CONICET | Buscador de Institutos y Recursos Humanos

The misfolding of proteins into a toxic conformation is proposed to be at the molecular foundation of neurodegenerative disorders including Alzheimer and Parkinson´s disease (PD). Neurodegeneration in PD is progressive and characterized by the loss of dopaminergic neurons in the substantia nigra and the presence of fibrillar cytoplasmatic aggregates of alpha-synuclein (AS) in multiple brain regions. Currently, no preventative therapy is available for PD and related synucleinopathies. Identification of therapeutic drugs is not only complicated by a lack of understanding of many of the key aspects of PD pathogenesis but also by the multifactorial etiology of the disease. The aggregation pathway of AS represents then an obvious target for therapeutic intervention in PD. Indeed, one approach to the development of therapeutic agents in neurodegenerative diseases has been the use of small molecules that specifically and efficiently inhibit the aggregation process. In this work we sought to delineate the structural basis of the interaction between one of the most studied aggregation inhibitor, the cyclic tetrapyrrole phthalocyanine tetrasulfonates (PcTS) and AS, as a first step towards the understanding of the molecular mechanism by which this compound inhibits AS filament assembly and lead to the formation of non-toxic oligomeric species. From the analysis of the fibrillation kinetics and the intensities profiles obtained by NMR it becomes evident that the inhibition of AS aggregation of this compound is a direct consequence of binding to the N-terminus of the protein. In order to elucidate the structural rules that direct the inhibitory mechanism of PcTS we designed site-directed mutants of AS, which demonstrated unequivocally the role of the aromatic moieties as anchoring groups for PcTS binding to AS. This was further confirmed by analyzing the effects of PcTS on the side chains of aromatic residues by 1D 1H-NMR and 2D 1H-13C HSQC. From these studies we propose that specific aromatic interactions with the Y39 residue provide a central mechanistic basis for the inhibitory process of PcTS on AS aggregation. This hypothesis was confirmed by measurement of the aggregation kinetics of the designed site-directed mutants of AS, that revealed also a critical role for the Y39 residue in the aggregation pathway of AS. The elucidation of the structural details of this interaction provided the basis for understanding the role of specific residues in the fibrillation pathway of AS and shed new light into the mechanistic basis that direct the inhibitory process of anti-amyloidogenic compounds.