Although human transthyretin (TTR) is associated with systemic amyloidoses, an anti-amyloidogenic effect that prevents Aβ fibril formation in vitro and in animal models has been observed. Here we studied the ability of three different types of TTR, namely human tetramers (hTTR), mouse tetramers (muTTR) and an engineered monomer of the human protein (M-TTR), to suppress the toxicity of oligomers formed by two different amyloidogenic peptides/proteins (HypF-N and Aβ42). muTTR is the most stable homotetramer, hTTR can dissociate into partially unfolded monomers, whereas M-TTR maintains a monomeric state. Preformed toxic HypF-N and Aβ42 oligomers were incubated in the presence of each TTR then added to cell culture media. hTTR, and to a greater extent M-TTR, were found to protect human neuroblastoma cells and rat primary neurons against oligomer-induced toxicity, whereas muTTR had no protective effect. The thioflavin T assay and site-directed labeling experiments using pyrene ruled out disaggregation and structural reorganization within the discrete oligomers following incubation with TTRs, while confocal microscopy, SDS-PAGE, and intrinsic fluorescence measurements indicated tight binding between oligomers and hTTR, particularly M-TTR. Moreover, atomic force microscopy (AFM), light scattering and turbidimetry analyses indicated that larger assemblies of oligomers are formed in the presence of M-TTR and, to a lesser extent, with hTTR. Overall, the data suggest a generic capacity of TTR to efficiently neutralize the toxicity of oligomers formed by misfolded proteins and reveal that such neutralization occurs through a mechanism of TTR-mediated assembly of protein oligomers into larger species, with an efficiency that correlates inversely with TTR tetramer stability.
Keywords: 2′,7′-dichlorodihydrofluorescein diacetate; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; AD; Alzheimer's disease; Aβ; BSA; CM-H(2)DCFDA; CSF; D-PBS; DMSO; DTT; Dulbecco's phosphate-buffered saline; ED; FAC; FBS; HypF-N; MTT; Molecular chaperone; N-terminal domain of the HypF protein from Escherichia coli; NBM; PMSF; PSD-95; RBP; SDS-PAGE; SSA; T4; TTR; TTR protective effect; TTR-mediated oligomer clustering; ThT; amyloid-beta peptide; bovine serum albumin; cerebrospinal fluid; dimethylsulfoxide; dithiothreitol; embryonic day; familial amyloid cardiomyopathy; fetal bovine serum; neurobasal medium; phenylmethylsulfonyl fluoride; postsynaptic density protein 95; retinol binding protein charged with retinol; senile systemic amyloidosis; sodium dodecylsulfate polyacrylamide gel electrophoresis; thioflavin T; thyroxine; transthyretin.
© 2013.