The modulation of transthyretin tetramer stability by cysteine 10 adducts and the drug diflunisal - Direct analysis by fluorescence-detected analytical ultracentrifugation
The modulation of transthyretin tetramer stability by cysteine 10 adducts and the drug diflunisal - Direct analysis by fluorescence-detected analytical ultracentrifugation
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DOI：
10.1074/jbc.m709638200
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发表时间：
2008-05-02
影响因子：
4.8
通讯作者：
Connors, Lawreen H.
中科院分区：
文献类型：
--
作者：
Kingsbury, Jonathan S.;Laue, Thomas M.;Connors, Lawreen H.
甲状腺素运载蛋白（TTR）通常是一种稳定的血浆蛋白。然而，在家族性TTR相关淀粉样变性和老年性系统性淀粉样变性（SSA）的情况下，TTR沉积为淀粉样纤维，导致器官功能障碍和可能的死亡。TTR经历从稳定的可溶性前体到不溶性淀粉样纤维的转变的机制以及促进这一过程的因素在很大程度上是不确定的。大多数模型涉及天然TTR四聚体的解离作为初始步骤。人们普遍认为，TTR基因突变与TTR相关的淀粉样变性导致表达的变异蛋白质是本质上不稳定的，易于聚集。已经提出，通过混合二硫键对单独的半胱氨酸残基（Cys（10））进行化学修饰，可以赋予野生型TTR（SSA中沉积的形式）淀粉样蛋白原性。S-磺化和S-半胱氨酸化是TTR生理学上常见的修饰，并且研究表明它们在变性条件下调节TTR结构的能力。在本研究中，我们已经使用荧光检测沉降速度，以确定在非变性条件下的四级结构稳定性的荧光团共轭的重组TTR的S-磺酸盐和S-半胱氨酸的效果。我们确定，S-磺化稳定TTR四聚体的稳定性的一个因素7，而S-半胱氨酸化增强解离2倍，相对于未修饰的形式。此外，我们报告了潜在的治疗化合物二氟尼柳的四聚体稳定的直接观察。最后，作为概念的证明，我们报告了血清中TTR的沉降和所得数据的定性评估。
Transthyretin (TTR) is normally a stable plasma protein. However, in cases of familial TTR-related amyloidosis and senile systemic amyloidosis (SSA), TTR is deposited as amyloid fibrils, leading to organ dysfunction and possibly death. The mechanism by which TTR undergoes the transition from stable, soluble precursor to insoluble amyloid fibril and the factors that promote this process are largely undetermined. Most models involve the dissociation of the native TTR tetramer as the initial step. It is largely accepted that the TTR gene mutations associated with TTR-related amyloidosis lead to the expression of variant proteins that are intrinsically unstable and prone to aggregation. It has been suggested that amyloidogenicity may be conferred to wild-type TTR (the form deposited in SSA) by chemical modification of the lone cysteine residue (Cys(10)) through mixed disulfide bonds. S-Sulfonation and S-cysteinylation are prevalent TTR modifications physiologically, and studies have suggested their ability to modulate the structure of TTR under denaturing conditions. In the present study, we have used fluorescence-detected sedimentation velocity to determine the effect of S-sulfonate and S-cysteine on the quaternary structural stability of fluorophore-conjugated recombinant TTR under nondenaturing conditions. We determined that S-sulfonation stabilized TTR tetramer stability by a factor of 7, whereas S-cysteinylation enhanced dissociation by 2-fold with respect to the unmodified form. In addition, we report the direct observation of tetramer stabilization by the potential therapeutic compound diflunisal. Finally, as proof of concept, we report the sedimentation of TTR in serum and the qualitative assessment of the resulting data.