COP9 Signalosome Subunit Research Articles

COPS8 in cutaneous melanoma: an oncogene that accelerates the malignant development of tumor cells and predicts poor prognosis.

This study aimed to investigate the roles of COP9 signalosome subunit 8 (COPS8) and its underlying mechanism in cutaneous melanoma. Bioinformatics tools were utilized to analyze the expression of COPS8 in cutaneous melanoma, while Kaplan-Meier analysis was employed to assess the correlation between COPS8 and patients' overall survival. The proliferation, migration, and invasion of cells were estimated by CCK8, colony formation, and Transwell assays. Western blot was used to check the expression of epithelial-mesenchymal transition (EMT)-related proteins. Results showed that COPS8 was up-regulated and predicted a poor clinical outcome for cutaneous melanoma patients. Knockdown of COPS8 inhibited cutaneous melanoma cell proliferation, migration and invasion, whereas overexpression of COPS8 resulted in the opposite outcomes. The up-regulation of E-cadherin and down-regulation of N-cadherin, vimentin, and snail were caused by silencing COPS8 while their expression showed contrary trends in cells with overexpressed COPS8. Collectively, COPS8 is up-regulated and promotes cutaneous melanoma progression via regulating EMT.

The Network of Interactions Between Classical Swine Fever Virus Nonstructural Protein p7 and Host Proteins.

Classical swine fever (CSF) is a highly contagious viral disease causing severe economic losses to the swine industry. As viroporins of viruses modulate the cellular ion balance and then take over the cellular machinery, blocking the activity of viroporin or developing viroporin-defective attenuated vaccines offers new approaches to treat or prevent viral infection. Non-structural protein p7 of CSF virus (CSFV) is a viroporin, which was highly involved in CSFV virulence. Deciphering the interaction between p7 and host proteins will aid our understanding of the mechanism of p7-cellular protein interaction affecting CSFV replication. In the present study, seven host cellular proteins including microtubule-associated protein RP/EB family member 1 (MAPRE1), voltage-dependent anion channel 1 (VDAC1), proteasome maturation protein (POMP), protein inhibitor of activated STAT 1 (PIAS1), gametogenetin binding protein 2 (GGNBP2), COP9 signalosome subunit 2 (COPS2), and contactin 1 (CNTN1) were identified as the potential interactive cellular proteins of CSFV p7 by using yeast two-hybrid (Y2H) screening. Plus, the interaction of CSFV p7 with MAPRE1 and VDAC1 was further evaluated by co-immunoprecipitation and GST-pulldown assay. Besides, the p7-cellular protein interaction network was constructed based on these seven host cellular proteins and the STRING database. Enrichment analysis of GO and KEGG indicated that many host proteins in the p7-cellular protein interaction network were mainly related to the ubiquitin-proteasome system, cGMP-PKG signaling pathway, calcium signaling pathway, and JAK-STAT pathway. Overall, this study identified potential interactive cellular proteins of CSFV p7, constructed the p7-cellular protein interaction network, and predicted the potential pathways involved in the interaction between CSFV p7 and host cells.

CUL4A regulates endometrial cancer cell proliferation, invasion and migration by interacting with CSN6

Endometrial cancer (EC) is a common malignant gynecological tumor arising from the endometrium, with an annually increasing morbidity and mortality. The present study aimed to investigate the functions of cullin 4A (CUL4A) in EC, as well as the underlying mechanisms. CUL4A expression was assessed in several human EC cells and normal human endometrial epithelial cells (hEECs) via reverse transcription-quantitative polymerase chain reaction and western blotting. Subsequently, short hairpin (sh)RNA-CULA4 was transfected into cells, and cell proliferation, invasion and migration were detected using Cell Counting kit-8, Transwell and wound healing assays, respectively. The STRING database identified that CSN6 interacted with CULA4, and immunoprecipitation was performed to verify the interaction. Subsequently, following CUL4A knockdown, pcDNA3.1-CSN6 was transfected into cells and its effects on cell proliferation, invasion and migration were assessed. The expression levels of matrix metallopeptidase (MMP)2, MMP9 and p53 were evaluated via western blotting. The results indicated that CUL4A was highly expressed in EC cells, compared with hEECs. CULA4-knockdown notably inhibited EC cell proliferation, invasion and migration. The expression levels of MMP2 and MMP9 were significantly decreased, while p53 expression was enhanced following CUL4A-knockdown. The immunoprecipitation assay verified that COP9 signalosome subunit 6 (CSN6) interacted with CULA4. Furthermore, CSN6-overexpression alleviated the inhibitory effects of CUL4A-knockdown on EC cell proliferation, invasion and migration. Similarly, CSN6 overexpression reversed CUL4A-knockdown-mediated effects on the expression of MMP2, MMP9 and p53. In summary, the results demonstrated that CUL4A regulated EC cell proliferation, invasion and migration by interacting with CSN6.

EGF Relays Signals to COP1 and Facilitates FOXO4 Degradation to Promote Tumorigenesis.

Forkhead‐Box Class O 4 (FOXO4) is involved in critical biological functions, but its response to EGF‐PKB/Akt signal regulation is not well characterized. Here, it is reported that FOXO4 levels are downregulated in response to EGF treatment, with concurrent elevation of COP9 Signalosome subunit 6 (CSN6) and E3 ubiquitin ligase constitutive photomorphogenic 1 (COP1) levels. Mechanistic studies show that CSN6 binds and regulates FOXO4 stability through enhancing the E3 ligase activity of COP1, and that COP1 directly interacts with FOXO4 through a VP motif on FOXO4 and accelerates the ubiquitin‐mediated degradation of FOXO4. Metabolomic studies demonstrate that CSN6 expression leads to serine and glycine production. It is shown that FOXO4 directly binds and suppresses the promoters of serine‐glycine‐one‐carbon (SGOC) pathway genes, thereby diminishing SGOC metabolism. Evidence shows that CSN6 can regulate FOXO4‐mediated SGOC gene expression. Thus, these data suggest a link of CSN6‐FOXO4 axis and ser/gly metabolism. Further, it is shown that CSN6‐COP1‐FOXO4 axis is deregulated in cancer and that the protein expression levels of CSN6 and FOXO4 can serve as prognostic markers for cancers. The results illustrate a pathway regulation of FOXO4‐mediated serine/glycine metabolism through the function of CSN6‐COP1 axis. Insights into this pathway may be strategically designed for therapeutic intervention in cancers.

Thrombin-Par1 signaling axis disrupts COP9 signalosome subunit 3-mediated ABCA1 stabilization in inducing foam cell formation and atherogenesis.

ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1) play a vital role in promoting cholesterol efflux. Although, the dysregulation of these transporters was attributed as one of the mechanisms of atherogenesis, what renders their dysfunction is not well explored. Previously, we have reported that thrombin without having any effect on ABCG1 levels depletes ABCA1 levels affecting cholesterol efflux. In this study, we explored the mechanisms underlying thrombin-induced depletion of ABCA1 levels both in macrophages and smooth muscle cells. Under normal physiological conditions, COP9 signalosome subunit 3 (CSN3) was found to exist in complex with ABCA1 and in the presence of proatherogenic stimulants such as thrombin, ABCA1 was phosphorylated and dissociated from CSN3, leading to its degradation. Forced expression of CSN3 inhibited thrombin-induced ABCA1 ubiquitination and degradation, restored cholesterol efflux and suppressed foam cell formation. In Western diet (WD)-fed ApoE-/- mice, CSN3 was also disassociated from ABCA1 otherwise remained as a complex in Chow diet (CD)-fed ApoE-/- mice. Interestingly, depletion of CSN3 levels in WD-fed ApoE-/- mice significantly lowered ABCA1 levels, inhibited cholesterol efflux and intensified foam cell formation exacerbating the lipid laden atherosclerotic plaque formation. Mechanistic studies have revealed the involvement of Par1-Gα12-Pyk2-Gab1-PKCθ signaling in triggering phosphorylation of ABCA1 and its disassociation from CSN3 curtailing cholesterol efflux and amplifying foam cell formation. In addition, although both CSN3 and ABCA1 were found to be colocalized in human non-lesion coronary arteries, their levels were decreased as well as dissociated from each other in advanced atherosclerotic lesions. Together, these observations reveal for the first time an anti-atherogenic role of CSN3 and hence, designing therapeutic drugs protecting its interactions with ABCA1 could be beneficial against atherosclerosis.

Hypoxia-induced downregulation of cyclooxygenase 2 leads to the loss of immunoprivilege of allogeneic mesenchymal stem cells.

Allogeneic mesenchymal stem cells (MSCs) from young and healthy donors are reported to hold the potential to treat several immunological and degenerative disorders. However, recent data from animal studies and clinical trials demonstrate that immunogenicity and poor survival of transplanted MSCs impaired the efficacy of cells for regenerative applications. It is reported that initially immunoprivileged under in vitro conditions, MSCs are targeted by the host immune system after transplantation in the ischemic tissues in vivo. We performed in vitro (in MSCs) and in vivo (in the rat model of myocardial infarction [MI]) studies to elucidate the mechanisms responsible for the change in the immunophenotype of MSCs from immunoprivileged to immunogenic under ischemic conditions. We have recently reported that a soluble factor prostaglandin E2 (PGE2) preserves the immunoprivilege of allogeneic MSCs. In the current study, we found that PGE2 levels, which were elevated during normoxia, decreased in MSCs following exposure to hypoxia. Further, we found that proteasome-mediated degradation of cyclooxygenase-2 (COX2, rate-limiting enzyme in PGE2 biosynthesis) in hypoxic MSCs is responsible for PGE2 decrease and loss of immunoprivilege of MSCs. While investigating the mechanisms of COX2 degradation in hypoxic MSCs, we found that in normoxic MSCs, COP9 signalosome subunit 5 (CSN5) binds to COX2 and prevents its degradation by the proteasome. However, exposure to hypoxia leads to a decrease in CSN5 levels and its binding to COX2, rendering COX2 protein susceptible to proteasome-mediated degradation. This subsequently causes PGE2 downregulation and loss of immunoprivilege of MSCs. Maintaining COX2 levels in MSCs preserves immunoprivilege in vitro and improves the survival of transplanted MSCs in a rat model of MI. These data provide novel mechanistic evidence that PGE2 is downregulated in hypoxic MSCs which is responsible for the post-transplantation rejection of allogeneic MSCs. Therefore, our data suggest that the new strategies that target CSN5-COX2 signaling may improve survival and utility of transplanted allogeneic MSCs in the ischemic heart.

New Insights From Imputed Whole-Genome Sequence-Based Genome-Wide Association Analysis and Transcriptome Analysis: The Genetic Mechanisms Underlying Residual Feed Intake in Chickens.

Poultry feed constitutes the largest cost in poultry production, estimated to be up to 70% of the total cost. Moreover, there is pressure on the poultry industry to increase production to meet the protein demand of humans and simultaneously reduce emissions to protect the environment. Therefore, improving feed efficiency plays an important role to improve profits and the environmental footprint in broiler production. In this study, using imputed whole-genome sequencing data, genome-wide association analysis (GWAS) was performed to identify single-nucleotide polymorphisms (SNPs) and genes associated with residual feed intake (RFI) and its component traits. Furthermore, a transcriptomic analysis between the high-RFI and the low-RFI groups was performed to validate the candidate genes from GWAS. The results showed that the heritability estimates of average daily gain (ADG), average daily feed intake (ADFI), and RFI were 0.29 (0.004), 0.37 (0.005), and 0.38 (0.004), respectively. Using imputed sequence-based GWAS, we identified seven significant SNPs and five candidate genes [MTSS I-BAR domain containing 1, folliculin, COP9 signalosome subunit 3, 5′,3′-nucleotidase (mitochondrial), and gametocyte-specific factor 1] associated with RFI, 20 significant SNPs and one candidate gene (inositol polyphosphate multikinase) associated with ADG, and one significant SNP and one candidate gene (coatomer protein complex subunit alpha) associated with ADFI. After performing a transcriptomic analysis between the high-RFI and the low-RFI groups, both 38 up-regulated and 26 down-regulated genes were identified in the high-RFI group. Furthermore, integrating regional conditional GWAS and transcriptome analysis, ras-related dexamethasone induced 1 was the only overlapped gene associated with RFI, which also suggested that the region (GGA14: 4767015–4882318) is a new quantitative trait locus associated with RFI. In conclusion, using imputed sequence-based GWAS is an efficient method to identify significant SNPs and candidate genes in chicken. Our results provide valuable insights into the genetic mechanisms of RFI and its component traits, which would further improve the genetic gain of feed efficiency rapidly and cost-effectively in the context of marker-assisted breeding selection.

CSN6\u2013TRIM21 axis instigates cancer stemness during tumorigenesis

BackgroundCancer stem cells (CSCs) are responsible for tumour initiation, metastasis and recurrence. However, the mechanism of CSC formation, maintenance and expansion in colorectal cancer (CRC) remains poorly characterised.MethodsThe role of COP9 signalosome subunit 6 (CSN6) in regulating cancer stemness was evaluated by organoid formation and limited dilution analysis. The role of CSN6–TRIM21–OCT1–ALDH1A1 axis in CSC formation was evaluated in vitro and in vivo. The association of CSN6, TRIM21 and ALDH1A1 expression was validated by a tissue microarray with 267 CRC patients.ResultsThe results showed that CSN6 is critical for sphere formation and maintaining the growth of patient-derived organoids (PDOs). We characterised the role of CSN6 in regulating cancer stemness, which involves the TRIM21 E3 ubiquitin ligase, transcription factor POU class 2 homeobox 1 (OCT1) and cancer stem cell marker aldehyde dehydrogenase 1 A1 (ALDH1A1). Our data showed that CSN6 facilitates ubiquitin-mediated degradation of TRIM21, which in turn decreases TRIM21-mediated OCT1 ubiquitination and subsequently stabilises OCT1. Consequently, OCT1 stabilisation leads to ALDH1A1expression and promotes cancer stemness. We further showed that the protein expression levels of CSN6, TRIM21 and ALDH1A1 can serve as prognostic markers for human CRC.ConclusionsIn conclusion, we validate a pathway for cancer stemness regulation involving ALDH1A1 levels through the CSN6–TRIM21 axis, which may be utilised as CRC molecular markers and be targeted for therapeutic intervention in cancers.

Basis for metabolite-dependent Cullin-RING ligase deneddylation by the COP9 signalosome

The Cullin-RING ligases (CRLs) are the largest family of ubiquitin E3s activated by neddylation and regulated by the deneddylase COP9 signalosome (CSN). The inositol polyphosphate metabolites promote the formation of CRL-CSN complexes, but with unclear mechanism of action. Here, we provide structural and genetic evidence supporting inositol hexakisphosphate (IP6) as a general CSN cofactor recruiting CRLs. We determined the crystal structure of IP6 in complex with CSN subunit 2 (CSN2), based on which we identified the IP6-corresponding electron density in the cryoelectron microscopy map of a CRL4A-CSN complex. IP6 binds to a cognate pocket formed by conserved lysine residues from CSN2 and Rbx1/Roc1, thereby strengthening CRL-CSN interactions to dislodge the E2 CDC34/UBE2R from CRL and to promote CRL deneddylation. IP6 binding-deficient Csn2K70E/K70E knockin mice are embryonic lethal. The same mutation disabled Schizosaccharomyces pombe Csn2 from rescuing UV-hypersensitivity of csn2-null yeast. These data suggest that CRL transition from the E2-bound active state to the CSN-bound sequestered state is critically assisted by an interfacial IP6 small molecule, whose metabolism may be coupled to CRL-CSN complex dynamics.

Cullin-RING Ligase Regulation by the COP9 Signalosome: Structural Mechanisms and New Physiologic Players.

The Cullin-RING E3 ligases (CRLs) are major ubiquitylation machineries regulated by reversible cycles of neddylation and deneddylation. The deneddylase COP9 Signalosome (CSN) terminates CRL catalytic cycle. CSN also provides a docking platform for several kinases and deubiquitinases that might play a role in regulating CRL. Recently, remarkable progress has been made in elucidating the biochemical principles and physiological implications of such exquisite regulation. The cryo-EM structures of CRL-CSN complexes provide the biochemical basis of their cognate interactions and reveal potential regulatory mechanisms during complex disassembly. Moreover, novel players beyond the canonical eight subunits of CSN were identified. This includes CSNAP, a potential 9th CSN subunit with regulatory functions, and the metabolite inositol hexakisphosphate (IP6), which enhances CRL-CSN complex formation, with IP6-metabolizing enzymes possibly instilling dynamics to the CRL-CSN system. Here, we review and summarize these new mechanistic insights along with progress in understanding CSN biology based on model organisms with genetically edited CSN subunits.

The Salix SmSPR1 Involved in Light-Regulated Cell Expansion by Modulating Microtubule Arrangement

Light signaling and cortical microtubule (MT) arrays are essential to the anisotropic growth of plant cells. Microtubule-associated proteins (MAPs) function as regulators that mediate plant cell expansion or elongation by altering the arrangements of the MT arrays. However, current understanding of the molecular mechanism of MAPs in relation to light to regulate cell expansion or elongation is limited. Here, we show that the MPS SPR1 is involved in light-regulated directional cell expansion by modulating microtubule arrangement. Overexpression of SmSPR1 in Arabidopsis results in right-handed helical orientation of hypocotyls in dark-grown etiolated seedlings, whereas the phenotype of transgenic plants was indistinguishable from those of wild-type plants under light conditions. Phenotypic characterization of the transgenic plants showed reduced anisotropic growth and left-handed helical MT arrays in etiolated hypocotyl cells. Protein interaction assays revealed that SPR1, CSN5A (subunits of COP9 signalosome, a negative regulator of photomorphogenesis), and ELONGATED HYPOCOTYL 5 (HY5, a transcription factor that promotes photomorphogenesis) interacted with each other in vivo. The phenotype of Arabidopsis AtSPR1-overexpressing transgenic lines was similar to that of SmSPR1-overexpressing transgenic plants, and overexpression of Salix SmSPR1 can rescue the spr1 mutant phenotype, thereby revealing the function of SPR1 in plants.

COP9 Signalosome CSN4 and CSN5 Subunits Are Involved in Jasmonate-Dependent Defense Against Root-Knot Nematode in Tomato.

COP9 signalosome (CSN) is an evolutionarily conserved regulatory component of the ubiquitin/proteasome system that plays crucial roles in plant growth and stress tolerance; however, the mechanism of COP9-mediated resistance to root-knot nematodes (RKNs, e.g. Meloidogyne incognita) is not fully understood in plants. In the present study, we found that RKN infection in the roots rapidly increases the transcript levels of CSN subunits 4 and 5 (CSN4 and CSN5) and their protein accumulation in tomato (Solanum lycopersicum) plants. Suppression of CSN4 or CSN5 expression resulted in significantly increased number of egg masses and aggravated RKN-induced lipid peroxidation of cellular membrane but inhibited RKN-induced accumulation of CSN4 or CSN5 protein in tomato roots. Importantly, the RKN-induced accumulation of jasmonic acid (JA) and JA-isoleucine (JA-Ile), as well as the transcript levels of JA-related biosynthetic and signaling genes were compromised by CSN4 or CSN5 gene silencing. Moreover, protein–protein interaction assays demonstrated that CSN4 and CSN5B interact with the jasmonate ZIM domain 2 (JAZ2), which is the signaling component of the JA pathway. Silencing of CSN4 or CSN5 also compromises RKN-induced JAZ2 expression. Together, our findings indicate that CSN4 and CSN5 play critical roles in JA-dependent basal defense against RKN.

Chemogenomic study of gemcitabine using Saccharomyces cerevisiae as model cell-molecular insights about chemoresistance.

Gemcitabine (GEM) is the drug used as first line to treat pancreatic cancer, one of the most devastating human tumors. This peculiar type of tumor develops resistance to several drugs, including GEM, due to its desmoplastic reaction and other features. The GEM chemoresistance has been investigated at molecular level aiming to find a pathway whose inhibition or activation should overcome it. Through next-generation sequencing was performed a chemogenomic assay of GEM using Saccharomyces cerevisiae as model cell and the results showed that more than 40% of genes related to GEM response in yeast possess unknown or dubious function. We choose two yeast mutants to individually validate the fitness defect results observed by chemogenomic assay, Δhmt1 and Δcsi1, and it was found that in addition to some already described pathways involved in GEM resistance, cells deficient in deneddylation enzyme Cop9 Signalosome Interactor 1 (Csi1p) presented a high sensitivity to GEM. This was confirmed by individual growth analyses of Δcsi1 cells exposed to GEM, and this phenotype was reverted with CSI1 complementation gene. Csi1p is a well-characterized homolog equivalent to human Csn6 subunit of COP9 signalosome (CSN) involved in deneddylation process. We highlighted too that epigenetic alterations, such as methylation mediated by protein arginine methyltransferase 1, play an important role in regulating gemcitabine treatment resistance. Our results point out new unexplored molecular pathways that can be used to overcome GEM resistance: the inhibition of CSN and the arginine methyltransferase activities.

COP9 Signalosome Interaction with UspA/Usp15 Deubiquitinase Controls VeA-Mediated Fungal Multicellular Development.

COP9 signalosome (CSN) and Den1/A deneddylases physically interact and promote multicellular development in fungi. CSN recognizes Skp1/cullin-1/Fbx E3 cullin-RING ligases (CRLs) without substrate and removes their posttranslational Nedd8 modification from the cullin scaffold. This results in CRL complex disassembly and allows Skp1 adaptor/Fbx receptor exchange for altered substrate specificity. We characterized the novel ubiquitin-specific protease UspA of the mold Aspergillus nidulans, which corresponds to CSN-associated human Usp15 and interacts with six CSN subunits. UspA reduces amounts of ubiquitinated proteins during fungal development, and the uspA gene expression is repressed by an intact CSN. UspA is localized in proximity to nuclei and recruits proteins related to nuclear transport and transcriptional processing, suggesting functions in nuclear entry control. UspA accelerates the formation of asexual conidiospores, sexual development, and supports the repression of secondary metabolite clusters as the derivative of benzaldehyde (dba) genes. UspA reduces protein levels of the fungal NF-kappa B-like velvet domain protein VeA, which coordinates differentiation and secondary metabolism. VeA stability depends on the Fbx23 receptor, which is required for light controlled development. Our data suggest that the interplay between CSN deneddylase, UspA deubiquitinase, and SCF-Fbx23 ensures accurate levels of VeA to support fungal development and an appropriate secondary metabolism.

Role of Cop9 Signalosome Subunits in the Environmental and Hormonal Balance of Plant.

The COP9 (Constitutive photomorphogenesis 9) signalosome (CSN) is a highly conserved protein complex that influences several signaling and developmental processes. The COP9 signalosome consists of eight subunits, among which two subunits, CSN5 and CSN6, contain an Mpr1/Pad1 N-terminal (MPN) domain and the remaining six subunits contain a proteasome, COP9 signalosome, and initiation factor 3 (PCI) domain. In plants, each MPN subunit is encoded by two genes, which is not the case in other organisms. This review aims to provide in-depth knowledge of each COP9 signalosome subunit, concentrating on genetic analysis of both partial and complete loss-of-function mutants. At the beginning of this review, the role of COP9 signalosome in the hormonal signaling and defense is discussed, whereas later sections deal in detail with the available partial loss-of-function, hypomorphic mutants of each subunit. All available hypomorphic mutants are compared based on their growth response and deneddylation activity.

Asrij/OCIAD1 suppresses CSN5-mediated p53 degradation and maintains mouse hematopoietic stem cell quiescence

AbstractInactivation of the tumor suppressor p53 is essential for unrestrained growth of cancers. However, only 11% of hematological malignancies have mutant p53. Mechanisms that cause wild-type p53 dysfunction and promote leukemia are inadequately deciphered. The stem cell protein Asrij/OCIAD1 is misexpressed in several human hematological malignancies and implicated in the p53 pathway and DNA damage response. However, Asrij function in vertebrate hematopoiesis remains unknown. We generated the first asrij null (knockout [KO]) mice and show that they are viable and fertile with no gross abnormalities. However, by 6 months, they exhibit increased peripheral blood cell counts, splenomegaly, and an expansion of bone marrow hematopoietic stem cells (HSCs) with higher myeloid output. HSCs lacking Asrij are less quiescent and more proliferative with higher repopulation potential as observed from serial transplantation studies. However, stressing KO mice with sublethal γ irradiation or multiple injections of 5-fluorouracil results in reduced survival and rapid depletion of hematopoietic stem/progenitor cells (HSPCs) by driving them into proliferative exhaustion. Molecular and biochemical analyses revealed increased polyubiquitinated protein levels, Akt/STAT5 activation and COP9 signalosome subunit 5 (CSN5)-mediated p53 ubiquitination, and degradation in KO HSPCs. Further, we show that Asrij sequesters CSN5 via its conserved OCIA domain, thereby preventing p53 degradation. In agreement, Nutlin-3 treatment of KO mice restored p53 levels and reduced high HSPC frequencies. Thus, we provide a new mouse model resembling myeloproliferative disease and identify a posttranslational regulator of wild-type p53 essential for maintaining HSC quiescence that could be a potential target for pharmacological intervention.

Discovery of Protein Degradation Machinery at the Desmosome Reveals Novel Triggers of the Desmosomal Disease, Arrhythmogenic Right Ventricular Cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic‐based cardiac disease which is characterized by right/bi‐ventricular dysfunction, fibro‐fatty replacement of the myocardium and ventricular arrhythmias, leading to sudden death in young people and athletes. ARVC is termed a “disease of the desmosome” (mechanical cell‐cell junction) as 40% of ARVC patients harbor mutations and/or loss of desmosomal components. However, limited information exists on how cardiac desmosomal protein loss/degradation triggers ARVC. Through a yeast‐two‐hybrid screen in an adult human heart cDNA library, we uncovered a new interaction between the desmosomal protein, desmoplakin (DSP, N terminus) and a protein degradation machinery component, CSN6 (COP9 signalosome subunit 6). Traditionally, CSN proteins are thought to prevent proteins from being degraded; however, a role for CSN6 in the heart is undefined. We show that CSN6 localizes to desmosomal junctions in cardiac muscle and structurally complex with desmoplakin, suggesting that the novel hypothesis that CSN6 may protect the cardiac desmosome from degradation and loss/destablization this pathway may lead to ARVC. Using two novel cardiac‐specific CSN6 knockout (CSN6‐KO) mice, we further show that CSN6 loss accelerates desmosome destruction and triggers ARVC, as CSN6‐KO hearts exhibit selective loss of desmosomal proteins and recapitulate the key cardiomyopathy features and sudden death associated with ARVC, similar to classic mouse model of ARVC (DSP‐cKO). CSN6‐KO and DSP‐cKO hearts also harbor underlying hyper‐accumulation of protein degradation machinery specifically at cell‐cell junctions. Restoration of CSN function by the protein degradation inhibitor (MLN4924) could rescue the loss of desmosomal protein expression associated with CSN6 loss in vitro, further highlighting that the cardiac desmosome is regulated by protein degradation. CSN6 pathways are also relevant to human ARVC as a N‐terminus desmoplakin mutation (R315C) identified in a human ARVC patient was sufficient to abrogate DSP‐CSN6 binding and result in loss of CSN6 localization at the cell‐cell junction in cardiac biopsies. DSP R315C knock‐in mice also exhibited cardiac CSN6 reduction and early electrical defects found in ARVC. We altogether identify CSN6 as a novel “brake” to prevent desmosomal protein degradation, and reveal that disruption of this complex is an important “accelerator” of sudden death and ARVC.Support or Funding InformationAmerican Heart Association Postdoctoral Fellowship 17POST33670205This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of COP9 Signalosome Subunit Genes in Bactrocera dorsalis and Functional Analysis of csn3 in Female Fecundity.

The COP9 signalosome (CSN) is an evolutionarily conserved multi-subunit complex that plays crucial roles in regulating various biological processes in plants, mammals, and the model insect Drosophila. However, it is poorly studied in non-model insects, whereas its role in fecundity remains unclear. In this study, all nine CSN subunits were identified and characterized in Bactrocera dorsalis, a major invasive agricultural tephritid pest. Each subunit gene, except for csn9x1, encoded a protein containing a PCI/PINT or MPN domain. Phylogenetic analysis revealed that all CSN subunits were individually clustered into a specific branch with their counterparts from other species. All CSN subunit genes were expressed in all detected developmental stages and tissues. Most subunits, except for csn8 and csn9x1, showed the highest expression level in the eggs. Notably, csn3 and csn5 were significantly enriched in mature female adults. Further analysis of csn3 revealed that it was enriched in the ovary and that its ovarian expression level gradually increased with the reproductive development process. RNAi-based knockdown of csn3 in female adults significantly reduced the number of laid eggs. The expression level of EcRB1 and USP, which encode the heterodimer receptors of 20E, and vitellogenin transcripts (Vg1 and Vg2) was suppressed in the fat body of female adults injected with csn3dsRNA. Decreased level of Vg1 protein was confirmed by means of Western blots. These data indicate that csn3 is involved in female reproduction by regulating 20E signaling and Vg synthesis. Overall, our study may facilitate the development of new strategies for controlling B. dorsalis since it provides insights into the evolution and expression patterns of all CSN subunit genes as well as the critical roles of csn3 in female fecundity.

CSN5 promotes the invasion and metastasis of pancreatic cancer by stabilization of FOXM1

COP9 signalosome subunit 5 (CSN5) has been involved in the progression of diverse human cancers. MMP2 plays an important role in the metastasis of cancer cells. However, the roles and relationship of in pancreatic cancer (PC) is still unknown. Here, our data shown that both CSN5 and MMP2 were significantly upregulated in PC compared with the corresponding adjacent tissues, where a positive correlation in their expression and associated malignant characteristics were found. Further, silencing of CSN5 expression markedly inhibited PC invasion and metastasis in vitro and in vivo, accompanied by decreased MMP2 expression. Moreover, the anti-metastasis role of CSN5 silence was reversed by MMP2 overexpression, whereas knockdown of MMP2 decreased PC metastasis driven by upregulation of CSN5. Further investigation revealed that CSN5 regulated MMP2 expression via activation of FOXM1 in PC cells. Mechanistically, CSN5 directly bound FOXM1 and decreased its ubiquitination to enhance the protein stability of FOXM1. Taken together, the results indicate that CSN5 can contribute to PC invasion and metastasis through activation of FOXM1/MMP2 axis.

Renal COP9 Signalosome Deficiency Alters CUL3-KLHL3-WNK Signaling Pathway.

The familial hyperkalemic hypertension (FHHt) cullin 3 (CUL3) mutant does not degrade WNK kinases normally, thereby leading to thiazide-sensitive Na-Cl cotransporter (NCC) activation. CUL3 mutant (CUL3Δ9) does not bind normally to the COP9 signalosome (CSN), a deneddylase involved in regulating cullin-RING ligases. CUL3Δ9 also caused increased degradation of the CUL3-WNK substrate adaptor kelch-like 3 (KLHL3). Here, we sought to determine how defective CSN action contributes to the CUL3Δ9 phenotype. The Pax8/LC1 mouse system was used to generate mice in which the catalytically active CSN subunit, Jab1, was deleted only along the nephron, after full development (KS-Jab1-/-). Western blot analysis demonstrated that Jab1 deletion increased the abundance of neddylated CUL3. Moreover, total CUL3 expression was reduced, suggesting decreased CUL3 stability. KLHL3 was almost completely absent in KS-Jab1-/- mice. Conversely, the protein abundances of WNK1, WNK4, and SPAK kinases were substantially higher. Activation of WNK4, SPAK, and OSR1 was indicated by higher phosphorylated protein levels and translocation of the proteins into puncta, as observed by immunofluorescence. The ratio of phosphorylated NCC to total NCC was also higher. Surprisingly, NCC protein abundance was low, likely contributing to hypokalemia and Na+ and K+ wasting. Additionally, long-term Jab1 deletion resulted in kidney damage. Together, the results indicate that deficient CSN binding contributes importantly to the FHHt phenotype. Although defective CUL3Δ9-faciliated WNK4 degradation likely contributes, dominant effects on KLHL3 may be a second factor that is necessary for the phenotype.