Thermodynamic and Kinetic Studies of the Interaction of Nuclear Receptor Coactivator-4 (NCOA4) with Human Ferritin

Authors:
Publisher website
Google Scholar
Abstract
Ferritinophagy is a ferritin autophagic degradation process mediated by the selective Nuclear Receptor Coactivator-4 (NCOA4). NCOA4 binds to ferritin and delivers it to nascent autophagosomes, which then merge with the lysosomes for ferritin degradation and 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. Using isothermal titration calorimetry (ITC), light absorption spectroscopy, and a soluble fragment (residues 383–522) of human NCOA4 expressed in E. coli, we show that the NCOA4 fragment specifically binds H-rich ferritins with a binding stoichiometry of 8 NCOA4 molecules per ferritin shell, and a Kd of ~0.4 M and ~2 M, for homopolymer H-chain ferritin and heteropolymer H-rich ferritin, respectively. The binding reaction was both enthalpically and entropically favored. Whereas the iron oxidation kinetics were not affected by the presence of NCOA4, iron mobilization from ferritin by two different reducing agents (FMN/NADH and sodium dithionite) showed a strong inhibition effect that was dependent on the concentration of NCOA4 present in solution. 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.
Funding Information
  • Camille and Henry Dreyfus Foundation (TH-16-007)
  • National Institute of General Medical Sciences (R15GM104879)
  • State University of New York

This publication has 26 references indexed in Scilit: