Identification of CCNF/Cyclin F targets

The founding member of the F-box protein family, Cyclin F, serves as substrate adaptor for the Ubiquitin E3 ligase Skp1-Cul1-F-box (SCF)Cyclin F which is responsible for ubiquitination of proteins involved in cell cycle progression, DNA damage and mitotic fidelity. Missense mutations in CCNF encoding for Cyclin F are associated with amyotrophic lateral sclerosis (ALS). However, it remains elusive whether CCNF mutations affect the substrate adaptor function of Cyclin F and whether altered SCFCyclin F mediated ubiquitination contributes to ALS pathogenesis in CCNF mutation carrier. To start addressing these questions, we set out to identify new SCFCyclin F targets in neuronal and patient-derived cells. Mass spectrometry-based ubiquitinome profiling of CCNF knockout and mutant cell lines as well as Cyclin F proximity and interaction proteomics converged on the HSP90 chaperone machinery as new substrate candidate. Biochemical analyses confirmed the Cyclin F dependent binding and ubiquitination of HSP90AB1 and unveiled a regulatory role of this ubiquitination in controlling the binding of a number of HSP90 clients and co-factors, thereby implying chaperone dysregulation and CCNF loss-of-function mechanism in ALS.