Activity-modulating monoclonal antibodies to the human serine protease HtrA3 provide novel insights into regulating HtrA proteolytic activities.
Activity-modulating monoclonal antibodies to the human serine protease HtrA3 provide novel insights into regulating HtrA proteolytic activities.
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DOI：
10.1371/journal.pone.0108235
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Nie G
中科院分区：
文献类型：
--
作者：
Singh H;Nero TL;Wang Y;Parker MW;Nie G
哺乳动物HtrA(高温需求因子A)蛋白酶由4个多结构域成员HtrA1-4组成，在许多正常的细胞过程以及癌症、关节炎、神经退行性疾病和妊娠障碍等病理条件下发挥重要作用。然而，HtrA活性是如何被调控的还不是很清楚，到目前为止还没有发现针对单个HtrA蛋白的抑制剂。在这里，我们研究了我们之前生产的五种HtrA3单抗，并证明其中两种以相反的方式调节HtrA3的活性：一种被抑制，另一种被刺激。该抑制单抗还可阻断HtrA3在滋养层细胞中的活性，促进滋养细胞的迁移和侵袭，证实了其在体内的潜在用途。为了了解这些单抗的结合是如何调节HtrA3蛋白酶活性的，我们用三维HtrA3同源模型对它们的表位进行了可视化。这一模型表明，抑制性HtrA3单抗可以阻断底物对蛋白酶催化部位的访问，而刺激性单抗可以单独与PDZ结构域结合，也可以与N-末端和蛋白酶结构域结合。由于HtrA1、HtrA3和HtrA4具有相同的结构域组织，我们的结果为开发针对这些HtrA蛋白的潜在疗法奠定了重要基础，这些蛋白质专门用于治疗许多疾病，包括癌症和妊娠障碍。
Mammalian HtrA (high temperature requirement factor A) proteases, comprising 4 multi-domain members HtrA1-4, play important roles in a number of normal cellular processes as well as pathological conditions such as cancer, arthritis, neurodegenerative diseases and pregnancy disorders. However, how HtrA activities are regulated is not well understood, and to date no inhibitors specific to individual HtrA proteins have been identified. Here we investigated five HtrA3 monoclonal antibodies (mAbs) that we have previously produced, and demonstrated that two of them regulated HtrA3 activity in an opposing fashion: one inhibited while the other stimulated. The inhibitory mAb also blocked HtrA3 activity in trophoblast cells and enhanced migration and invasion, confirming its potential in vivo utility. To understand how the binding of these mAbs modulated HtrA3 protease activity, their epitopes were visualized in relation to a 3-dimensional HtrA3 homology model. This model suggests that the inhibitory HtrA3 mAb blocks substrate access to the protease catalytic site, whereas the stimulatory mAb may bind to the PDZ domain alone or in combination with the N-terminal and protease domains. Since HtrA1, HtrA3 and HtrA4 share identical domain organization, our results establish important foundations for developing potential therapeutics to target these HtrA proteins specifically for the treatment of a number of diseases, including cancer and pregnancy disorders.
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