Investigation of the molecular mechanism of the radical reaction to induce the amyloid fibril derived from the transthyretin

老年系统性淀粉样变性和家族性淀粉样多发性神经病是由构象改变的甲状腺素运载蛋白（TTR）氧化沉积引起的。我们在体外通过S-磺化鉴定了TTR的第10个半胱氨酸的氧化修饰。基于质谱分析，我们确定了分光光度法，蛋白质印迹法，和荧光显微镜下的TTR孵育和不含半胱氨酸-S-磺酸盐在酸性（pH 4）和碱性（pH 8）条件下，在37度。聚集TTR分子的吸收随孵育时间和半胱氨酸-S-磺酸盐的浓度在pH 4下比在pH 8下增加更多。在pH 4下，刚果红与S-磺化TTR的结合饱和，表观Bmax为2.01 mol/mol S-磺化TTR，表观KD为7.75 × 10（-6）M。半胱氨酰TTR的Bmax为1.38，KD为3.52 × 10 ~（-6）M;还原TTR的Bmax为0.86，KD为2.86 × 10 ~（-6）M。此外，通过显微荧光分析，我们检测到使用硫磺素T和刚果红与S-磺化TTR的淀粉样蛋白纤维染色阳性，但未处理或还原的TTR未检测到。TTR在体外修饰后，寡聚体抵抗还原和变性被不可逆地诱导，这导致了用四种抗TTR抗体获得的Western印迹模式的差异。总之，本研究表明，通过氧化修饰TTR的第10个半胱氨酸上的巯基残基形成TTR的S-磺化是甲状腺素运载蛋白相关淀粉样纤维形成的重要触发步骤。

Senile systemic amyloidosis and familial amyloid polyneuropathy are caused by oxidative deposition of conformationally altered transthyretin (TTR). We identified oxidative modification of the 10th cysteine of TTR through S-sulfonation in vitro. Based on mass spectrometric analysis, we determined the spectrophotometric, western blotting, and fluorescent microscopic properties of TTR incubated with and without cysteine-S-sulfonate in acidic (pH 4) and alkaline (pH 8) conditions at 37 degrees. The absorption of the aggregated TTR molecules increased more with incubation time and the concentration of cysteine-S-sulfonate at pH 4 than at pH 8. The Congo red binding to the S-sulfonated TTR at pH 4 was saturated with an apparent Bmax of 2.01 mol per mole of the S-sulfonated TTR and apparent KD of 7.75x10(-6) M. On the other hand, the Bmax of cysteinyl TTR was1.38, and its KD was 3.52x10(-6) M while the Bmax of reduced TTR was 0.86, and its KD was 2.86x10(-6) M. Moreover, we detected poitive amyloid fibril staining using Thioflavin T and Congo red with the S-sulfonated TTR but not with untreated or reduced TTR by microscopic fluorescent analysis. After modification of TTR in vitro, oligomers resisted reduction and denaturation was irreversibly induced, and which contributed differences in the Western blotting patterns obtained with four anti-TTR antibodies. In conclusion, thisstudy showed that the formation of S-sulfonation of TTR through oxidative modifications of the thiol residue on the 10th cysteine of TTR is an important trigger step in the formation of transthyretin-related amyloid fibril.