COP9 Signalosome Research Articles

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.

Computational exploration of the significance of COPS6 in cancer: Functional and clinical relevance across tumor types.

The COP9 signalosome subunit 6 (COPS6) has been implicated in cancer progression, while its precise role in most types of cancer remains elusive. To investigate the functional and clinical relevance of COPS6 across various tumor types using publicly available databases. We used R software and online analysis databases to analyze the differential expression, prognosis, mutation and related functions of COPS6 in pan-cancer. Differential expression analysis and survival analysis demonstrated that COPS6 was highly expressed and associated with high-risk profiles in the majority of cancer types. Possible associations between COPS6 expression level and prognostic outcomes were found using data from public databases. Mutational analysis revealed that missense mutations were the predominant type of COPS6 mutation. Additionally, positive correlations were identified between COPS6 expression level and tumor mutational burden and microsatellite instability in most types of cancer. Immune infiltration analysis demonstrated a negative correlation between COPS6 expression level and CD8+ T cell infiltration in certain types of cancer. The correlation between COPS6 expression level and cancer-associated fibroblast infiltration exhibited heterogeneity, in which a positive correlation was found in head and neck squamous cell carcinoma and tenosynovial giant cell tumor, and a negative correlation was identified in diffuse large B-cell lymphoma and thymoma. The correlation between COPS6 expression level and macrophage infiltration was closely related to macrophage type. Gene co-expression and enrichment analysis highlighted transcription elongation factor B polypeptide 2 and G protein pathway suppressor 1 were significantly and positively associated with COPS6 expression level. These genes were predominantly involved in processes, such as ubiquitin-mediated proteolysis and human immunodeficiency virus 1 infection. In conclusion, this study systematically explored the significance of COPS6 across different tumor types, providing a solid foundation for considering COPS6 as a novel biomarker in cancer research.

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.

Extracellular macrophage migration inhibitory factor (MIF) downregulates adipose hormone-sensitive lipase (HSL) and contributes to obesity

Attenuation of adipose hormone sensitive lipase (HSL) may impair lipolysis and exacerbate obesity. We investigate the role of cytokine, macrophage migration inhibitory factor (MIF) in regulating adipose HSL and adipocyte hypertrophy. Extracellular MIF downregulates HSL in an autocrine fashion, by activating the AMPK/JNK signaling pathway upon binding to its membrane receptor, CD74. WT mice fed high fat diet (HFD), as well as mice overexpressing MIF, both had high circulating MIF levels and showed suppression of HSL during the development of obesity. Blocking the extracellular action of MIF by a neutralizing MIF antibody significantly reduced obesity in HFD mice. Interestingly, intracellular MIF binds with COP9 signalosome subunit 5 (Csn5) and JNK, which leads to an opposing effect to inhibit JNK phosphorylation. With global MIF deletion, adipocyte JNK phosphorylation increased, resulting in decreased HSL expression, suggesting that the loss of MIF's intracellular inhibitory action on JNK was dominant in Mif−/− mice. Adipose tissue from Mif−/− mice also exhibited higher Akt and lower PKA phosphorylation following HFD feeding compared with WT, which may contribute to the downregulation of HSL activation during more severe obesity. Both intracellular and extracellular MIF have opposing effects to regulate HSL, but extracellular actions predominate to downregulate HSL and exacerbate the development of obesity during HFD.

A plant cytorhabdovirus modulates locomotor activity of insect vectors to enhance virus transmission

Transmission of many plant viruses relies on phloem-feeding insect vectors. However, how plant viruses directly modulate insect behavior is largely unknown. Barley yellow striate mosaic virus (BYSMV) is transmitted by the small brown planthopper (SBPH, Laodelphax striatellus). Here, we show that BYSMV infects the central nervous system (CNS) of SBPHs, induces insect hyperactivity, and prolongs phloem feeding duration. The BYSMV accessory protein P6 interacts with the COP9 signalosome subunit 5 (LsCSN5) of SBPHs and suppresses LsCSN5-regulated de-neddylation from the Cullin 1 (CUL1), hereby inhibiting CUL1-based E3 ligases-mediated degradation of the circadian clock protein Timeless (TIM). Thus, virus infection or knockdown of LsCSN5 compromises TIM oscillation and induces high insect locomotor activity for transmission. Additionally, expression of BYSMV P6 in the CNS of transgenic Drosophila melanogaster disturbs circadian rhythm and induces high locomotor activity. Together, our results suggest the molecular mechanisms whereby BYSMV modulates locomotor activity of insect vectors for transmission.

Fungal COP9 signalosome assembly requires connection of two trimeric intermediates for integration of intrinsic deneddylase

The conserved eight-subunit COP9 signalosome (CSN) is required for multicellular fungal development. The CSN deneddylase cooperates with the Cand1 exchange factor to control replacements of E3 ubiquitin cullin RING ligase receptors, providing specificity to eukaryotic protein degradation. Aspergillus nidulans CSN assembles through a heptameric pre-CSN, which is activated by integration of the catalytic CsnE deneddylase. Combined genetic and biochemical approaches provided the assembly choreography within a eukaryotic cell for native fungal CSN. Interactomes of functional GFP-Csn subunit fusions in pre-CSN deficient fungal strains were compared by affinity purifications and mass spectrometry. Two distinct heterotrimeric CSN subcomplexes were identified as pre-CSN assembly intermediates. CsnA-C-H and CsnD-F-G form independently of CsnB, which connects the heterotrimers to a heptamer and enables subsequent integration of CsnE to form the enzymatically active CSN complex. Surveillance mechanisms control accurate Csn subunit amounts and correct cellular localization for sequential assembly since deprivation of Csn subunits changes the abundance and location of remaining Csn subunits.

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.

Systematic analysis of the Frazzled receptor interactome establishes previously unreported regulators of axon guidance.

The Netrin receptor Dcc and its Drosophila homolog Frazzled play crucial roles in diverse developmental process, including axon guidance. In Drosophila, Fra regulates midline axon guidance through a Netrin-dependent and a Netrin-independent pathway. However, what molecules regulate these distinct signaling pathways remain unclear. To identify Fra-interacting proteins, we performed affinity purification mass spectrometry to establish a neuronal-specific Fra interactome. In addition to known interactors of Fra and Dcc, including Netrin and Robo1, our screen identified 85 candidate proteins, the majority of which are conserved in humans. Many of these proteins are expressed in the ventral nerve cord, and gene ontology, pathway analysis and biochemical validation identified several previously unreported pathways, including the receptor tyrosine phosphatase Lar, subunits of the COP9 signalosome and Rho-5, a regulator of the metalloprotease Tace. Finally, genetic analysis demonstrates that these genes regulate axon guidance and may define as yet unknown signaling mechanisms for Fra and its vertebrate homolog Dcc. Thus, the Fra interactome represents a resource to guide future functional studies.

The C-terminal tail of CSNAP attenuates the CSN complex.

Protein degradation is one of the essential mechanisms that enables reshaping of the proteome landscape in response to various stimuli. The largest E3 ubiquitin ligase family that targets proteins to degradation by catalyzing ubiquitination is the cullin-RING ligases (CRLs). Many of the proteins that are regulated by CRLs are central to tumorigenesis and tumor progression, and dysregulation of the CRL family is frequently associated with cancer. The CRL family comprises ∼300 complexes, all of which are regulated by the COP9 signalosome complex (CSN). Therefore, CSN is considered an attractive target for therapeutic intervention. Research efforts for targeted CSN inhibition have been directed towards inhibition of the complex enzymatic subunit, CSN5. Here, we have taken a fresh approach focusing on CSNAP, the smallest CSN subunit. Our results show that the C-terminal region of CSNAP is tightly packed within the CSN complex, in a groove formed by CSN3 and CSN8. We show that a 16 amino acid C-terminal peptide, derived from this CSN-interacting region, can displace the endogenous CSNAP subunit from the complex. This, in turn, leads to a CSNAP null phenotype that attenuates CSN activity and consequently CRLs function. Overall, our findings emphasize the potential of a CSNAP-based peptide for CSN inhibition as a new therapeutic avenue.

Phytophthora RxLR effector PcSnel4B promotes degradation of resistance protein AtRPS2.

Phytophthora capsici deploys effector proteins to manipulate host immunity and facilitate its colonization. However, the underlying mechanisms remain largely unclear. In this study, we demonstrated that a Sne-like (Snel) RxLR effector gene PcSnel4 is highly expressed at the early stages of P. capsici infection in Nicotiana benthamiana. Knocking out both alleles of PcSnel4 attenuated the virulence of P. capsici, while expression of PcSnel4 promoted its colonization in N. benthamiana. PcSnel4B could suppress the hypersensitive reaction (HR) induced by Avr3a-R3a and RESISTANCE TO PSEUDOMONAS SYRINGAE 2 (AtRPS2), but it did not suppress cell death elicited by Phytophthora infestin 1 (INF1) and Crinkler 4 (CRN4). COP9 signalosome 5 (CSN5) in N. benthamiana was identified as a host target of PcSnel4. Silencing NbCSN5 compromised the cell death induced by AtRPS2. PcSnel4B impaired the interaction and colocalization of Cullin1 (CUL1) and CSN5 in vivo. Expression of AtCUL1 promoted the degradation of AtRPS2 and disrupted HR, while AtCSN5a stabilized AtRPS2 and promoted HR, regardless of the expression of AtCUL1. PcSnel4 counteracted the effect of AtCSN5 and enhanced the degradation of AtRPS2, resulting in HR suppression. This study deciphered the underlying mechanism of PcSnel4-mediated suppression of HR induced by AtRPS2.

Associations of circulating proteins with lipoprotein profiles: proteomic analyses from the OmniHeart randomized trial and the Atherosclerosis Risk in Communities (ARIC) Study

BackgroundWithin healthy dietary patterns, manipulation of the proportion of macronutrient can reduce CVD risk. However, the biological pathways underlying healthy diet-disease associations are poorly understood. Using an untargeted, large-scale proteomic profiling, we aimed to (1) identify proteins mediating the association between healthy dietary patterns varying in the proportion of macronutrient and lipoproteins, and (2) validate the associations between diet-related proteins and lipoproteins in the Atherosclerosis Risk in Communities (ARIC) Study.MethodsIn 140 adults from the OmniHeart trial, a randomized, cross-over, controlled feeding study with 3 intervention periods (carbohydrate-rich; protein-rich; unsaturated fat-rich dietary patterns), 4,958 proteins were quantified at the end of each diet intervention period using an aptamer assay (SomaLogic). We assessed differences in log2-transformed proteins in 3 between-diet comparisons using paired t-tests, examined the associations between diet-related proteins and lipoproteins using linear regression, and identified proteins mediating these associations using a causal mediation analysis. Levels of diet-related proteins and lipoprotein associations were validated in the ARIC study (n = 11,201) using multivariable linear regression models, adjusting for important confounders.ResultsThree between-diet comparisons identified 497 significantly different proteins (protein-rich vs. carbohydrate-rich = 18; unsaturated fat-rich vs. carbohydrate-rich = 335; protein-rich vs. unsaturated fat-rich dietary patterns = 398). Of these, 9 proteins [apolipoprotein M, afamin, collagen alpha-3(VI) chain, chitinase-3-like protein 1, inhibin beta A chain, palmitoleoyl-protein carboxylesterase NOTUM, cathelicidin antimicrobial peptide, guanylate-binding protein 2, COP9 signalosome complex subunit 7b] were positively associated with lipoproteins [high-density lipoprotein (HDL)-cholesterol (C) = 2; triglyceride = 5; non-HDL-C = 3; total cholesterol to HDL-C ratio = 1]. Another protein, sodium-coupled monocarboxylate transporter 1, was inversely associated with HDL-C and positively associated with total cholesterol to HDL-C ratio. The proportion of the association between diet and lipoproteins mediated by these 10 proteins ranged from 21 to 98%. All of the associations between diet-related proteins and lipoproteins were significant in the ARIC study, except for afamin.ConclusionsWe identified proteins that mediate the association between healthy dietary patterns varying in macronutrients and lipoproteins in a randomized feeding study and an observational study.Trial registrationNCT00051350 at clinicaltrials.gov.

Hormone profiling and the root proteome analysis of itpk1 mutant seedlings of barley (Hordeum vulgare) during the red-light induced photomorphogenesis

Inositol phosphates represent crucial primary metabolites, known as phosphate storage molecules or molecules enabling protein interactions. Inositol phosphates and their pyrophosphate forms direct the assembly of the COP9 signalosome and Cullin Ring Ligase complexes. Thus, inositol phosphates participate on protein degradation, which is a means of signal transduction for several plant hormone classes and light signaling cascade. In plants, production of inositol phosphates is maintained by several inositol phosphate kinases including the group of inositol-1,3,4-trisphosphate 5/6 kinases (ITPKs). The members of ITPKs connect plant phosphate status with light perception and hormonal balance. Photomorphogenesis in red light was investigated using allelic variants of barley Hvitpk1 mutants produced by gene editing. Barley ITPK promoter regions, conserved protein motifs, and expression profiles were investigated to characterize their evolutionary diversification and transcriptional regulation. We focused on ITPK1 relation to auxins, cytokinins, gibberellins, jasmonates, abscisic acid, and salicylic acid in red light-induced photomorphogenesis. Global proteome analyses was conducted in the mutant and wild-type (WT) seedlings grown under continuous red light. Our results demonstrate that different allelic variants of itpk1 mutants manifested defects in producing important phytohormones with consequences for root growth. Mutations in itpk1 affected hormone biosynthesis and metabolite, while proteome analysis revealed significant differences in seedling root proteomes between mutant lines. Based on our results and published data, a model of ITPK activity is proposed depicting them as regulators of hormone signal transduction at the level of protein degradation.

Screen natural terpenoids to identify potential Jab1 inhibitors for treating breast cancer

Jab1 (c-Jun activation domain-binding protein-1) overexpression has been extensively linked to cancer development (or metastasis) in various malignancies by positively regulating cancer cell proliferation or inactivating several tumor suppressors. Recent research has focused on utilizing plant products to target crucial elements of dysregulated signaling pathways to elucidate a potent cancer therapeutic approach. Terpenoids have shown significant anti-inflammatory and anti-cancerous properties in a broader range of carcinomas by inducing apoptosis. Through an extensive literature search, we have selected only those terpenoids (from the NPACT database) that have not been explored against Jab1 (CSN5, COP9 signalosome subunit 5) in breast cancer for our research study. We have used two docking servers, PATCH DOCK, and CB DOCK, to find the binding interaction between selected terpenoids and Jab1. Further, we have also used SWISS ADME to investigate the pharmacokinetics of selected ligands. Amongst all selected ligands, lutein (belongs to the xanthophylls class) has displayed maximum binding energy in both CB Dock and Patch Dock analysis. Hence, our preliminary in silico results have shown lutein as the potent lead candidate for developing a better drug against breast cancer. However, more in silico and in vitro studies are still needed to validate the inhibitory potential of lutein terpenoid against Jab1 in breast cancer.

FBXW7β loss-of-function enhances FASN-mediated lipogenesis and promotes colorectal cancer growth

Continuous de novo fatty acid synthesis is required for the biosynthetic demands of tumor. FBXW7 is a highly mutated gene in CRC, but its biological functions in cancer are not fully characterized. Here, we report that FBXW7β, a FBXW7 isoform located in the cytoplasm and frequently mutated in CRC, is an E3 ligase of fatty acid synthase (FASN). Cancer-specific FBXW7β mutations that could not degrade FASN can lead to sustained lipogenesis in CRC. COP9 signalosome subunit 6 (CSN6), an oncogenic marker of CRC, increases lipogenesis via interacting with and stabilizing FASN. Mechanistic studies show that CSN6 associates with both FBXW7β and FASN, and antagonizes FBXW7β’s activity by enhancing FBXW7β autoubiquitination and degradation, which in turn prevents FBXW7β-mediated FASN ubiquitination and degradation, thereby regulating lipogenesis positively. Both CSN6 and FASN are positively correlated in CRC, and CSN6-FASN axis, regulated by EGF, is responsible for poor prognosis of CRC. The EGF-CSN6-FASN axis promotes tumor growth and implies a treatment strategy of combination of orlistat and cetuximab. Patient-derived xenograft experiments prove the effectiveness of employing orlistat and cetuximab combination in suppressing tumor growth for CSN6/FASN-high CRC. Thus, CSN6-FASN axis reprograms lipogenesis to promote tumor growth and is a target for cancer intervening strategy in CRC.

COPS3 inhibition promotes cell proliferation blockage and anoikis via regulating PFKFB3 in osteosarcoma cancer cells

As a key component of the COP9 signalosome complex, which participates in a variety of physiological processes, COPS3 is intimately related to multiple cancers. It promotes cell proliferation, progression and metastasis in several cancer cells. However, whether COPS3 participates in regulating anoikis, a specific kind of apoptosis and functions as an essential modulator of cell metastasis, has not yet been studied. Here, we found COPS3 is highly expressed in several cancers especially in osteosarcoma (OS). Overexpression of COPS3 promoted cell proliferation, cell viability and migration/invasion in both control cells and oxaliplatin (Oxa) treated cells. On the contrary, knockdown of COPS3 further enhanced the cytotoxicity of Oxa. Utilizing bioinformatics analysis, we found that COPS3 was higher expressed in the metastatic group, and associated with the extra-cellular matrix (ECM) receptor interaction pathway, which involve in regulating anoikis. In an anoikis model, COPS3 expression varied and genetic modification of COPS3 influenced the cell death enhanced by Oxa. PFKFB3, an essential modulator of glycolysis, was found to interact with COPS3. Inhibition of PFKFB3 promoted apoptosis and anoikis enhanced by Oxa, and COPS3 overexpression failed to rescue this cell death. On the contrary, in the COPS3 knockdown cells, overexpression of PFKFB3 recovered the anoikis resistance, indicating COPS3 function upstream of PFKFB3. In summary, our results elucidated that COPS3 modulated anoikis via affecting PFKFB3 in OS cancer cells.

Analysis of distal convoluted tubule (DCT) cell de-differentiation due to COP9 signalosome (CSN) dysfunction via enriched DCT single-nucleus RNA-sequencing

Cullin-RING-ligases (CRLs) are a group of E3 ubiquitin ligases that mediate the regulated degradation of proteins. All CRLs are regulated by the COP9 signalosome (CSN), which interacts with the CRL and turns off ubiquitin ligase activity. To investigate the role of impaired CSN dysfunction in the kidney, we inactivated the CSN by deleting Jab1 (the key CSN catalytic subunit) along the entire nephron. Even though the CSN is expressed all along the nephron, these mice showed remodeling only along the distal nephron, with shortening of the distal convoluted tubule (DCT) and a large reduction in DCT-specific proteins. The DCT is known to remodel due to changes in dietary salt intaked, drug administration, and human disease. Yet, the cellular and molecular basis for this plasticity is unclear. We hypothesized that CSN dysfunction causes de-differentiation of DCT cells leading to DCT remodeling. To test our hypothesis, inducible DCT-specific NCC-Cre-ERT2 mice were bred to INTACT (Isolation of Nuclei TAgged in specific Cell Types, which fluorescently labels nuclei) and Jab1 floxed mice to generate DCT-INTACT- Jab1 -/- mice. One adult male DCT-INTACT- Jab1 -/- mouse and one DCT-INTACT control mouse were treated with tamoxifen to induce nuclear GFP expression and to induce deletion of Jab1 only in DCT cells. Six-weeks post treatment, nuclei from one kidney cortex was isolated and the DCT was enriched using Fluorescence-Activated Nuclei Sorting (FANS). GFP-positive nuclei were used for single-nucleus RNA-sequencing (NovaSeq). Initial results showed that unbiased clustering and UMAP visualization revealed 7 clusters with most cells from the DCT (99.0% for control and 97.6% for Jab1 -/- mice), indicating that the enrichment process was highly successful. Both the control and Jab1 -/- mouse had similar proportions of DCT1 and DCT2 cells; however, we identified two specialized cell types (dDCT1 and dDCT2) which comprised a much larger proportion of the cells in the Jab1 -/- mouse (dDCT1: 2.9% in control vs 41.1% in Jab1 -/- ; dDCT2: 2.0% in control vs 13.9% in Jab1 -/- ). These cells were highly de-differentiated with lower expression of the canonical DCT identifier genes such as Egf and Umod for DCT1, Calb1 and S100g for DCT2, as well as Slc12a3. Both dDCT1 and dDCT2 cell populations had induced expression of the disease- or carcinoma-related genes Ano3, Nrg1, and Pvt1. In conclusion, Jab1 deletion causes de-differentiation of a population of DCT cells. Further examination of CSN dysfunction via DCT-enriched single-nucleus RNA-sequencing could lead to a better understanding of the molecular mechanisms involved in DCT remodeling. NIH K01DK120790, NIH R01DK51496, Foundation LeDucq: Transatlantic Network of Excellence 17CVD05 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A global genetic interaction network by single-cell imaging and machine learning.

Cellular and organismal phenotypes are controlled by complex gene regulatory networks. However, reference maps of gene function are still scarce across different organisms. Here, we generated synthetic genetic interaction and cell morphology profiles of more than 6,800 genes in cultured Drosophila cells. The resulting map of genetic interactions was used for machine learning-based gene function discovery, assigning functions to genes in 47 modules. Furthermore, we devised Cytoclass as a method to dissect genetic interactions for discrete cell states at the single-cell resolution. This approach identified an interaction of Cdk2 and the Cop9 signalosome complex, triggering senescence-associated secretory phenotypes and immunogenic conversion in hemocytic cells. Together, our data constitute a genome-scale resource of functional gene profiles to uncover the mechanisms underlying genetic interactions and their plasticity at the single-cell level.

The COP9 signalosome complex regulates fungal development and virulence in the wheat scab fungus Fusarium graminearum.

The COP9 signalosome (Csn) complex is an evolutionarily conserved complex that regulates various important cellular processes. However, the function of the Csn complex in pathogenic fungi remains elusive. Here, the distribution of Csn subunits in the fungal kingdom was surveyed, and their biological functions were systematically characterized in the fungal pathogen Fusarium graminearum, which is among the top 10 plant fungal pathogens. The results obtained from bioinformatic analyses suggested that the F. graminearum Csn complex consisted of seven subunits (Csn1-Csn7) and that Csn5 was the most conserved subunit across the fungi kingdom. Yeast two-hybrid assays demonstrated that the seven Csn subunits formed a complex in F. graminearum. The Csn complex was localized to both the nucleus and cytoplasm and necessary for hyphal growth, asexual and sexual development and stress response. Transcriptome profiling revealed that the Csn complex regulated the transcription abundance of TRI genes necessary for mycotoxin deoxynivalenol (DON) biosynthesis, subsequently regulating DON production to control fungal virulence. Collectively, the roles of the Csn complex in F. graminearum were comprehensively analyzed, providing new insights into the functions of the Csn complex in fungal virulence and suggesting that the complex may be a potential target for combating fungal diseases.

Tespa1 facilitates hematopoietic and leukemic stem cell maintenance by restricting c-Myc degradation

Hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs) have robust self-renewal potential, which is responsible for sustaining normal and malignant hematopoiesis, respectively. Although considerable efforts have been made to explore the regulation of HSC and LSC maintenance, the underlying molecular mechanism remains obscure. Here, we observe that the expression of thymocyte-expressed, positive selection-associated 1 (Tespa1) is markedly increased in HSCs after stresses exposure. Of note, deletion of Tespa1 results in short-term expansion but long-term exhaustion of HSCs in mice under stress conditions due to impaired quiescence. Mechanistically, Tespa1 can interact with CSN subunit 6 (CSN6), a subunit of COP9 signalosome, to prevent ubiquitination-mediated degradation of c-Myc protein in HSCs. As a consequence, forcing c-Myc expression improves the functional defect of Tespa1-null HSCs. On the other hand, Tespa1 is identified to be highly enriched in human acute myeloid leukemia (AML) cells and is essential for AML cell growth. Furthermore, using MLL-AF9-induced AML model, we find that Tespa1 deficiency suppresses leukemogenesis and LSC maintenance. In summary, our findings reveal the important role of Tespa1 in promoting HSC and LSC maintenance and therefore provide new insights on the feasibility of hematopoietic regeneration and AML treatment.

Latent CSN-CRL complexes are crucial for curcumin-induced apoptosis and recruited during adipogenesis to lipid droplets via small GTPase RAB18

The COP9 signalosome (CSN) and cullin-RING ubiquitin ligases (CRLs) form latent CSN-CRL complexes detectable in cells. We demonstrate that the CSN variants CSNCSN7A and CSNCSN7B preferentially bind to CRL3 or CRL4A and CRL4B, respectively. Interestingly, the interacting protein ubiquitin-specific protease 15 exclusively binds to latent CSNCSN7A-CRL3, while p27KIP attaches to latent CSNCSN7B-CRL4A complex. Inhibition of deneddylation by CSN5i-3 or neddylation by MLN4924 do not impede the formation of latent complexes. Latent CSNCSN7A-CRL3 and latent CSNCSN7B-CRL4A/B particles are essential for specific cellular functions. We found that curcumin-induced cell death requires latent CSNCSN7B-CRL4A. Knockout of CSN7B in HeLa cells leads to resistance against curcumin. Remarkably, the small GTPase RAB18 recruits latent CSNCSN7A-CRL3 complex to lipid droplets (LDs), where CRL3 is activated by neddylation, an essential event for LD formation during adipogenesis. Knockdown of CSN7A or RAB18 or destabilization of latent complexes by cutting off CSN7A C-terminal 201-275 amino acids blocks adipogenesis.