Engineering botulinum neurotoxin to extend therapeutic intervention
Engineering botulinum neurotoxin to extend therapeutic intervention
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DOI：
10.1073/pnas.0903111106
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发表时间：
2009-06-09
影响因子：
11.1
通讯作者：
Barbieri, Joseph T.
中科院分区：
文献类型：
--
作者：
Chen, Sheng;Barbieri, Joseph T.
肉毒梭菌神经毒素 (BoNT) 是治疗多种神经系统疾病的有效药物，如斜视、眼睑痉挛、半侧痉挛和颈肌张力障碍，因为该毒素对神经元有趋向性，并且能特异性裂解神经元可溶性 N-乙基马来酰亚胺敏感融合蛋白-附着蛋白受体 （圈套）蛋白质。已经尝试修饰 BoNT 以结合非神经元细胞来扩展治疗应用。然而，开发非神经元疗法的先决条件是将 BoNT 的催化活性重新定位到非神经元 SNARE 亚型。在此，我们报道了一种 BoNT 衍生物的工程设计，该衍生物可切割 SNAP23（一种非神经元 SNARE 蛋白）。 SNAP23 介导囊泡-质膜融合过程，包括气道粘液、抗体、胰岛素、胃酸和离子的分泌。这种突变的 BoNT/E 轻链 LC/E((KD)-D-224) 显示出扩展的底物特异性，可以裂解 SNAP23 和天然底物 SNAP25，但不能裂解 SNAP29 或 SNAP47。将蛋白质直接递送至培养的人上皮细胞后，LC/E((KD)-D-224) 裂解内源性 SNAP23，从而抑制粘蛋白和 IL-8 的分泌。这些研究表明对 LC 进行基因改造以靶向非神经元 SNARE 蛋白的可行性，该蛋白可扩展治疗人类分泌过多疾病的治疗潜力。
Clostridium botulinum neurotoxins (BoNTs) are effective therapeutics for a variety of neurological disorders, such as strabismus, blepharospam, hemificial spasm, and cervical dystonia, because of the toxin's tropism for neurons and specific cleavage of neuronal soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors (SNARE) proteins. Modifying BoNT to bind nonneuronal cells has been attempted to extend therapeutic applications. However, prerequisite to develop nonneuronal therapies requires the retargeting the catalytic activity of BoNTs to nonneuronal SNARE isoforms. Here, we reported the engineering of a BoNT derivative that cleaves SNAP23, a nonneuronal SNARE protein. SNAP23 mediates vesicle-plasma membrane fusion processes, including secretion of airway mucus, antibody, insulin, gastric acids, and ions. This mutated BoNT/E light chain LC/E((KD)-D-224) showed extended substrate specificity to cleave SNAP23, and the natural substrate, SNAP25, but not SNAP29 or SNAP47. Upon direct protein delivery into cultured human epithelial cells, LC/E((KD)-D-224) cleaved endogenous SNAP23, which inhibited secretion of mucin and IL-8. These studies show the feasibility of genetically modifying LCs to target a nonneuronal SNARE protein that extends therapeutic potential for treatment of human hypersecretion diseases.