Outlook

Unified nomenclature for the COP9 signalosome and its subunits: an essential regulator of development

The COP9 signalosome (CSN) and cullin-RING ubiquitin ligases (CRLs) form latent CSN-CRL complexes detectable in cells. We demonstrate that the CSN variants CSN^CSN7A and CSN^CSN7B preferentially bind to CRL3 or CRL4A and CRL4B, respectively. Interestingly, the interacting protein ubiquitin-specific protease 15 exclusively binds to latent CSN^CSN7A-CRL3, while p27^KIP attaches to latent CSN^CSN7B-CRL4A complex. Inhibition of deneddylation by CSN5i-3 or neddylation by MLN4924 do not impede the formation of latent complexes. Latent CSN^CSN7A-CRL3 and latent CSN^CSN7B-CRL4A/B particles are essential for specific cellular functions. We found that curcumin-induced cell death requires latent CSN^CSN7B-CRL4A. Knockout of CSN7B in HeLa cells leads to resistance against curcumin. Remarkably, the small GTPase RAB18 recruits latent CSN^CSN7A-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.

The COP9 signalosome (CSN) is a universal regulator of Cullin-RING ubiquitin ligases (CRLs) – a family of modular enzymes that control various cellular processes via timely degradation of key signaling proteins. The CSN, with its eight-subunit architecture, employs multisite binding of CRLs and inactivates CRLs by removing a small ubiquitin-like modifier named neural precursor cell–expressed, developmentally downregulated 8 (Nedd8). Besides the active site of the catalytic subunit CSN5, two allosteric sites are present in the CSN, one of which recognizes the substrate recognition module and the presence of CRL substrates, and the other of which can ‘glue’ the CSN-CRL complex by recruitment of inositol hexakisphosphate. In this review, we present recent findings on the versatile regulation of CSN-CRL complexes.

A variety of biological processes are regulated by posttranslational modifications. Posttranslational modifications including phosphorylation, ubiquitination, glycosylation, and proteolytic cleavage, control diverse physiological functions in the gastrointestinal tract. Therefore, a better understanding of their implications in intestinal diseases, including inflammatory bowel disease, irritable bowel syndrome, celiac disease, and colorectal cancer would provide a basis for the identification of novel biomarkers as well as attractive therapeutic targets. Posttranslational modifications can be common denominators, as well as distinct biomarkers, characterizing pathological differences of various intestinal diseases. This review provides experimental evidence that identifies changes in posttranslational modifications from patient samples, primary cells, or cell lines in intestinal disorders, and a summary of carefully selected information on the use of pharmacological modulators of protein modifications as therapeutic options.

Notch signaling pathway is a highly evolutionary conserved signaling pathway, which modulates many biological processes such as cell differentiation, tissue development and immune response. Our previous study revealed that Litopenaeus vannamei Notch (LvNotch) was involved in immune response by regulating reactive oxygen species (ROS) production in hemocytes. However, the immune regulatory networks mediated by LvNotch remain unclear in shrimp. In this study, 21 proteins that potentially interact with LvNotch were identified by GST pull-down and liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses. Among these proteins, COP9 signalosome complex subunit 1 (CSN1) was chosen for further studies due to its putative role in immune response. The interaction between LvNotch and LvCSN1 was confirmed by Far-Western blot and GST pull-down analyses. In vivo knockdown of LvNotch resulted in an increase in LvCSN1 expression in hemocytes, which suggest that the COP9 signalosome complex might be negatively regulated by LvNotch. In addition, in vivo silencing of LvNotch upregulated the expression of LvDorsal, LvTNFSF and LvCrustin2 (NF-κB pathway related-genes), while their expression decreased after LvCSN1 depletion. Collectively, the current results indicate that LvNotch negatively regulates the NF-κB pathway by modulating LvCSN1 in shrimp.

Although the Notch signaling pathway has been implicated in the regulation of immune response in vertebrates and invertebrates, the functions and immune-related interacting networks of Notch in shrimp immune response remain unknown. In this study, twenty-one proteins including COP9 signalosome complex subunit 1 (CSN1) were identified as potential interacting partners of LvNotch. Further analysis revealed that LvNotch negatively regulates the NF-κB pathway by binding to CSN1 and modulating its expression. These findings for the first time suggest that the Notch signaling pathway has cross-talk with the NF-κB pathway in shrimp as part of the immune response.

Mammalian COP9 signalosome (CSN) exists as two variant complexes containing either CSN7A or CSN7B paralogs of unknown functional specialization. Constructing knockout cells, we found that CSN7A and CSN7B have overlapping functions in the deneddylation of cullin-RING ubiquitin ligases. Nevertheless, CSN^CSN7B has a unique function in DNA double-strand break (DSB) sensing, being selectively required for ataxia telangiectasia mutated (ATM)-dependent formation of NBS1^S343p and γH2AX as well as DNA-damage-induced apoptosis triggered by mitomycin C and ionizing radiation. Live-cell microscopy revealed rapid recruitment of CSN7B but not CSN7A to DSBs. Resistance of CSN7B knockout cells to DNA damage is explained by the failure to deneddylate an upstream DSB signaling component, causing a switch in DNA repair pathway choice with increased utilization of non-homologous end joining over homologous recombination. In mice, CSN7B knockout tumors are resistant to DNA-damage-inducing chemotherapy, thus providing an explanation for the poor prognosis of tumors with low CSN7B expression.

Up-regulation of mammalian COP9 signalosome subunit 6 (CSN6) and consequent reduction of SCF ubiquitin ligase substrate receptor β-transduction repeat-containing protein (β-TrCP) have been shown to be associated with cancer cells proliferation. However, it is unclear whether CSN6 and β-TrCP are also involved in PDGF-induced pulmonary arterial smooth muscle cells (PASMCs) proliferation. This study aims to address this issue and further explore its potential mechanisms. Our results indicated that PDGF phosphorylated Akt, stimulated PASMCs proliferation; while inhibition of PDGF receptor (PDGFR) by imatinib prevented these effects. PDGF further up-regulated CSN6 protein expression, this was accompanied with β-TrCP reduction and increase of Cdc25A. Inhibition of PDGFR/PI3K/Akt signaling pathway reversed PDGF-induced such changes and cell proliferation. Prior transfection of CSN6 siRNA blocked PDGF-induced β-TrCP down-regulation, Cdc25A up-regulation and cell proliferation. Furthermore, pre-treatment of cells with MG-132 also abolished PDGF-induced β-TrCP reduction, Cdc25A elevation and cell proliferation. In addition, pre-depletion of Cdc25A by siRNA transfection suppressed PDGF-induced PASMCs proliferation. Taken together, our study indicates that up-regulation of CSN6 by PDGFR/PI3K/Akt signaling pathway decreases β-TrCP by increasing its ubiquitinated degradation, and thereby increases the expression of Cdc25A, which promotes PDGF-induced PASMCs proliferation.