Mechanism of Ferroportin Ubiquitination and its Therapeutic Potential in Anemia of Chronic Disease

• Applicant: Dr. Lisa Schrader
• Subject Area: Anaesthesiology ; Cell Biology
• Term: from 2018 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 413746767

• Final Report Year: 2021

Final Report Abstract

Hepcidin regulates iron homeostasis by controlling the level of ferroportin, the only membrane channel that facilitates export of iron from within cells. Binding of hepcidin to ferroportin induces the ubiquitination of ferroportin at multiple lysine residues and subsequently causes the internalization and degradation of the ligand-channel complex within lysosomes. The objective of this study was to identify the specific E2 and E3 enzymes that are involved in ferroportin degradation. The ability of siRNAs directed against components of components of the ubiquitin pathway to prevent BMP6- and exogeneous hepcidin-induced ferroportin degradation was tested in a HepG2 cell line, which inducibly expresses ferroportin-GFP (FPN-GFP) in the presence of doxycycline. In total, 108 siRNAs directed against components of the ubiquitin pathway were tested in vitro. siRNA-mediated depletion of the alternative E1 enzyme UBA6 as well as the adaptor protein NDFIP1 prevented BMP6- and hepcidininduced degradation of ferroportin in vitro. The E3 enzyme MARCH8 impaired BMP6- and exogenous hepcidin induced FPN-GFP localization to lysosomes and subsequent degradation of the fusion protein. A second E3 enzyme, ARIH1, indirectly inhibited ferroportin degradation by impairing BMP6 mediated induction of hepcidin and ID1, another target of the BMP signaling pathway. In mice, the AAV-mediated silencing of Ndfip1 in the murine liver increased the level of hepatic ferroportin and increased circulating iron suggesting that Ndfip1 regulates hepatic ferroportin in vivo. In conclusion, the results suggest that the E1 enzyme UBA6, the adaptor protein NDFIP1 and the E3 enzyme MARCH8 are involved in iron homeostasis by regulating the degradation of ferroportin. In addition, the study identified the E3 enzyme ARIH1 as a novel regulator of hepcidin expression. These specific components of the ubiquitin system may be promising targets for the treatment of iron related diseases, including iron overload and anemia of inflammation.

Publications

• HFE and ALK3 act in the same signaling pathway. Free Radical Biology and Medicine, 2020 Aug 27;160:501-505
  Traeger L, Schnittker J, Dogan DY, Oguama D, Kuhlmann T, Muckenthaler MU, et al.
  
• The role of hepcidin and iron homeostasis in atherosclerosis. Pharmacological Research, 2020 March; 153:104664
  Wunderer F, Traeger L, Sigurslind HH, Meybohm P, Bloch DB, Malhotra R
  
• Antimicrobial effects of nitric oxide in murine models of Klebsiella pneumonia. Redox Biology, 2021 Feb; 39:101826
  Wiegand SB, Traeger L, Nguyen HK, Rouillard KR, Fischbach A, Zadek F, et al.
  
• Sulfide catabolism ameliorates hypoxic brain injury. Nature Communications, 2021, May 25;12(1):3108
  Marutani E, Morita M, Shuichi H, Grange RMH, Miyazaki Y, Nagashima F, Traeger L, et al.