Characterization of Photo-induced Oxidative Modifications of Parkinson´s Disease Related Protein α-Synuclein

GIAN FRANCO CAVAZZUTTI; HENNING URLAUB; ANDRÉS MARTÍN TOSCANI; LISANDRO J. FALOMIR LOCKHART; EZEQUIEL GIMÉNEZ; THOMAS M. JOVIN

Sao Paulo

Congreso; 20th International Congress of the International Union for Pure Applied Biophysics (IUPAB); 2021

Introduction and Objectives: A gain of toxic function of the protein alpha-Synuclein (aSyn) has been largely linked to the development and progress of Parkinson?s Disease (PD) and related Synucleinopathies. However, the identity of aSyn toxic species remains elusive. In this way, several post-translational modifications (PTMs) that derived from oxidative stress could be possible modulators of the physiopathology of aSyn in neurons. In this work we investigated the susceptibility of aSyn to be modified when this protein adopts different conformations, allowing us to identify potential pathological modifications. Materials and Methods: Recombinantly expressed aSyn was modified employing optimized photochemical methods that uses Rutheniun (II) Tris(bipyridine) as photosensitizer to produce either Tyrosine (Tyr) crosslinking or, in the presence of NaNO2, nitration. The reactions were characterized and quantified by Fluorescence emission, UV-Vis absorption and Mass Spectrometry. Results and Conclusions: Three conditions were analyzed: disordered aSyn free in solution, bound to membranes (helix-rich structure) and fibril conformation (cross β-sheets). Tyrosine crosslinking of disordered aSyn mainly involved Tyr39 and C-term residues Tyr133 or Tyr136. On the other hand, Tyrosine nitration were identified in all Tyr residues, being Tyr125 the most nitrated during photo-reaction. When bound to negatively charged SUVs, modification of Tyr39 is highly restricted. Similar outcome was found in the fibrillary form. Nitration of C-term residues is only reduced when aSyn interacts with membranes with a negative net charged, not in the unstructured C-term in fibrils. In conclusion, particular modifications, such Tyr39 nitration or Tyr39-Tyr133/136 crosslinking, could only emerge in pathological conditions and their identification in complex biological samples could be employed for the development of innovative tools for the early diagnosis of Synucleopathies.Keywords: alpha-Synclein, Oxidative Stress, Post-translational ModificationsFunding: CONICET (Argentina), ANPCyT (Argentina), UNLP (Argentina), Bunge & Born, Max Planck Society and Williams Foundations (Argentina-Germany), DFG (Germany)