6PPD-Q interaction with human apo-transferrin accompanies hepatic iron dysregulation and pro-ferroptotic responses: Evidence from in vitro and zebrafish studies.

As an important mechanism of cellular homeostasis, ferroptosis depends on apo-transferrin (apo‑TRF)-mediated iron homeostasis. However, how emerging pollutants disrupting this balance by interfering with apo‑TRF remains unclear. Although the highly toxic N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6PPD‑Q) is known to induce oxidative stress and systemic toxicity, whether it triggers ferroptosis by targeting apo‑TRF is unknown. Therefore, this study investigated the potential mechanism of interaction between 6PPD‑Q and apo‑TRF using multispectral techniques, molecular simulations, and in vivo zebrafish exposure experiments. Spectroscopy combined with molecular docking indicated the moderate affinity complex formation (Kₐ = 1.6 × 104 M-1, 298 K). Moreover, this binding occurred spontaneously, driven by hydrogen bonding (H-bonding) and van der Waals forces. The decreased α-helix content (17.3% to 14.3%) indicated a more relaxed conformation of apo-TRF, consistent with the molecular dynamics (MD) simulation results. Furthermore, molecular docking implicated amino acid residues associated with iron binding (Tyr426, Tyr517, Asp392, and His585) in the interaction with 6PPD‑Q. Zebrafish exposure experiments exhibited abnormal expression of transferrin and ferroptosis-related genes (e.g., gpx4a, gpx4b, slc7a11, tfr1a, hamp et al.), accompanied by elevated ferrous iron deposition and lipid peroxidation in the liver. Meanwhile, competitive binding analysis of 6PPD-Q and Fe3+ to apo-TRF suggests that 6PPD-Q interacts with the iron-binding pockets and impairs Fe3+ loading. This study offered a new perspective on the apo‑TRF-6PPD‑Q interaction, revealing interaction‑driven structural alterations in apo‑TRF that affect its normal physiological function, thereby disrupting iron homeostasis and triggering pro-ferroptotic responses.