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

老年性全身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转甲状腺蛋白(TTR)氧化沉积引起的。我们在体外通过S磺化反应鉴定了TTR第10位半胱氨酸的氧化修饰。在37℃的酸性(PH 4)和碱性(PH 8)条件下，我们测定了半胱氨酸-S-磺酸盐与非半胱氨酸-S-磺酸盐孵育的TTR的分光光度、免疫印迹和荧光显微镜性质。在pH 4条件下，随着孵育时间的延长和半胱氨酸-S磺酸盐浓度的增加，聚集态TTR分子的吸收增加更明显。在pH值为4的条件下，S磺化TTR与刚果红结合的表观Bmax为2.01摩尔/摩尔，表观Kd值为7.75×10~(-6)M。半胱氨基TTRBmax为1.38，Kd值为3.52×10~(-6)M，还原TTRBmax为0.86，Kd值为2.86×10~(-6)M。我们使用硫代黄素T和刚果红与S磺化的TTR一起检测到阳性的淀粉样纤维染色，但没有通过显微荧光分析检测到未经处理或还原的TTR。体外修饰TTR后，寡聚体抵抗还原和不可逆地诱导变性，这导致了四种抗TTR抗体获得的蛋白质印迹图谱的不同。综上所述，本研究表明，TTR10位半胱氨酸上的硫醇残基的氧化修饰形成TTR10位硫醇残基的S磺化反应是转甲状腺素相关淀粉样原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.