α1-antitrypsin Portland, a bioengineered serpin highly selective for furin:: Application as an antipathogenic agent
α1-antitrypsin Portland, a bioengineered serpin highly selective for furin:: Application as an antipathogenic agent
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DOI：
10.1073/pnas.95.13.7293
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发表时间：
1998-06-23
影响因子：
11.1
通讯作者：
Thomas, G
中科院分区：
文献类型：
--
作者：
Jean, F;Stella, K;Thomas, G
弗林蛋白酶在许多致病分子的蛋白水解激活中的重要作用使得这种内切蛋白酶成为开发有效和选择性抗蛋白水解剂的靶点。在这里，我们证明了基于蛋白质的抑制剂α（1）-抗胰蛋白酶波特兰（α（1）-PDX）作为抗病原体药物的实用性，可以预防性地用于阻断假单胞菌外毒素A对弗林蛋白酶依赖性细胞的杀伤。细菌表达的His-和FLAG-标记的α（1）-PDX（α（1）-PDX/hf）的特异性的生物化学分析揭示了α（1）-PDX/hf反应位点环对弗林蛋白酶（Ki，600 pM）的选择性，但对其他前蛋白转化酶家族成员或其他不相关的内切蛋白酶没有选择性。动力学研究表明，α（1）-PDX/hf抑制弗林蛋白酶的缓慢紧结合机制的丝氨酸蛋白酶抑制剂分子和功能作为一种自杀底物抑制剂。一旦与弗林蛋白酶的活性位点结合，α（1）-PDX/hf以相等的概率在反应中心-Arg（355）-Ile-Pro-Arg（358）->的C-末端侧通过弗林蛋白酶进行蛋白水解或与酶形成动力学捕获的SDS稳定复合物。复合物形成和蛋白水解途径之间的这种分配有助于α（1）-PDX/hf差异抑制前蛋白转化酶家族成员的能力。最后，我们提出了一个结构模型的α（1）-PDX反应位点环，解释了这种丝氨酸蛋白酶抑制剂的高度酶的选择性，并可用于产生小分子弗林蛋白酶抑制剂。
The important role of furin in the proteolytic activation of many pathogenic molecules has made this endoprotease a target for the development of potent and selective antiproteolytic agents. Here, we demonstrate the utility of the protein-based inhibitor alpha(1)-antitrypsin Portland (alpha(1)-PDX) as an antipathogenic agent that can be used prophylactically to block furin-dependent cell killing by Pseudomonas exotoxin A. Biochemical analysis of the specificity of a bacterially expressed His- and FLAG-tagged alpha(1)-PDX (alpha(1)-PDX/hf) revealed the selectivity of the alpha(1)-PDX/hf reactive site loop for furin (K-i, 600 pM) but not for other proprotein convertase family members or other unrelated endoproteases. Kinetic studies show that alpha(1)-PDX/hf inhibits furin by a slow tight-binding mechanism characteristic of serpin molecules and functions as a suicide substrate inhibitor. Once bound to furin's active site, alpha(1)-PDX/hf partitions with equal probability to undergo proteolysis by furin at the C-terminal side of the reactive center -Arg(355)-Ile-Pro-Arg(358)- --> or to form a kinetically trapped SDS-stable complex with the enzyme. This partitioning between the complex-forming and proteolytic pathways contributes to the ability of alpha(1)-PDX/hf to differentially inhibit members of the proprotein convertase family. Finally, we propose a structural model of the alpha(1)-PDX-reactive site loop that explains the high degree of enzyme selectivity of this serpin and which can be used to generate small molecule furin inhibitors.