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.

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Osteogenesis imperfecta (OI) is characterized primarily by susceptibility to fractures with or without bone deformation. OI is genetically heterogeneous: over 20 genetic causes are recognized. We identified bi-allelic pathogenic KDELR2 variants as a cause of OI in four families. KDELR2 encodes KDEL endoplasmic reticulum protein retention receptor 2, which recycles ER-resident proteins with a KDEL-like peptide from the cis-Golgi to the ER through COPI retrograde transport. Analysis of patient primary fibroblasts showed intracellular decrease of HSP47 and FKBP65 along with reduced procollagen type I in culture media. Electron microscopy identified an abnormal quality of secreted collagen fibrils with increased amount of HSP47 bound to monomeric and multimeric collagen molecules. Mapping the identified KDELR2 variants onto the crystal structure of G. gallus KDELR2 indicated that these lead to an inactive receptor resulting in impaired KDELR2-mediated Golgi-ER transport. Therefore, in KDELR2-deficient individuals, OI most likely occurs because of the inability of HSP47 to bind KDELR2 and dissociate from collagen type I. Instead, HSP47 remains bound to collagen molecules extracellularly, disrupting fiber formation. This highlights the importance of intracellular recycling of ER-resident molecular chaperones for collagen type I and bone metabolism and a crucial role of HSP47 in the KDELR2-associated pathogenic mechanism leading to OI.Osteogenesis imperfecta (OI) (MIM: PS166200) is a clinically and genetically heterogeneous connective tissue disorder characterized by liability to fractures with or without bone deformation. Secondary features include blue sclerae, dentinogenesis imperfecta (DI), progressive hearing loss, and joint hypermobility. OI is divided into five clinical types, and it has long been estimated that 90% of individuals with OI have dominant pathogenic variants in COL1A1 (MIM: 120150) or COL1A2 (MIM: 120160) encoding the a1 and a2 chains of collagen type I. However, there is growing evidence that autosomal-recessive forms of OI can be more common in consanguineous populations. 1,2 OI type 1 (MIM: 166200) is usually caused by pathogenic variants resulting in haploinsufficiency of COL1A1, and OI type 5 (MIM: 610967) is always caused by the dominant c.À14C>T variant in the 5 0 UTR of IFITM5. 1 To date,

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.