Fe2+-Sensing α-Synuclein Iron-Responsive Messenger RNA/eIF4F Complex Binding and Regulating mRNA Translation Activation and Repression

Alpha-synuclein (α-Syn) protein plays a crucial role in the pathophysiology of Parkinson's disease (PD). In the 5'-untranslated regions (5'-UTRs) of α-Syn, mRNA has a structured iron-responsive element (IRE) with a stem loop that regulates translation. Iron (labile as Fe²⁺) enhances protein synthesis rates through an IRE mRNA. This investigation aimed to describe the way in which α-Syn IRE interacts with eIF4F and establish a relationship between binding affinity and translation efficiency. The strong binding affinity of α-Syn IRE with eIF4F was demonstrated by a fluorescence-based experiment, with <i>K</i>_a = 8.4 × 10⁶ M^-1 at 25 °C. Fe²⁺ further increased (~three-fold) the affinity of α-Syn IRE with eIF4F, outcompeting binding with IRP1. With an increase in temperature (10-30 °C), <i>K</i>_d values increased from 35.8 ± 1.6 nM to 158 ± 8.7 nM for the interaction of α-Syn IRE with eIF4F; however, adding Fe²⁺ demonstrated significantly increased affinity throughout the same temperature range. Thermodynamic analyses demonstrated that α-Syn IRE/eIF4F binding occurred spontaneously, with the presence of van der Waals and hydrogen bonding. Fe²⁺ enhanced the α-Syn IRE/eIF4F complex's change in enthalpic and binding free energy contributions, which led to a more stable complex formation through the involvement of more hydrogen bonding. Exogenous addition of eIF4F in depleted WG or RR lysates restored α-Syn protein synthesis. Fe²⁺ further boosted α-Syn mRNA translation. IRP1 repressed α-Syn translation, although the addition of Fe²⁺ reversed this effect by boosting activator eIF4F binding and decreasing repressor IRP1 binding. These findings reveal the significance of iron in the α-synuclein mRNA regulatory process and validate its contribution as a strong enhancer of α-Syn mRNA translation.