Ferritinophagy is an autophagic degradation process that involves the iron storage protein ferritin. It is mediated by the selective Nuclear Receptor Coactivator-4 (NCOA4) which binds to ferritin and delivers it to nascent autophagosomes for lysosomal degradation and ultimately iron release. Earlier studies have demonstrated a specific association of NCOA4 with ferritin H-subunits, but not L-subunits. However, neither the thermodynamics of this interaction, nor the effect of NCOA4 on iron oxidation, iron mineral core formation, or iron mobilization in ferritin have been explored. In this study, we examined the thermodynamics of the complexation reaction between a soluble fragment of human NCOA4 and homopolymer and heteropolymer ferritin using isothermal titration calorimetry (ITC) and light absorption spectroscopy. Our results show a molecular association that is both enthalpically and entropically favored, with a binding stoichiometry of ~ 8 NCOA4 fragments per ferritin shell and a dissociation constant in the range of 0.4 – 2 uM. Whereas iron uptake and oxidation by ferritin is not affected by the presence of NCOA4, a strong NCOA4-dependent inhibition was observed for iron mobilization. Our results suggest that NCOA4 binding to ferritin may interfere in the electron transfer pathway through the ferritin shell and may have important biological implications on cellular iron homeostasis.

Thermodynamic and kinetic studies of the interaction of nuclear receptor coactivator 4 (NCOA4) with human ferritin

ACS Spring Meeting 2021

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