Engineering Neprilysin Activity and Specificity to Create a Novel Therapeutic for Alzheimer’s Disease
Engineering Neprilysin Activity and Specificity to Create a Novel Therapeutic for Alzheimer’s Disease
复制标题
改造脑啡肽酶活性和特异性以创造一种治疗阿尔茨海默病的新疗法
作者：
C. Webster;M. Burrell;Lise;Susan B. Fowler;S. Digby;Alan M. Sandercock;A. Snijder;J. Tebbe;U. Haupts;J. Grudzinska;L. Jermutus;Christin Andersson
Neprilysin是一种跨膜锌金属肽酶，可降解多种肽底物。由于其降解β淀粉样蛋白的能力，它作为一种潜在的治疗阿尔茨海默病的药物受到了关注。然而，其广泛的肽底物有可能限制其治疗用途，因为其他肽底物的降解严格调节许多生理过程。我们试图生成一种可溶性的neprilysin外畴，它对淀粉样蛋白β具有更好的活性和特异性，作为阿尔茨海默病的潜在治疗药物。在活性位点周围的位置和酶的内表面进行了广泛的氨基酸替换，并对淀粉样蛋白β 1 - 40,1 - 42和各种其他生理相关肽的活性进行了筛选。我们确定了几个突变，调节和改善酶的选择性和内在活性。Neprilysin变体G399V/G714K对淀粉样蛋白β 1-40的活性提高了约20倍，对其他肽的活性降低了3200倍。随着肽底物特异性的改变，与野生型酶相比，突变酶产生了一系列明显改变的β淀粉样蛋白裂解产物。突变酶的结晶表明，与野生型酶相比，氨基酸取代导致含有活性位点的口袋的形状和大小发生改变。这种突变酶提供了在体内更有效地降解淀粉样蛋白的潜力，作为治疗阿尔茨海默病的一种治疗方法。
Neprilysin is a transmembrane zinc metallopeptidase that degrades a wide range of peptide substrates. It has received attention as a potential therapy for Alzheimer’s disease due to its ability to degrade the peptide amyloid beta. However, its broad range of peptide substrates has the potential to limit its therapeutic use due to degradation of additional peptides substrates that tightly regulate many physiological processes. We sought to generate a soluble version of the ectodomain of neprilysin with improved activity and specificity towards amyloid beta as a potential therapeutic for Alzheimer’s disease. Extensive amino acid substitutions were performed at positions surrounding the active site and inner surface of the enzyme and variants screened for activity on amyloid beta 1–40, 1–42 and a variety of other physiologically relevant peptides. We identified several mutations that modulated and improved both enzyme selectivity and intrinsic activity. Neprilysin variant G399V/G714K displayed an approximately 20-fold improved activity on amyloid beta 1–40 and up to a 3,200-fold reduction in activity on other peptides. Along with the altered peptide substrate specificity, the mutant enzyme produced a markedly altered series of amyloid beta cleavage products compared to the wild-type enzyme. Crystallisation of the mutant enzyme revealed that the amino acid substitutions result in alteration of the shape and size of the pocket containing the active site compared to the wild-type enzyme. The mutant enzyme offers the potential for the more efficient degradation of amyloid beta in vivo as a therapeutic for the treatment of Alzheimer’s disease.
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影响因子：
6
作者：
El-Amouri, Salim S.;Zhu, Hong;Kindy, Mark S.
通讯作者：
Kindy, Mark S.
DOI：
10.1073/pnas.0500063102
发表时间：
2005-05-10
影响因子：
11.1
作者：
Varadarajan, N;Gam, J;Iverson, BL
通讯作者：
Iverson, BL
影响因子：
2.9
作者：
M. Pozsgay;C. Michaud;M. Liebman;M. Orłowski
通讯作者：
M. Pozsgay;C. Michaud;M. Liebman;M. Orłowski
在没有底物的情况下，嗜热菌蛋白酶具有开放构象。
DOI：
10.1107/s090744490200584x
发表时间：
2002
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Hausrath,AndrewC;Matthews,BrianW
通讯作者：
Matthews,BrianW