| Transthyretin(TTR)aggregation and amyloid formation are associated with several TTR amyloidosis(ATTR)diseases.However,what triggers the initial pathologic aggregation process of TTR remains largely elusive.A growing number of experiments have shown that numerous proteins in diseases may undergo liquid–liquid phase separation(LLPS)and subsequent liquid-to-solid phase transition previous to amyloid fibrils formation.TTR tetramer disassociates into misfolded monomer and self-assembles into amyloid fibrils via p H-mediated denaturation pathway in vitro.To study the pathological aggregation process of TTR,phase separation and aggregation behavior were explored under acidic conditions.We constructed p ET-28a-TTR,p ET-28a-EGFP-TTR and mutants(V30M,R34 T,K35T)recombinant plasmids by molecular cloning.Eight kinds of TTR and mutant proteins were purified by Ni affinity chromatography after induced protein expression in Escherichia coli.Applying multiple biophysical techniques,such as confocal microscope and fluorescence recovery after photobleaching(FRAP)to study protein phase separation,thioflavin T(Th T)fluorescence aggregation kinetics and transmission electron microscope(TEM)to study protein aggregation.The experimental results showed that TTR underwent LLPS under acidic p H in vitro,which was driven by electrostatic interactions.The droplets converted from liquid-like to solid-like state and eventually formed amyloid fibrils.Furthermore,pathogenic mutations(V30M,R34 T,K35T)and heparin promoted the process of phase transition and facilitated the formation of fibrillar aggregates.In addition,S-cysteinylation reduced the kinetic stability of TTR and increased propensity for aggregation while S-sulfonation stabilized TTR tetramer and reduced aggregation rate.S-cysteinylation and S-sulfonation all dramatically underwent the process of phase transition,providing a foundation for post-translational modifications that could modulate TTR LLPS in the context of pathological interactions.These novel findings revealed molecular insights into the mechanism of TTR from initial LLPS to liquid-to-solid phase transition and finally to aggregates,providing a new direction dimension for ATTR therapy. |
