Kinetic Instability of the Serpin Z α1-Antitrypsin Promotes Aggregation
Kinetic Instability of the Serpin Z α1-Antitrypsin Promotes Aggregation
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DOI：
10.1016/j.jmb.2009.11.048
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发表时间：
2010-02-19
影响因子：
5.6
通讯作者：
Bottomley, Stephen P.
中科院分区：
文献类型：
--
作者：
Knaupp, Anja S.;Levina, Vita;Bottomley, Stephen P.
丝氨酸蛋白酶抑制剂（serpin）分子不适当的构象改变和自结合（聚合）引起的大量疾病。最常见的丝状病变是α(1)-抗胰蛋白酶（α (1)AT）缺乏，这与肝硬化、肝细胞癌和早发性肺气肿的风险增加有关。α (1)AT的Z变异占所有α (1)AT缺乏症病例的95%，在合成和分泌后发生聚合。在这里，我们使用内禀和外禀荧光探针表明，Z α (1)AT以非天然构象存在。我们通过横向尿素梯度凝胶电泳、热变性和平衡盐酸胍展开来检测热力学稳定性，发现尽管两种蛋白在结构上存在差异，但野生型α (1)AT和Z α (1)AT表现出相似的展开途径和热力学稳定性。远紫外圆二色性和双ans（4,4'-二苯胺-1,1'-二萘基-5,5'-二磺酸-二钾盐）荧光表明野生型α (1)AT和Z α (1)AT展开过程中形成的中间系簇具有相似的结构特征。展开转变的动力学分析表明，Z α (1)AT的展开速度比野生型至少快1.5倍。讨论了这些数据的生物学意义。2009爱思唯尔有限公司版权所有。
The serpinopathies encompass a large number of diseases caused by inappropriate conformational change and self-association (polymerization) of a serpin (serine proteinase inhibitor) molecule. The most common serpinopathy is alpha(1)-antitrypsin (alpha(1)AT) deficiency, which is associated with an increased risk for liver cirrhosis, hepatocellular carcinoma and early-onset emphysema. The Z variant of alpha(1)AT, which accounts for 95% of all cases of alpha(1)AT deficiency, polymerizes during synthesis and after secretion. Here, we show using intrinsic and extrinsic fluorescence probes that Z alpha(1)AT exists in a non-native conformation. We examined the thermodynamic stability by transverse urea gradient gel electrophoresis, thermal denaturation and equilibrium guanidine hydrochloride unfolding and found that, despite structural differences between the two proteins, wild-type alpha(1)AT and Z alpha(1)AT display similar unfolding pathways and thermodynamic stabilities. Far-UV circular dichroism and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid, dipotassium salt) fluorescence suggest that the intermediate ensembles formed during unfolding of wild-type of alpha(1)AT and Z alpha(1)AT are characterized by similar structural features. Kinetic analysis of the unfolding transition showed that Z alpha(1)AT unfolds at least 1.5-fold faster than the wild type. The biological implications of these data are discussed. (C) 2009 Elsevier Ltd. All rights reserved.