Department of Chemistry, State University of New York at Potsdam, Potsdam, New York 13676, United States.
Department of Molecular and Translational Medicine, University of Brescia, 25123 Brescia, Italy.
Biochemistry. 2020 Jul 28;59(29):2707-2717. doi: 10.1021/acs.biochem.0c00246. Epub 2020 Jul 8.
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 has been explored. Using isothermal titration calorimetry, light absorption spectroscopy, and a soluble fragment (residues 383-522) of human NCOA4 expressed in , we show that the NCOA4 fragment specifically binds H-rich ferritins with a binding stoichiometry of ∼8 NCOA4 molecules per ferritin shell, and values of ∼0.4 and ∼2 μM 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 inhibitory effect that was dependent on the concentration of NCOA4 present in solution. Our results suggest that the binding of NCOA4 to ferritin may interfere in the electron transfer pathway through the ferritin shell and may have important biological implications on cellular iron homeostasis.
铁蛋白噬铁作用是一种由选择性核受体共激活因子-4（NCOA4）介导的铁蛋白自噬降解过程。NCOA4 与铁蛋白结合，并将其递送至新生的自噬体，然后自噬体与溶酶体融合，使铁蛋白降解并释放铁。早期的研究表明，NCOA4 与铁蛋白 H 亚基特异性结合，但不与 L 亚基结合。然而，这种相互作用的热力学以及 NCOA4 对铁氧化、铁矿物核心形成或铁蛋白中铁动员的影响都尚未得到探索。使用等温滴定量热法、光吸收光谱法和在大肠杆菌中表达的人 NCOA4 的可溶性片段（残基 383-522），我们表明 NCOA4 片段特异性结合富含 H 的铁蛋白，结合比约为每个铁蛋白壳 8 个 NCOA4 分子，对于同聚物 H 链铁蛋白和杂聚物富含 H 的铁蛋白， 值分别约为 0.4 和 2 μM。结合反应在焓和熵上都是有利的。虽然 NCOA4 的存在并不影响铁氧化动力学，但两种不同还原剂（FMN/NADH 和连二亚硫酸钠）从铁蛋白中动员铁显示出强烈的抑制作用，该抑制作用依赖于溶液中存在的 NCOA4 浓度。我们的结果表明，NCOA4 与铁蛋白的结合可能干扰铁蛋白壳中的电子传递途径，并可能对细胞内铁稳态具有重要的生物学意义。
