Characterization of botulinum neurotoxin A subtypes

肉毒杆菌神经毒素 A 亚型的表征

• 批准号: 9542546
• 负责人: Joseph T Barbieri
• 金额: $ 56.41万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9542546
• 关键词: Affect; Affinity; Amino Acid Substitution; Amino Acids; Animals; Antitoxins; Area; Behavior; Binding; Biological; Bontoxilysin; Botulinum Toxin Type A; Botulism; C-terminal; CCRL2 gene; Caring; Categories; Cell model; Cells; Characteristics; Chimera organism; Data; Development; Disease; Elements; Engineering; Esthetics; Exhibits; GTP-Binding Protein alpha Subunits, Gs; Gangliosides; Human; Hybrids; Immunologics; In Vitro; Individual; Industry; Intoxication; Investigation; Kinetics; Laboratories; Medical; Medicine; Membrane; Methods; Modeling; Molecular; Mus; Mutagenesis; Mutate; Neuromuscular Diseases; Neurons; Paralysed; Pathologic; Pathology; Patients; Pharmacologic Substance; Pharmacology; Property; Protein Family; Proteins; Receptor Cell; Recombinants; Role; SNAP receptor; Serotyping; Severity of illness; Stem cells; Structural Models; Supportive care; Synaptic Vesicles; System; Testing; Therapeutic; Time; Toxic effect; Toxin; Toxin Conjugates; Variant; Work; base; cost; experimental study; field study; holotoxins; immunocytochemistry; in vivo; insight; mouse model; nervous system disorder; novel; physical symptom; rare variant; receptor; receptor binding; respiratory; symptomatology; therapy development; trafficking; vaccine development; weapons

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.

摘要： 肉毒神经毒素（BotanicNeurotoxins，BoNT）是蛋白质毒素的一个大家族，并且由于它们的生物活性而具有重要意义。 极端的效力和它们在人类和动物中引起的疾病的严重性。肉毒杆菌中毒是一种 持续时间长的神经麻痹性疾病，可持续数月。如果没有适当的医疗护理， 肉毒杆菌中毒在高达50%的病例中是致命的，即使有呼吸和其他支持性护理， 抗毒素给药后，高达5%的患者死亡。虽然自然发生的肉毒杆菌中毒是罕见的， 由于其潜在的生物恐怖主义武器的威胁，被归类为第1级A类选择代理。令人惊讶的是， BoNT还广泛用于医学中以治疗100多种神经肌肉疾病和用于美学目的。 目前，这是一个价值超过20亿美元的行业，而且还在不断增长。BoNT在免疫学上分为 分为7个血清型，其进一步细分为亚型。今天，已经有100多个BoNT变体 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变体中，目前只有两种用于医学领域， 使用其他变体的概率很低。我们的RO 1项目“BoNT/A亚型分析”已经确定了第一个 BoNT/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、效力和免疫学变化。这个更新项目建议将这些数据 在分子和结构水平上确定这些独特性质的机制。 具体而言，本项目将研究BoNT/A3作用持续时间较短的机制， BoNT/A2进入细胞的速度更快，效率更高，而BoNT/A4的效力低1,000倍。 我们的合作努力在这一领域是独一无二的，因为我们能够将联合收割机的详细机理研究结合起来， 在全毒素水平上进行基于细胞和体内研究。Barbieri实验室将使用 结构建模，以指导对BoNTs功能结构域的广泛诱变研究，并研究 使用神经元细胞模型的受体结合和细胞运输机制。约翰逊/佩利特实验室 将利用这些数据在其天然宿主中创建靶向全毒素构建体，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，根据这些数据， 研究中，将在小鼠中研究选择的全毒素构建体，以确定病理学和药理学 结构变化的后果。使用这种方法，我们能够调查大量的 氨基酸取代和选择特定的改变，用于更费力和成本密集的构建， 重组全毒素通过在进行体内研究之前利用几种细胞模型，我们能够 减少所需动物的数量，并使用人类特定的模型。最后，结合在 体外，细胞为基础的，和在体内的研究将提供新的洞察机制的基础上观察到的 这些毒素的病理学和药理学特性。