Kinetic stabilization of an oligomeric protein under physiological conditions demonstrated by a lack of subunit exchange: implications for transthyretin amyloidosis.
Kinetic stabilization of an oligomeric protein under physiological conditions demonstrated by a lack of subunit exchange: implications for transthyretin amyloidosis.
复制标题
通过缺乏亚基交换证明生理条件下寡聚蛋白的动力学稳定性：对转甲状腺素蛋白淀粉样变性的影响。
DOI：
10.1021/bi050352o
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发表时间：
2005
期刊：
影响因子：
2.9
通讯作者：
Kelly,JefferyW
中科院分区：
文献类型：
--
作者：
Wiseman,RLuke;Green,NoraS;Kelly,JefferyW
转甲状腺素(TTR)的动力学稳定化是为了防止人类神经退变。因此，小分子介导的自然状态的动力学稳定是一种有吸引力的策略，以防止与TTR淀粉样病相关的错误折叠和错误组装。由于导致人类TTR淀粉样变的生理微环境尚不清楚，保守的方法是确定在各种条件下发挥作用的抑制剂。通过浓度依赖地抑制酸诱导的淀粉样蛋白合成和尿素诱导的四聚体解离，建立了TTR的小分子动力学稳定。由于变性条件降低了抑制剂的结合亲和力，使得在生理条件下预测抑制剂的效果变得困难，我们介绍了一种定量测定生理条件下的动力学稳定性的方法。野生型TTR同源异构体和标记有N-末端酸性标记的野生型TTR同质异构体之间的亚基交换速率由重组前四聚体到其单体亚基的解离速率决定，这使得该方法非常适合于评估小分子结合赋予TTR的动力学稳定性和评估小分子结合常数。在这种交换反应中加入淀粉样蛋白生成抑制剂以浓度依赖的方式减缓四聚体的解离，在溶液中至少有一个抑制剂与每个四聚体结合的浓度下停止解离。亚基交换使四聚体的解离速率和小分子结合所带来的动力学稳定性能够在不因聚集或不可逆解离而降低TTR浓度的生理条件下被评估。
Kinetic stabilization of transthyretin (TTR) is established to prevent human neurodegeneration. Therefore, small molecule-mediated kinetic stabilization of the native state is an attractive strategy to prevent the misfolding and misassembly associated with TTR amyloid disease. Since the physiological microenvironment resulting in human TTR amyloidogenesis remains unclear, the conservative approach is to identify inhibitors that function under a variety of conditions. Small molecule kinetic stabilization of TTR has been established by concentration-dependent inhibition of acid-mediated amyloidogenesis and urea-induced tetramer dissociation. Since denaturing conditions reduce the binding affinity of inhibitors making it difficult to predict inhibitor efficacy under physiological conditions, we introduce a method for quantifying kinetic stabilization under physiological conditions. The rate of subunit exchange between wild-type TTR homotetramers and wild-type TTR homotetramers tagged with an N-terminal acidic flag tag is dictated by the rate of tetramer dissociation to its monomeric subunits prior to reassembly, rendering this method ideally suited for assessing the kinetic stabilization of TTR imparted by small molecule binding and evaluating small molecule binding constants. Addition of amyloidogenesis inhibitors to this exchange reaction slows tetramer dissociation in a concentration-dependent manner, stopping dissociation at concentrations where at least one inhibitor is bound to each tetramer in solution. Subunit exchange enables the rate of tetramer dissociation and the kinetic stabilization imparted by small molecule binding to be evaluated under physiological conditions in which the TTR concentration is not reduced by aggregation or irreversible dissociation.