Advances in the prerequisite and consequence of STING downstream signalosomes

The cyclic GMP-AMP synthase(cGAS)-stimulator of interferon genes(STING)pathway is an evolving DNA-sensing mechanism involved in innate immunity and pathogen defense that has been optimized while remaining conserved.Aside from recognizing pathogens through conserved motifs,these receptors also detect aberrant or misplaced self-molecules as possible signs of perturbed homeostasis.Upon binding external or self-derived DNA,a mobile sec-ondary messenger 2′3′-cyclic GMP-AMP(cGAMP)is produced by cGAS and in turn activates its adapter STING in the endo-plasmic reticulum(ER).Resting-state or activated STING protein isfinely restricted by multiple degradation machin-eries.The post-translational changes of the STING protein,along with the regulatory machinery ofthe secret routes,limit the onset,strength and sustention of STING signal.STING experiences a conformational shift and relocates with TBK1 from the ER to perinuclear vesicles containing transcription factors,provoking the transcription activity of IRF3/IFN-I and NF-κB pathways,as well as to initiate a number of cellular processes that have been shown to alter the immune landscape in cancer,such as autophagy,NLRP3 inflamma-some,ER stress,and cell death.STING signal thus serves as a potent activator for immune mobilization yet also triggers immune-mediated pathology in tissues.Recent advances have established the vital role of STING in immune surveil-lance as well as tumorigenic process.This review provides an overview of the disparate outcomes of cancer attributed to the actions of pleiotropic and coordinated STING downstream signalosomes,along with the underlying mechanisms of STING function in pathologies,providing therapeutic impli-cations for new approaches in hunt for the next generation of cancer immunotherapy base on STING.

Evolution of COP9 Signalosome and Proteasome Lid Complex

The COP9 signalosome and the regulatory lid of the 26S proteasome are both eight-subunit protein complexes which are present in most eukaryotes. There is a one-to-one relationship between the corresponding subunits of the two protein complexes in terms of their size and amino acid sequences. Eight groups of subunits from the COP9 signalosome and the proteasome lid complex of different organisms are collected from all the databases at the NCBI website. The corresponding subunits of COP9 signalosome and proteasome lid complex share at least 12% amino acid identity and some conserved regions, and the conserved sites spread evenly over the entire length of the subunits, suggesting that the two complexes have a common evolutionary ancestor. Phylogenetic analyses based on the amino acid sequences of the corresponding subunits of two protein complexes indicate that every tree consists of two clades. The subunits from one of the two protein complexes of different organisms are grouped into one of the two clades respectively. The sequences of single-cell organisms are always the basal groups to that of multi-cell animal and plant species. These results imply that the duplication/divergence events of COP9 signalosome and regulatory lid of the proteasome genes have occurred before the divergence of single-cell and multi-cell eukaryotes, and the genes of the two complexes are independently evolved. The analyses of dN/dS correlation show significant Pearson's correlations between 21 and 15 pairs of subunit-encoding sequences within the COP9 signalosome and the proteasome lid complex respectively, suggesting that those subunits pairs might have related functions and interacted with one another, and resulted in co-evolution.

Targeting the COP9 signalosome for cancer therapy

The COP9 signalosome(CSN)is a highly conserved protein complex composed of 8 subunits(CSN1 to CSN8).The individual subunits of the CSN play essential roles in cell proliferation,tumorigenesis,cell cycle regulation,DNA damage repair,angiogenesis,and microenvironmental homeostasis.The CSN complex has an intrinsic metalloprotease that removes the ubiquitin-like activator NEDD8 from cullin-RING ligases(CRLs).Binding of neddylated CRLs to CSN is sensed by CSN4 and communicated to CSN5 with the assistance of CSN6,thus leading to the activation of deneddylase.Therefore,CSN is a crucial regulator at the intersection between neddylation and ubiquitination in cancer progression.Here,we summarize current understanding of the roles of individual CSN subunits in cancer progression.Furthermore,we explain how the CSN affects tumorigenesis through regulating transcription factors and the cell cycle.Finally,we discuss individual CSN subunits as potential therapeutic targets to provide new directions and strategies for cancer therapy.

The COP9 Signalosome Controls Adipocyte Differentiation by Regulating CHOP Protein Stability

Obesity is a serious health problem of our time. Dysfunction of adipogenesis, the differentiation of adipocytes, is a hallmark of obesity. Therefore here we investigate the role of the COP9 signalosome and of CHOP in the differentiation of LiSa-2 preadipocytes.

Berberine diminishes cancer cell PD-L1 expression and facilitates antitumor immunity via inhibiting the deubiquitination activity of CSN5

Programmed cell death-1(PD-1)/programmed cell death ligand-1(PD-L1)blocking therapy has become a major pillar of cancer immunotherapy.Compared with antibodies targeting,small-molecule checkpoint inhibitors which have favorable pharmacokinetics are urgently needed.Here we identified berberine(BBR),a proven anti-inflammation drug,as a negative regulator of PDL1 from a set of traditional Chinese medicine(TCM)chemical monomers.BBR enhanced the sensitivity of tumour cells to co-cultured T-cells by decreasing the level of PD-L1 in cancer cells.In addition,BBR exerted its antitumor effect in Lewis tumor xenograft mice through enhancing tumorinfiltrating T-cell immunity and attenuating the activation of immunosuppressive myeloid-derived suppressor cells(MDSCs)and regulatory T-cells(Tregs).BBR triggered PD-L1 degradation through ubiquitin(Ub)/proteasome-dependent pathway.Remarkably,BBR selectively bound to the glutamic acid76 of constitutive photomorphogenic-9 signalosome 5(CSN5)and inhibited PD-1/PD-L1 axis through its deubiquitination activity,resulting in ubiquitination and degradation of PD-L1.Our data reveals a previously unrecognized antitumor mechanism of BBR,suggesting BBR is small-molecule immune checkpoint inhibitor for cancer treatment.

The Arabidopsis COP9 SIGNALOSOME INTERACTING F-BOX KELCH 1 Protein Forms an SCF Ubiquitin Ligase and Regulates Hypocotyl Elongation

The regulation of protein turnover by the ubiquitin proteasome system （UPS） is a major posttranslational mechanism in eukaryotes. One of the key components of the UPS, the COP9 signalosome （CSN）, regulates ＇cullin-ring＇ E3 ubiquitin ligases. In plants, CSN participates in diverse cellular and developmental processes, ranging from light signaling to cell cycle control. In this work, we isolated a new plant-specific CSN-interacting F-box protein, which we denominated CFK1 （COP9 INTERACTING F-BOX KELCH 1）. We show that, in Arabidopsis thaliana, CFK1 is a component of a functional ubiquitin ligase complex. We also show that CFK1 stability is regulated by CSN and by proteasome-dependent proteoly- sis, and that light induces accumulation of the CFK1 transcript in the hypocotyl. Analysis of CFK1 knockdown, mutant, and overexpressing seedlings indicates that CFK1 promotes hypocotyl elongation by increasing cell size. Reduction of CSN levels enhances the short hypocotyl phenotype of CFKl-depleted seedlings, while complete loss of CSN activity sup- presses the Iong-hypocotyl phenotype of CFKl-overexpressing seedlings. We propose that CFK1 （and its regulation by CSN） is a novel component of the cellular mechanisms controlling hypocotyl elongation.

CSN1 inhibits c-Jun phosphorylation and down-regulates ectopic expression of JNK1

CSN1 is a component of the COP9 signalosome(CSN),a conserved protein complex with pleiotropic functions in many organs and cell types.CSN regulates ubiquitinproteasome dependent protein degradation via the deneddylation and the associated deubiquitination activities.In addition,CSN associates with protein kinases and modulates cell signaling,particularly the activator protein 1(AP-1)pathway.We have shown previously that CSN1 suppresses AP-1 transcription activity and inhibits ultraviolet(UV)and serum activation of c-fos expression.Here we show that CSN1 can inhibit phosphorylation of proto-oncogene c-Jun product and repress c-Jun dependent transcription.Further,CSN1 dramatically downregulates ectopic expression of c-Jun N-terminal kinase 1(JNK1)in cultured cells.The decline in JNK1 is not caused by excessive proteolysis or by 3′UTR-dependent mRNA instability,but by CSN1-dependent repression of one or multiple steps in transcriptional and posttranscriptional mechanisms.Thus,in contrast to CSN5/Jab1,which promotes AP-1 activity,CSN1 displays a negative effect on the AP-1 pathway.Finally,we discuss about the dynamic equilibrium of the CSN complexes in regulation of the AP-1 pathway.

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

NEDD8 conjugation of Cullin has an important role in ubiquitin-mediated protein degradation. The COP9 signalosome, of which CSN5 is the major catalytic subunit, is a major Cullin deneddylase, Another deneddylase, Deneddylase 1, has also been shown to process the Nedd8 precursor. In Drosophila, the DEN1 mutants do not have increased levels of Cullin neddylation, but instead show a significant decrease in neddylated Cullin. This characteristic decrease in neddylated Cullins in the DEN1^null background can be rescued by UAS-dDEN1^WT overexpression but not by overexpression of mature NEDDS, indicating that this phenotype is distinct from the NEDD8-processing function of DENI. We examined the role of DEN 1-CSN interaction in regulating Cullin neddylation. Overexpression of DEN1 in a CSN5^hypo background slightly reduced unneddylated Cullin levels. The CSN5, DEN1 double mutation partially rescues the premature lethality associated with the CSN5 single mutation. These results suggest that DEN1 regulates Cullin neddylation by suppressing CSN deneddylase activity.

Higher-order assemblies in immune signaling:supramolecular complexes and phase separation

Signaling pathways in innate and adaptive immunity play vital roles in pathogen recognition and the functions of immune cells.Higher-order assemblies have recently emerged as a central principle that governs immune signaling and,by extension,cellular communication in general.There are mainly two types of higher-order assemblies:1)ordered,solid-like large supramolecular complexes formed by stable and rigid protein-protein interactions,and 2)liquid-like phase-separated condensates formed by weaker and more dynamic intermolecular interactions.This review covers key examples of both types of higher-order assemblies in major immune pathways.By placing emphasis on the molecular structures of the examples provided,we discuss how their structural organization enables elegant mechanisms of signaling regulation.

The COP9 SIGNALOSOME Is Required for Postembryonic Meristem Maintenance in Arabidopsis thaliana

Cullin-RING E3 ligases （CRLs） regulate different aspects of plant development and are activated by modification of their cullin subunit with the ubiquitin-like protein NEDD8 （NEural precursor cell expressed Developmentally Down-regulated 8） （neddylation） and deactivated by NEDD8 removal （deneddylation）. The CONSTITUTIVELY PHOTOMORPHOGENIC9 （COP9） signalosome （CSN） acts as a molecular switch of CRLs activity by reverting their neddylation status, but its contribution to embryonic and early seedling development remains poorly characterized. Here, we analyzed the phenotypic defects of csn mutants and monitored the cullin deneddylation/neddylation ratio during embryonic and early seedling development. We show that while csn mutants can complete embryogenesis （albeit at a slower pace than wildtype） and are able to germinate （albeit at a reduced rate）, they progressively lose meristem activity upon germination until they become unable to sustain growth. We also show that the majority of cullin proteins are progressively neddylated during the late stages of seed maturation and become deneddylated upon seed germination. This developmentally regulated shift in the cullin neddylation status is absent in csn mutants. We conclude that the CSN and its cullin deneddylation activity are required to sustain postembryonic meristem function in Arabidopsis.

COP9 signalosome subunit PfCsnE regulates secondary metabolism and conidial formation in Pestalotiopsis fici

The COP9 signalosome(CSN) is a highly conserved multiprotein complex in all eukaryotes and involved in regulation of organism development. In filamentous fungi, several lines of evidence indicate that fungal development and secondary metabolism(SM) are mediated by the fifth subunit of CSN, called CsnE. Here we uncover a connection with CsnE and conidial formation as well as SM regulation in the plant endophytic fungus Pestalotiopsis fici. A homology search of the P. fici genome with CsnE, involved in sexual development and SM in Aspergillus nidulans, identified PfCsnE. Deletion of PfcsnE resulted in a mutant that stopped conidial production, but the conidia are recovered in a PfcsnE complemented strain. This indicates that PfCsnE is required for the formation of conidia. Secondary metabolite analysis demonstrated that the ΔPfcsnE strain produced more chloroisosulochrin, less ficiolide A production in comparison to wild type(WT). Transcriptome analysis of WT andΔPfcsnE strains indicated that PfcsnE impacts the expression levels of 8.37% of 14,797 annotated genes. Specifically, nine biosynthetic gene clusters(BGCs) were up-regulated and three BGCs were down-regulated by PfCsnE. Our results suggest that PfCsnE plays major roles in SM regulation and conidial development in P. fici.