Characterization of botulinum neurotoxin A subtypes

摘要： 肉毒杆菌神经毒素(BoNTs)是一大类蛋白质毒素，具有重要的意义。 它们在人类和动物中引起的疾病的极端效力和严重程度。肉毒杆菌中毒是一种 一种持续时间较长的神经性瘫痪疾病，可持续数月。如果没有适当的医疗护理，自然会 发生肉毒杆菌中毒在高达50%的病例中是致命的，即使在呼吸和其他支持性护理和 服用抗毒素药物，高达5%的患者死亡。虽然自然发生的肉毒杆菌中毒很少见，但BoNTs是 由于其潜在的生物恐怖武器的威胁，被归类为一级A类精选特工。令人惊讶的是， BoNTs还被广泛用于医学，治疗100多种神经肌肉疾病和美容 用途，它现在是一个价值超过20亿美元的行业，而且还在增长。BoNTs在免疫上是分开的 分为7个血清型，并进一步细分为亚型。今天，已经有100种BONT变种 通过测序工作鉴定，但只有少数在蛋白质水平上进行了研究。值得注意的是 在所有这些BONT变种中，只有两种目前用于医疗领域，研究人员正在研究其益处 使用其他变种的情况很少见。我们的RO1项目‘BONT/A亚型分析’首次确定 ONT/A血清型中的亚型具有不同生物学特性的时间，包括细胞进入动力学， 作用持续时间、效力和免疫变异。该更新项目建议扩展这些数据 在分子和结构水平上确定这些独特性质背后的机制。 具体地说，该项目将调查BONT/A3作用时间较短的机制， BONT/A2输入细胞的速度更快、效率更高，而BONT/A4的效力降低了1,000倍。 我们的合作努力在这一领域是独一无二的，因为我们能够结合详细的机械研究 全毒素水平的亚域和基于细胞的体内研究。巴比耶里实验室将使用 用结构模型指导BoNTs功能域的诱变研究和研究 用神经细胞模型研究受体结合和细胞转运的机制。约翰逊/佩莱特实验室 将利用这些数据在其本机主机中创建靶向全毒素结构，并进行详细的 在各种细胞模型中的研究，包括人类细胞模型。最后，根据来自这些公司的数据 研究，选择全毒素结构将在小鼠身上进行调查，以确定病理和药理学 结构变化的后果。使用这种方法，我们能够调查大量的 氨基酸替代和选择特定的改变，以进行更费力和成本更密集的建设 重组全毒素。在进行体内研究之前，通过利用几种细胞模型，我们能够 减少所需动物的数量，也使用人类特有的模型。最后，In的组合 体外、基于细胞和体内的研究将提供对所观察到的 这些毒素的病理和药理特性。

Abstract: Botulinum Neurotoxins (BoNTs) are a large family of protein toxins and are of great significance due to their extreme potency and the severity of the disease they cause in humans and animals. Botulism is a neuroparalytic disease of long duration, lasting up to several months. Without proper medical care, naturally occurring botulism is lethal in up to 50% of cases, and even with respiratory and other supportive care and antitoxin administration, up to 5 % of patients die. While naturally occurring botulism is rare, BoNTs are classified as a Tier 1 Category A Select Agent due to their threat as potential bioterrorist weapons. Amazingly, BoNTs are also widely used in medicine to treat more than 100 neuromuscular disorders and for aesthetic purposes, which is now an over 2 billion dollar industry and is growing. BoNTs are immunologically divided into 7 serotypes, which are further subdivided into subtypes. Today, 100s of BoNT variants have been identified by sequencing efforts, but only few have been investigated at the protein level. It is remarkable that out of all these BoNT variants only two are currently used in the medical field, and studies examining benefits of using other variants are rare. Our RO1 project `Analysis of BoNT/A subtypes' has determined for the first time that subtypes within the BoNT/A serotype have distinct biologic properties, including cell entry kinetics, duration of action, potency, and immunological variations. This renewal project proposes to extend these data to determine on a molecular and structural level the mechanisms underlying these unique properties. Specifically, this project will investigate the mechanisms underlying the shorter duration of action of BoNT/A3, the faster and more efficient cell entry by BoNT/A2, and the 1,000 fold lower potency of BoNT/A4. Our collaborative efforts are unique in this area, as we are able to combine detailed mechanistic studies on subdomains with cell-based and in vivo studies on the holotoxin level. The Barbieri laboratory will use structural modeling to guide extensive mutagenesis studies on functional domains of BoNTs and investigate mechanisms of receptor binding and cell trafficking using neuronal cell models. The Johnson/Pellett laboratory will utilize these data to create targeted holotoxin constructs in their native hosts and conduct detailed investigations in various cell models, including human cell models. Finally, based on the data from these studies, select holotoxin constructs will be investigated in mice to determine the pathologic and pharmacologic consequences of structural alterations. Using this approach, we are able to investigate a large number of amino acid substitutions and select specific alterations for the more effort- and cost-intensive construction of recombinant holotoxins. By utilizing several cell models before conducting in vivo studies, we are able to reduce the number of required animals and also use human specific models. Finally, the combination of in vitro, cell-based, and in vivo studies will provide novel insight into the mechanisms underlying the observed pathologic and pharmacologic properties of these toxins.