Deneddylases remove the ubiquitin-like protein Nedd8 from modified proteins. An increased deneddylase activity has been associated with various human cancers. In contrast, we show here that a mutant strain of the model fungus Aspergillus nidulans deficient in two deneddylases is viable but can only grow as a filament and is highly impaired for multicellular development. The DEN1/DenA and the COP9 signalosome (CSN) deneddylases physically interact in A. nidulans as well as in human cells, and CSN targets DEN1/DenA for protein degradation. Fungal development responds to light and requires both deneddylases for an appropriate light reaction. In contrast to CSN, which is necessary for sexual development, DEN1/DenA is required for asexual development. The CSN-DEN1/DenA interaction that affects DEN1/DenA protein levels presumably balances cellular deneddylase activity. A deneddylase disequilibrium impairs multicellular development and suggests that control of deneddylase activity is important for multicellular development.

SummaryThe eight-subunit COP9 signalosome (CSN) is conserved from filamentous fungi to humans and functions at the interface between cellular signalling and protein half-life control. CSN consists of six PCI and two MPN domain proteins and forms a scaffold for additional interacting proteins. CSN controls protein stability in the ubiquitin-proteasome system where the MPN domain CSN5/CsnE subunit inactivates cullin-RING ligases. The CSN5/CsnE isopeptidase functions as deneddylase and removes the ubiquitinlike protein Nedd8. The six PCI domain proteins of human CSN form a horseshoe-like ring and all eight subunits are connected by a bundle of C-terminal α-helices. We show that single deletions of any csn subunit of Aspergillus nidulans resulted in the lack of deneddylase activity and identical defects in the coordination of development and secondary metabolism. The CSN1/CsnA N-terminus is dispensable for deneddylase activity but required for asexual spore formation. Complex analyses in mutant strains revealed the presence of a seven-subunit pre-CSN without catalytic activity. Reconstitution experiments with crude extracts of deletion strains and recombinant proteins allowed the integration of CSN5/CsnE into pre-CSN resulting in an active deneddylase. This supports a stable seven subunit pre-CSN intermediate where deneddylase activation in vivo can be controlled by CSN5/CsnE integration as final assembly step.

Background: In a post-hoc analysis of a pediatric asthma study, we identified the predictors of asthma exacerbations (AEs) and related them to forced expiratory volume (FEV₁), the FEV₁/FVC ratio, and bronchial hyperresponsiveness (BHR). Objectives: We sought to detect predictors of AEs in a prospective study that utilizes impulse oscillometry (IOS) and to compare the results to previously determined predictors. Methods: A moderate AE was defined as an increased use of salbutamol during coughing episodes. Pulmonary function and BHR were measured during symptom- and medication-free periods. Additionally, allergen testing and IOS were included. To calculate the sensitivity and specificity of AE detection, a receiver-operating characteristic (ROC) curve was plotted, and accuracy was measured with the area under the ROC curve (AUC). A logistic regression analysis was used to predict the probability of an exacerbation. Results: Seventy-five pediatric patients (4-7 years of age) with intermittent asthma were included. In 69 patients, the following cut-off values demonstrated the best sensitivity and specificity combination for predicting an AE: FEV₁ 103.2% (AUC 0.62), BHR (PD₂₀methacholine) 0.13 mg (AUC 0.61), and, in 54 children, Rrs5 0.78 kPa × l^-1 × s (AUC 0.80). Logistic regression analysis demonstrated that the combination of all parameters predicted the individual risk of AEs with an accuracy of 86%. Conclusions: IOS, a simple method, predicted the probability of AEs in young children. Airway resistance, measured by IOS, was superior to FEV₁ and methacholine testing. The current data suggest that peripheral airway obstruction is present during symptom-free periods and that these children more likely experience AEs.

SummaryDefects in the COP9 signalosome (CSN) impair multicellular development, including embryonic plant or animal death or a block in sexual development of the fungus Aspergillus nidulans. CSN deneddylates cullin-RING ligases (CRLs), which are activated by covalent linkage to ubiquitin-like NEDD8. Deneddylation allows CRL disassembly for subsequent reassembly. An attractive hypothesis is a consecutive order of CRLs for development, which demands repeated cycles of neddylation and deneddylation for reassembling CRLs. Interruption of these cycles could explain developmental blocks caused by csn mutations. This predicts an accumulation of neddylated CRLs exhibiting developmental functions when CSN is dysfunctional. We tested this hypothesis in A. nidulans, which tolerates reduced levels of neddylation for growth. We show that only genes for CRL subunits or neddylation are essential, whereas CSN is primarily required for development. We used functional tagged NEDD8, recruiting all three fungal cullins. Cullins are associated with the CSN1/CsnA subunit when deneddylation is defective. Two CRLs were identified which are specifically involved in differentiation and accumulate during the developmental block. This suggests that an active CSN complex is required to counteract the accumulation of specific CRLs during development.

Aim:The Fontan circulation is highly dependent on ventilation, improving pulmonary blood flow and cardiac output. A reduced ventilatory function is reported in these patients. The extent of this impairment and its relation to exercise capacity and quality of life is unknown and objective of this study.Methods: This multicenter retrospective/cross-sectional study included 232 patients (140 females, age 25.6 ± 10.8 years) after Fontan palliation (19.8% atrioventricular connection; 20.3% atriopulmonary connection; 59.9% total cavopulmonary connection). Resting spirometry, cardiopulmonary exercise tests, and quality-of-life assessment (SF-36 questionnaire) were performed between 2003 and 2015.Results: Overall, mean forced expiratory volume in one second (FEV 1 ) was 74.7 ± 17.8%predicted (%pred). In 59.5% of the patients, FEV 1 was <80%pred., and all of these patients had FEV 1 /forced vital capacity (FVC) > 80%, suggestive of a restrictive ventilatory pattern. Reduced FEV 1 was associated with a reduced peakVO 2 of 67.0 ± 17.6%pred. (r = 0.43, P < .0001), even if analyzed together with possible confounding factors (sex, BMI, age, years after palliation, number of interventions, scoliosis, diaphragmatic paralysis). Synergistically to exercise capacity, FEV 1 was associated to quality of life in terms of physical component summary (r = 0.30, P = .002), physical functioning (r = 0.25, P = .008), bodily pain (r = 0.22, P = .02), and general health (r = 0.16, P = .024). Lower FEV 1 was associated with diaphragmatic paralysis (P = .001), scoliosis (P = .001), higher number of interventions (P = .002), and lower BMI (P = .01). No correlation was found to ventricular morphology, type of surgeries, or other perioperative/long-term complications.Conclusions: This study shows that the common restrictive ventilatory pattern in Fontan patients is associated with lower exercise capacity and quality of life. Risk factors are diaphragmatic paralysis, scoliosis, a high total number of interventions and low BMI. K E Y W O R D SCPET, Fontan, quality of life, spirometry, ventilatory function