Formation of cytotoxic transthyretin is not dependent on inter-molecular disulphide bridges commonly found within the amyloid form

Abstract

Familial amyloidotic polyneuropathy (FAP) is linked to destabilising point mutations in the human plasma protein transthyretin (TTR). Consistent with similar amyloid disorders, low molecular weight TTR oligomers have been shown to exert the major cytotoxic effect. The amyloid structure of TTR contains non-native inter-molecular disulphide linkages via the cysteine at position 10 (Cys10). Moreover, substitution of Cys10 in a mouse model for TTR-amyloidosis abolishes TTR deposits, indicating an important role of Cys10 in FAP pathogenesis. However, the role of disulphide bridges in TTR cytotoxicity has not been elucidated. By probing Cys10Ser TTR variants to the human neuroblastoma SH-SY5Y cell line, we have addressed this question, and our results clearly show that formation of an inter-molecular disulphide bridge is not a pre-requisite for TTR cytotoxicity. This finding suggests that prevention of inter-molecular TTR disulphide bridges as a therapeutic intervention will not impair the cytotoxic potential of TTR.

Mechanisms of transthyretin cardiomyocyte toxicity inhibition by resveratrol analogs

“…Consistent with previous observations [19; 24; 25], WT TTR purified at room temperature was not toxic to AC16; however, when it was purified at 4°C, WT TTR was toxic to the cells in a concentration-dependent manner. In contrast, the amyloidogenic V122I TTR variant was cytotoxic to AC16 cells regardless of the temperature of purification (Supplemental Figure S1).…”

The Flavonoid Luteolin, but Not Luteolin-7-O-Glucoside, Prevents a Transthyretin Mediated Toxic Response

“…This suggests that the toxic effect does not correlate well with the total load of aggregates. Such result is further supported by cell studies, where the stability of a tetramer instead correlates with toxic effect [ 29 , 42 ]. Measuring the stability of the TTR tetramer within the fly is however technically challenging and could not be performed.…”

“…In this work, we have used a cell-based viability assay to assess the ability of luteolin to suppress TTR-induced toxicity. The cytotoxicity of recombinant human TTR (TTR) in SH-SY5Y neuroblastoma cells has previously been demonstrated, and this was the experimental system used throughout the present investigation [ 29 ]. The cells were incubated with TTR at a concentration corresponding to 18 μM tetrameric TTR for a total time of 72 h. The response to TTR—as evaluated by a decrease in the conversion of resazurin to resorufin—was a 30% reduction in cell survival ( Fig 2 ).…”

“…Expression of TTR was performed according to a previously published protocol [ 29 ]. Briefly, after transformation of the plasmid into Escherichia coli BL21, the cells were grown until an OD 600 of 0.6 followed by induction with 0.4 mM isopropyl thiogalactopyranoside at 37°C.…”

Statistical molecular design of a focused salicylidene acylhydrazide library and multivariate QSAR of inhibition of type III secretion in the Gram-negative bacterium Yersinia

“…Cell viability was evaluated using a resazurin (blue and non-fluorescent, Sigma 40 lM final concentration), that is, reduced to resorufin (pink and highly fluorescent) in living cells. 63,64 Fluorescence was measured using a Tecan Safire plate reader with excitation at 535 nm and emission at 595 nm. Resazurin was added four hours prior to measurement and cell-free wells with medium and compound were used to rule out chemical reduction of resazurin by the compounds.…”