Abstract：
Transthyretin (TTR) is a homotetrameric protein involved in the transport of thyroxine. More than 150 different mutations have been described in the TTR gene, several of them associated with familial amyloid cardiomyopathy (FAC). Recently, our group described a new variant of TTR in Brazil, namely A39D-TTR, which causes a severe cardiac condition. Position 39 is in the AB loop, a region of the protein that is located within the thyroxine-binding channels and is involved in tetramer formation. In the present study we solved the structure and characterize the thermodynamic stability of this new variant of TTR using urea and high hydrostatic pressure (HHP). Interestingly, during the process of purification, A39D-TTR turned out to be a dimer and not a tetramer, a variation that might be explained by the close contact of the four aspartic acids at position 39, where they face each other inside the thyroxine channel. In the presence of sub-denaturing concentrations of urea, bis-ANS binding and dynamic light scattering revealed A39D-TTR in the form of a molten-globule dimer. Co-expression of A39D and WT isoforms in the same bacterial cell did not produce heterodimers or heterotetramers, suggesting that somehow a negative charge at the AB loop precludes tetramer formation. A39D-TTR proved to be highly amyloidogenic, even at mildly acidic pH values where WT-TTR does not aggregate. Interestingly, despite being a dimer, aggregation of A39D-TTR was inhibited by diclofenac, which binds to the thyroxine channel in the tetramer, suggesting the existence of other pockets in A39D-TTR able to accommodate this molecule.
摘要：
转甲状腺素蛋白(TTR)是参与甲状腺素转运的同四聚体蛋白。在TTR基因中已经描述了超过150种不同的突变，其中一些与家族性淀粉样心肌病（FAC）有关。最近,我们的研究小组在巴西描述了一种新的TTR变体，即A39D-TTR,导致严重的心脏病.位置39在AB环路中，位于甲状腺素结合通道内并参与四聚体形成的蛋白质区域。在本研究中，我们使用尿素和高静水压（HHP）解决了TTR的这种新变体的结构并表征了其热力学稳定性。有趣的是,在净化过程中，A39D-TTR原来是二聚体而不是四聚体，这种变化可以解释为四种天冬氨酸在39位的紧密接触，它们在甲状腺素通道内彼此面对。在亚变性浓度的尿素存在下，bis-ANS结合和动态光散射显示A39D-TTR为熔融小球二聚体。A39D和WT同种型在同一细菌细胞中的共表达不产生异二聚体或异四聚体，这表明AB环处的负电荷以某种方式阻止了四聚体的形成。A39D-TTR被证明是高度淀粉样蛋白，即使在轻度酸性pH值，WT-TTR不会聚集。有趣的是,尽管是二聚体，双氯芬酸抑制A39D-TTR的聚集，它与四聚体中的甲状腺素通道结合，这表明A39D-TTR中存在能够容纳该分子的其他口袋。
