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Regulation of ferroportin degradation
ubiquitin to a small number of E2 enzymes.35 Although some E2 enzymes interact with both UBA1 and UBA6, one E2 enzyme (USE1, also known as UBE2Z) is exclusively charged by UBA635,38. In this study, we found that inhibi- tion of USE1 did not interfere with hepcidin-induced FPN degradation (data not shown), indicating that an E2 enzyme other than (or in addition to) USE1 is involved in FPN regu- lation. In UBA6-depleted cells, UBA1 failed to induce hep- cidin-mediated FPN degradation, indicating non-redundant functions of UBA1 and UBA6 in FPN regulation. The results suggest that an as yet unidentified E2 enzyme, exclusively charged by UBA6, plays a role in FPN degradation.
Members of the NEDD4 family of HECT-type E3 ligases contain a “WW” domain that interacts with a proline rich PPXY (PY) motif in the target protein. However, some target proteins lack a PY domain and ubiquitination of these pro- teins requires the presence of adaptor proteins NDFIP1 or NDFIP2 to act as a scaffold between the two proteins. NDFIP proteins contain three transmembrane domains as well as two PY motifs, which interact with the WW domain of several members of the NEDD4 family of E3 ligases.28 In this study, NDFIP1 was shown to interact with FPN in HepG2 cells in vitro and regulates the level of FPN in the liver in vivo. None of the WW domain-containing NEDD4 family members that were tested individually or in pair-wise com- bination prevented BMP6-induced FPN degradation. The results suggest that several NEDD4 family members may have a redundant role in FPN degradation. Another possibil- ity is that an as yet unknown E3 ligase interacts with the adaptor protein NDFIP1 to ubiquitinate FPN.
ARIH1 is a member of the Ariadne family of E3 RBR lig- ase. ARIH1 is highly expressed in the nucleus, where it interacts with Cajal and PML nuclear bodies.39 ARIH1 associates with neddylated Cullin-RING E3 ligases (CRL) and monoubiquitinates CRL targets.40 In this study, ARIH1 was shown to indirectly regulate FPN stability by altering BMP6-mediated hepcidin induction through a non-canonical pathway. Depletion of ARIH1 blunted basal, as well as BMP6-mediated, hepcidin and ID1 mRNA expression without altering the phosphorylation of SMAD 1/5/8 proteins in response to BMP6. Further studies are needed to elucidate the mechanism as to how ARIH1 regulates hepcidin expression in response to BMP6.
NDFIP1 was previously shown to have a role in iron
homeostasis.41–43 NDFIP1 binds to divalent metal trans- porter 1 (DMT1), the major iron transporter for non-heme iron import.44 NDFIP1 recruits the NEDD4 family member WWP2 to ubiquitinate DMT143. In vivo, Ndfip1 is involved in the regulation of DMT1 in enterocytes.41 The expres- sion of Dmt1 in enterocytes of Ndfip1 deficient mice is increased under normal iron conditions as well as during iron deficiency. The increased level of Dmt1 leads to increased iron absorption, and under normal dietary iron conditions Ndfip1-deficient mice develop a phenotype resembling classic hereditary hemochromatosis, with increased hepatic, duodenal and serum iron levels.43,45 In this study we show that depletion of Ndfip1 in the liver increased the level of FPN. Ndfip1 appears to regulate two steps in iron metabolism: iron import by DMT1 in entero- cytes and iron export by FPN in the liver. Ndfip1-deficient mice were not used in this study, because Ndfip1 deficien- cy results in a severe inflammatory phenotype caused by hyperactivation of T cells.45,46
In summary, this study demonstrated that the E1 enzyme UBA6 and the adaptor protein NDFIP1 are impor- tant for iron homeostasis, regulating the degradation of hepatic FPN. In the future, it may be possible to target spe- cific components of the ubiquitin pathway with small molecules;47 the results of this study may offer novel approaches to treating disorders of iron metabolism.
Disclosures
No conflicts of interest to disclose.
Contributions
LT, FW and DBB designed and conceived the study; LT and DBB wrote and edited the manuscript; LT, SBW, AJS, BHPC, KP, AF and DBB performed experiments; LT, RM, AB and DBB ana- lyzed and interpreted the data; DBB and WMZ supervised the study. All authors approved the final version for submission.
Funding
This study was supported by Luisa Hunnewell and Larry Newman (DBB), the German Research Foundation (TR 1642/1- 1 to LT, WI 5162/2-1 to SBW, Wu 841/1-1 to FW, FI 2429/1-1 to AF), NIH (R01HL142809 to RM, R01DK125786 to AB) and the American Heart Association (18TPA34230025 to RM) 20IOA35360009 to AB).
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