STRUCTURAL INSIGHTS INTO THE MECHANISM OF PHOX2B VARIANTS AGGREGATION LEADING TO AMYLOID FORMATION

Congenital central hypoventilation syndrome (CCHS) is a rare neonatal disorder characterized by impaired autonomic control of respiration, resulting in abnormal ventilatory responses to hypoxia and hypercapnia [1]. The disease is primarily caused by mutations in the PHOX2B gene, including polyAla expansions (95%) and, less frequently, frameshift mutations (5%). Phox2B is a transcription factor essential for the development and function of the autonomic nervous system [2]. In this scenario, we investigated the structural, dynamical and functional properties of Phox2B and its pathological variants [3]. In details, we determined the high-resolution structure of Phox2B DNA-binding HD by using NMR spectroscopy [4]. We also demonstrated that the HD is structurally coupled, via transient interactions, to the intrinsically disordered C-terminal domain, in which the polyAla tract adopts a stable α-helical conformation [4]. This highlighted inter-domain coupling mechanism may play a protective role in preventing misfolding and aggregation, analogous to molecular mechanisms described in other aggregation-prone proteins [5]. This hypothesis is supported by ThT aggregation experiments showing that pathological variants, such as the +7Ala mutant, exhibit a strong propensity to aggregate into insoluble amyloid fibrils, suggesting a potential contribution to disease development. Building on these findings, we characterized, using a multidisciplinary approach combining biochemical, spectroscopic and computational techniques, the molecular determinants driving in the initial stages of the aggregation process of Phox2B variants. In addition, advanced real-time NMR experiments provided atomic resolution structural insights into the assembly mechanism identifying key HD residues involved in the early steps of the nucleation process. These structural insights will guide the rational design of peptide-based inhibitors targeting aggregation-prone HD regions, with the goal of preventing aggregation processes by which Phox2B variants form, through the formation of soluble oligomeric species, amyloid fibrils that in turn may have a crucial role in CCHS disease progression.