THESE SARS-COV-2 SPIKE FRAGMENTS INDUCE THE AGGREGATION OF NEURODEGENERATIVE PROTEINS

COVID-19 presents manifestations commonly associated with inflammatory and respiratory processes. However, late neurological complications may occur, possibly related to neurodegenerative processes. Previous in vitro and in silico studies suggest that SARS-CoV-2 infection induces the aggregation of proteins related to neurodegenerative diseases, such as α-synuclein (α-Syn). Therefore, we aim to identify and characterize the Spike (S) protein fragments s194, s192, and s532, which are generated following the digestion of the protein by neutrophil elastase during the inflammatory process. These fragments were selected due to their amyloidogenic potential, as observed in in silico and in vitro assays. Furthermore, we investigate how these fragments, alongside the viral nucleocapsid (N) protein, interact with and induce the aggregation of α-Syn (which possesses a "prion-like" conversion mechanism) and the prion protein (PrP), related to Spongiform Encephalopathies. Aiming at the proposed objectives, peptide-protein interactions were evaluated through aggregation kinetics using the thioflavin T (ThT) fluorescent probe; experiments were performed in replicates using a single protocol. The Spike peptides 194, 192, and 532 were obtained commercially by solid-phase synthesis with >95% purity (GenScript). Full-length recombinant PrP (PrP23-231) was expressed and purified by nickel affinity chromatography. Initially, aggregation controls were performed for 2 µM PrP23-231 using a 1% seed (pre-aggregated PrP23-231) and for monomeric s532 at 2 µM and 10 µM. Subsequently, 1:1 and 1:5 conditions of PrP23-231 to s532 (monomeric and pre-aggregated) were tested using a 1% seed. Finally, the pre-aggregated s532 fragment was used as a seed at 1% and 10%. All conditions were evaluated with and without the 1% PrP23-231 seed. The results indicated an increase in ThT fluorescence in the presence of the s532 peptide, suggesting an increase in the amount of amyloid fibrils, as well as a reduction in t½, indicating a change in the PrP aggregation kinetics mediated by the Spike peptide. These findings are promising for the project's continuation, justifying further experiments such as isothermal titration calorimetry (ITC), complete morphological characterization of the aggregates, and cellular toxicity assays with other S protein fragments, in addition to testing different conditions to expand and deepen the research.