Structural Mechanisms of Botulinum Neurotoxin Pathogenesis

描述(申请人提供)：肉毒杆菌神经毒素(BoNTs)是已知的最有毒的物质，已被疾控中心列为生物恐怖主义的六种最高风险威胁因子之一。他们是肉毒杆菌中毒的罪魁祸首，肉毒杆菌中毒是一种神经麻痹的疾病，如果不治疗，可能会进展为迟缓性瘫痪和死亡。BoNTs是七种不同的免疫血清型之一(A-G)。它们通过进入神经元并分解蛋白质来调节神经递质的胞吐作用。它们被认为通过双受体机制结合神经元，其中包括与神经节苷脂和蛋白质受体的相互作用。蛋白质受体的同一性取决于七种BONT血清型中的哪一种，但受体特异性和双受体结合的分子决定因素目前尚不清楚。这项建议描述了两个具体的目标，以探索BONT-受体相互作用的结构和功能特性。最近的证据表明，BoNT/A与神经节苷脂结合后改变构象，并与突触囊泡蛋白(SV2)受体的三种亚型具有不同的亲和力。在目标1中，我们将使用X射线结晶学、定点突变和结合分析来定义BoNT/A、神经节苷脂和SV2之间的相互作用。在BONT/B和BONT/G血清型的发病机制中，对神经节苷脂的要求可能不那么严格。这些BoNTs非常相似，但在它们与两种突触素亚型(SytI和II)相互作用的方式上有显著的差异。在目标2中，我们将使用X射线结晶学、定点突变和结合分析来描述BONT/G-SytI相互作用的特异性和亲和力要求。与BoNT/B-SytII相互作用的比较将提供一个分子答案，说明BoNT/B和/G与两种SYT亚型相互作用的不同之处，同时阐明可用于抑制两种血清型受体结合的共同特征。总之，这些研究将提供对BONT进入神经元的基本机制的洞察，并为设计和改进抗肉毒杆菌中毒疗法和预防药物产生新的战略。与公共卫生相关：肉毒杆菌神经毒素是疾控中心的A类毒素，因为它很容易生产和调制成生物武器，可能用于战争和恐怖主义。与其破坏力形成鲜明对比的是，肉毒杆菌神经毒素的效力使其能够在临床上用于治疗肌张力障碍、脑瘫、多发性硬化症、肌萎缩侧索硬化症、帕金森氏症和偏头痛。了解不同形式的肉毒杆菌神经毒素如何识别神经细胞的分子基础将揭示这种毒素的致病和治疗潜力的重要新见解。

DESCRIPTION (provided by applicant): The botulinum neurotoxins (BoNTs) are the most poisonous substances known and have been classified by the CDC as one of the six highest-risk threat agents for bioterrorism. They are responsible for the disease botulism, a neuroparalytic condition that can progress to flaccid paralysis and death if left untreated. The BoNTs exist as one of seven immunologically distinct serotypes (A-G). They act by entering neurons and cleaving proteins that mediate the exocytosis of neurotransmitters. They are thought to bind neurons through a dual-receptor mechanism that includes interactions with both ganglioside and protein receptors. The identity of the protein receptor depends on which of the seven BoNT serotypes is present, but the molecular determinants of receptor specificity and dual-receptor binding are currently unknown. This proposal describes two specific aims that explore the structural and functional properties of BoNT-receptor interactions. Recent evidence suggests that BoNT/A changes conformation upon binding ganglioside and has different affinities for the three isoforms of the synaptic vesicle protein (SV2) receptor. In Aim 1, we will define the interactions between BoNT/A, ganglioside, and SV2 using X-ray crystallography, site-directed mutagenesis, and binding assays. The requirement for ganglioside may not be as stringent in the pathogenesis of the BoNT/B and BoNT/G serotypes. These BoNTs are highly similar but have notable differences in how they interact with the two isoforms of synaptotagmin (SytI and II). In Aim 2, we will use X-ray crystallography, site-directed mutagenesis and binding assays to delineate the specificity and affinity requirements for BoNT/G-SytI interactions. Comparison to BoNT/B-SytII interactions will provide a molecular answer to how BoNT/B and /G differ in their interactions with the two Syt isoforms, while simultaneously illuminating the common features that could be exploited for inhibiting receptor binding in both serotypes. Together, these studies will provide insight into the fundamental mechanisms by which BoNT gains access to neurons and generate new strategies for the design and improvement of anti-botulism therapeutics and preventatives. PUBLIC HEALTH RELEVANCE: Botulinum neurotoxin is a CDC category A toxin because it is easily produced and concocted into a bio- weapon for potential use in warfare and terrorism. In striking contrast to its destructive power, the potency of botulinum neurotoxin allows it to be used clinically in the treatment of dystonias, cerebral palsy, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's, and migraines. Understanding the molecular basis of how different forms of botulinum neurotoxin recognize neuronal cells will reveal important new insights into the pathogenic and therapeutic potential of this toxin.