A POTENTIAL ROLE OF PHOSPHORYLATION IN CERULOPLASMIN STABILITY AND ACTIVITY

Aceruloplasminemia (ACP) is a rare disease caused by mutations in ceruloplasmin (CP) gene, resulting in insufficient ferrous iron oxidation and accumulation in various organs, including the brain. Accordingly, neurodegeneration is the main hallmark for ACP, together with microcytic anemia and high serum ferritin levels. Moreover, CP is neuroprotective by inhibiting the neuroinflammation mediator myeloperoxidase (MPO) (Kono and Miyajima 2006). Previous work demonstrated that plasma-derived CP administration ameliorated neurodegeneration in an ACP mouse model (Zanardi, Conti et al. 2018). In this work, we sought to characterize effects of CP phosphorylation on its stability and activity (Bielli, Bellenchi et al. 2001, Hellman, Kono et al. 2002). We propose that steric hindrance of the negatively charged phosphate groups close to protease cleavage sites may hamper the protease docking, protecting CP against degradation. Inactivating mutations of two trypsin cleavage sites on CP, R450 and K906 (2APC mutation), allowed us to focus solely on the R720 site, that is in proximity to S722 and S725 residues which are subject of phosphorylation (Bielli, Bellenchi et al. 2001, Tagliabracci, Wiley et al. 2015). Using a combination of phospho-mimetic and phospho-abrogative mutations and kinase overexpression we demonstrated that phosphorylation at these residues influences the ability of trypsin to cleave at R720. As other serine/threonine phosphorylation sites are in proximity of known protease cleavage sites in CP, we will apply the same approach to define the role of phosphorylation on CP proteolytic cleavage with the aim to develop more stable CP variants as improved protein replacement therapeutics for ACP.