From phosphorylation-driven stability of ceruloplasmin to functional prediction of missense variants in aceruloplasminemia: recombinant ceruloplasmin as a prognostic tool

Iron is crucial micronutrient, and it plays a key role in oxygen transport, mitochondrial respiration, DNA synthesis, among other biological functions. However, the same properties that make iron so essential, also lead to oxidative stress-mediated toxicity. Thus, our body developed a tightly controlled homeostatic mechanism to ensure that enough iron is absorbed, sorted and stored to meet the metabolic need and avoid its toxic accumulation. Ceruloplasmin (CP), a multicopper ferroxidase secreted by hepatocytes, guarantees iron balance by oxidizing Fe²⁺ to Fe³⁺ for transferrin (TF)-mediated transport in the bloodstream. Loss-of-function (LoF) mutations in the CP gene are known to cause aceruloplasminemia (ACP), a rare autosomal recessive disease marked by systemic and brain iron accumulation, anemia, diabetes, and progressive neurodegeneration. Current therapies, including iron chelation and fresh frozen plasma (FFP) transfusions provide limited benefit, whereas preclinical studies in CP-KO mice demonstrated that CP replacement mitigates neurodegeneration, supporting enzyme replacement as a rational strategy in ACP. However, this strategy has drawbacks related to CP relatively limited half-life in-vivo (approx. 5 days), impacted by various factors including proteolytic degradation by plasmatic serine proteases which reduce its in-vivo stability and ferroxidase activity. Here, we show that physiologically relevant phosphorylation in proximity of R701 and R481 residues in CP confers protection against proteolysis. This mechanism was validated via several in-vitro biochemical assays, where both synthetic peptides and full-length recombinant CP (R-CP) were proteolyzed under controlled conditions, also providing a rationale for engineering therapeutic R-CP prototypes with enhanced half-life through phospho-mimesis. Much to be explored remains in our understanding of the biology and biochemistry of CP in normal physiology. Even less is known on CP mechanistic aspects with respect to ACP, and new CP missense variants impacting its function are continuously discovered in the human population (e.g. Ziliotto, Lencioni et al. 2025). The paucity of clinical data associated with these functionally relevant, potentially pathogenic variants limits the possibility of establishing clear genotype-phenotype correlations. To address this problem, we expressed in HEK293T/17 cells ten ACP- associated CP missense variants derived from patients with available clinical data. Then, we purified and functionally characterized these variants. Genotype-phenotype correlations highlighted that copper-binding or buried CP variants drive severe LoF, whereas surface-exposed variants showed an intermediate phenotype, suggesting that structurally impacting variants affect CP function more severely than variants affecting the surface of CP. The characterization of ACP patients sera confirmed that biallelic missense LoF alleles lead to undetectable CP, causing severe iron overload, and, importantly for a replacement therapy approach in ACP, heterozygous CP carriers maintain near-normal iron homeostasis and a generally subclinical phenotype. This study provides a significant contribution to the understanding of the molecular mechanisms underlying ACP. We show that our human cell-based CP functional characterization platform effectively clarifies how specific CP missense variants lead to dysfunctional alleles, revealing a clear correlation between functional variant effects and the clinical presentation of ACP. Overall, this PhD thesis refines the understanding of ACP pathogenesis by uncovering the regulation of CP stability by phosphorylation and highlighting the need to assess both CP quantity and quality in patients. By establishing a pipeline for the functional evaluation of variants, this work provides a biochemical foundation for the future development of recombinant enzyme replacement therapies (ERT) aimed at restoring CP function.