Transthyretin Amyloidosis: Proteolytic cleavage accelerates G53A TTR misfolding and aggregation

Abstract

The proteolytic cleavage of the peptide bond Lys48-Thr49 of the CD loop in TTR pathological mutants S52P and E51_S52dup was previously demonstrated to promote aggregation as a result of tetrameric structure destabilization. Type A and B fibrils were detected in vivo, suggesting alternative TTR misfolding and aggregation mechanisms. The main component of the fibrils was the residue 49-127 fragment. In the proceeding studies, the misfolding and aggregation of G53A TTR, whose mutation is nearby the K48-T49 peptide bond and also associated with TTR amyloidosis, was investigated in thepresence and absence of proteolytic agent, trypsin. Fragmented G53A TTR misfolded and aggregated via a similar mechanism as full-length TTR, but at a faster rate. Similar morphology was exhibited by fragmented and full-length G53A TTR oligomers, as revealed by TEM images, suggesting similar aggregation pathway. G53A TTR in the presence and absence of trypsin generated similar CD and FT-IR profiles, suggesting similar transthyretin morphology during the early and late stages of amyloidosis. Aggregation kinetics was accomplished by monitoring the optical density of G53A TTR in the presence and absence of trypsin and by comparing the ThT fluorescence signal produced. The differences in TTR solubility and ThT enhancement imply G53A TTR aggregation is promoted in the presence of trypsin. Oligomeric effects on mammalian cell line, SH-SY5Y, was probed employing the MTT assay, which determined G53A TTR was more toxic to the cells in the presence of trypsin.