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Ferritin is a 24-mer protein nanocage that stores iron and regulates intracellular iron homeostasis. The nuclear receptor coactivator-4 (NCOA4) binds specifically to ferritin H subunits and facilitates the autophagic trafficking of ferritin to the lysosome for degradation and iron release. Using isothermal titration calorimetry (ITC), we studied the thermodynamics of the interactions between ferritin and the soluble fragment of NCOA4 (residues 383–522), focusing on the effects of the recently identified Fe–S cluster bound to NCOA4, ferritin subunit composition, and ferritin-iron loading. Our findings show that in the presence of the Fe–S cluster, the binding is driven by a more favorable enthalpy change and a decrease in entropy change, indicating a key role for the Fe–S cluster in the structural organization and stability of the complex. The ferritin iron core further enhances this association, increasing binding enthalpy and stabilizing the NCOA4-ferritin complex. The ferritin subunit composition primarily affects binding stoichiometry of the reaction based on the number of H subunits in the ferritin H/L oligomer. Our results demonstrate that both the Fe–S cluster and the ferritin iron core significantly affect the binding thermodynamics of the NCOA4-ferritin interactions, suggesting regulatory roles for the Fe–S cluster and ferritin iron content in ferritinophagy.
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This content will become publicly available on February 27, 2026
Characterization of the Iron–Sulfur Cluster in the NCOA4 Fragment (383–522) and Its Interaction with Ferritin
Ferritin degradation pathways, particularly NCOA4-mediated ferritinophagy, are crucial for maintaining iron homeostasis. Here, we demonstrate the coexistence of two NCOA4 isoforms, one iron−sulfur cluster-free and one iron−sulfur cluster-bound, in oxygenated cell cultures. Using a combination of spectroscopic and analytical techniques, in vitro characterization of the NCOA4 fragment (383−522), denoted NCOA4-D, revealed a predominance of monomeric species with a relatively stable [2Fe-2S] cluster under normoxic conditions. The results demonstrate distinct interactions between NCOA4-D isoforms and ferritin, underscoring the influence of cellular oxygen and iron concentrations on NCOA4’s regulatory functions, pathways, and ferritin’s fate. Our findings suggest that different NCOA4-initiated degradation pathways may concurrently occur in cells and highlight the necessity of further exploring the role of the Fe−S cluster in NCOA4 as an iron-sensing mechanism for maintaining cellular iron homeostasis.
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- Award ID(s):
- 2231900
- PAR ID:
- 10659274
- Publisher / Repository:
- American Chemical Society
- Date Published:
- Journal Name:
- ACS Chemical Biology
- Volume:
- 20
- Issue:
- 3
- ISSN:
- 1554-8929
- Page Range / eLocation ID:
- 731 to 745
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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