs from various origins. We also demonstrated that the yeast catalytic subunit, CSN5/Jab1, is targeted by an inhibitor that was selected for the human orthologue. Treatment of yeast by the inhibitor led to the accumulation of neddylated cullins and the formation of reactive oxygen species. Overall, our data revealed S. cerevisiae as a general platform that can be used for studies of CSN deneddylation and for testing the efficacy of selected CSN inhibitors.

Abrogation of USP7 is an alternative strategy to downregulate PD-L1 and sensitize gastric cancer cells to T cells killing.

Targeting immune checkpoints such as programmed cell death protein 1 (PD-1) and programmed death ligand-1 (PD-L1) have been approved for treating melanoma, gastric cancer (GC) and bladder cancer with clinical benefit. Nevertheless, many patients failed to respond to anti-PD-1/PD-L1 treatment, so it is necessary to seek an alternative strategy for traditional PD-1/PD-L1 targeting immunotherapy. Here with the data from The Cancer Genome Atlas (TCGA) and our in-house tissue library, PD-L1 expression was found to be positively correlated with the expression of ubiquitin-specific processing protease 7 (USP7) in GC. Furthermore, USP7 directly interacted with PD-L1 in order to stabilize it, while abrogation of USP7 attenuated PD-L1/PD-1 interaction and sensitized cancer cells to T cell killing in vitro and in vivo. Besides, USP7 inhibitor suppressed GC cells proliferation by stabilizing P53 in vitro and in vivo. Collectively, our findings indicate that in addition to inhibiting cancer cells proliferation, USP7 inhibitor can also downregulate PD-L1 expression to enhance anti-tumor immune response simultaneously. Hence, these data posit USP7 inhibitor as an anti-proliferation agent as well as a novel therapeutic agent in PD-L1/PD-1 blockade strategy that can promote the immune response of the tumor.

CSN5 Promotes Carcinogenesis of Thyroid Carcinoma Cells Through ANGPTL2.

COP9 signalosome subunit 5 (CSN5) plays a key role in carcinogenesis of multiple cancers and contributes to the stabilization of target proteins through deubiquitylation. However, the underlying role of CSN5 in thyroid carcinoma has not been reported. In this research, our data showed that CSN5 was overexpressed in thyroid carcinoma tissues compared with paracancerous tissues. Furthermore, a series of gain/loss functional assays were performed to demonstrate the role of CSN5 in facilitating thyroid carcinoma cell proliferation and metastasis. Additionally, we found there was a positive correlation between CSN5 and angiopoietin-like protein 2 (ANGPTL2) protein levels in thyroid carcinoma tissues and that CSN5 promoted thyroid carcinoma cell proliferation and metastasis through ANGPTL2. We also identified the underlying mechanism that CSN5 elevated ANGPTL2 protein level by directly binding it, decreasing its ubiquitination and degradation. Overall, our results highlight the significance of CSN5 in promoting thyroid carcinoma carcinogenesis and implicate CSN5 as a promising candidate for thyroid carcinoma treatment.