Proteína humana Cofilina-1: estudo plasmônico e eletroquímico do estado redox e da agregação induzida por oxidação

Abstract

The dysregulation of human cofilin-1 (CFL-1) protein is increasingly associated with the pathophysiology of neurodegenerative diseases. This pathogenicity is linked to cysteine oxidation mechanisms, which induce disulfide bond formation and confer toxic function, as well as to its intrinsic propensity for aggregation. Although physiological oxidants such as chlorotaurine (TauCl) are known to promote cellular stress, their specific role in inducing CFL-1 oligomers or aggregates remains unexplored. To address these gaps, this thesis aimed to monitor the redox state and interfacial aggregation kinetics of this protein in real-time. Initially, a platform on a gold substrate modified with 3-mercaptopropionic acid (MPA) and activated via EDC/NHS was developed for CFL-1 immobilization. Characterization was performed using surface plasmon resonance (SPR), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). This platform enabled the novel adaptation of the Ellman assay (DTNB) for a heterogeneous environment, with SPR monitoring. Results demonstrated that the immobilized protein predominantly maintained its native (reduced) conformation, presenting a stoichiometric ratio consistent with 4 free thiols per protein molecule at the interface. In the second phase, TauCl-induced CFL-1 aggregation kinetics were monitored in a novel manner via direct electrochemistry on screen-printed carbon electrodes (SPCEs). Using the intrinsic oxidation of tyrosine (Tyr) residues as a molecular marker, a time-dependent aggregation process was evidenced, manifested by a significant reduction in the anodic current. Aggregate formation was corroborated by complementary techniques, with DLS and AFM confirming increased hydrodynamic diameter and surface morphological changes, respectively. In conclusion, the developed platforms offer robust analytical tools for CFL-1 investigation. The SPR-based methodology offers perspectives for drug screening targeting specific redox forms of the protein, while the electrochemical approach elucidates redox state-dependent aggregation mechanisms, providing essential insights into the molecular bases of CFL-1-associated neurodegeneration.