N-homocysteinylation of ferritin and associated changes in iron metabolism as potential drivers of vascular endothelial dysfunction in hyperhomocysteinemia.

Hyperhomocysteinemia contributes to various diseases, including cardiovascular and neurodegenerative disorders. While previous research indicates iron's role in hyperhomocysteinemia, the underlying molecular interactions remain poorly understood. Our previous study demonstrated that homocysteine significantly increases ferritin levels, linked to disruptions in the Akt-FOXO3a pathway, though the reasons for these increases are unclear. In the present study, we investigated these mechanisms, evidencing that homocysteine thiolactone (HcyT) similarly increases the levels of Ferritin L and H in HUVEC, but not in SH-SY5Y cells. This ferritin upregulation was accompanied by elevations labile iron pools. Furthermore, in cells exposed to HcyT, there was an increase in proteins responsible for exporting iron, such as ferroportin and APP. Additionally, intracellular iron chaperones, such as PCBP1 and PCBP2, were significantly dysregulated, while the level of the transferrin receptor-the protein responsible for importing iron into the cell-was decreased. These results suggest a compensatory protective response to iron accumulation in HcyT-treated cells. Pre-incubating with H2O2 increased cellular sensitivity to HcyT-induced toxic effects, providing indirect evidence for the involvement of an iron-dependent cell death mechanism in endothelial cells HUVEC. We discovered that HcyT triggers the process of N-homocysteinylation of ferritin H, which most likely impairs its function. This explains the steady increase in ferritin and LIP levels in the studied cell model. In conclusion, we are the first to demonstrate that HcyT disrupt iron metabolism at multiple levels, including storage, export, import, and intracellular transport. Our findings indicate that these disturbances are caused by N-homocysteinylation of ferritin.