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.

F-box蛋白家族的创始成员——细胞周期蛋白F，充当泛素化E3连接酶Skp1-Cul1-F-box（SCF细胞周期蛋白F）的底物适配器。该连接酶负责泛素化参与细胞周期进展、DNA损伤和有丝分裂保真的蛋白质。编码细胞周期蛋白F的CCNF基因中的错义突变与肌萎缩侧索硬化症（ALS）相关。然而，关于CCNF突变是否影响细胞周期蛋白F的底物适配器功能，以及SCF细胞周期蛋白F介导的泛素化改变是否导致CCNF突变携带者中ALS的发病机制，这一问题仍然扑朔迷离。为了解答这些问题，本研究旨在识别神经元和患者来源细胞中新的SCF细胞周期蛋白F靶点。基于质谱技术的泛素组学分析以及细胞周期蛋白F的邻近和相互作用蛋白质组学分析，均聚焦于HSP90伴侣机器作为新的底物候选物。生化分析证实了细胞周期蛋白F依赖性的HSP90AB1结合和泛素化，并揭示了这种泛素化在控制众多HSP90客户和辅助因子结合中的调控作用，从而暗示了伴侣蛋白失调和CCNF功能丧失机制在ALS中的作用。