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.

抽象的： 肉毒杆菌神经毒素（BONTS）是大型蛋白质毒素家族，由于它们 它们在人类和动物中引起的疾病的极端效力和严重程度。肉毒杆菌是一个 长持续时间的神经分析疾病，长达几个月。没有适当的医疗服务，自然而然 肉毒杆菌发生在多达50％的病例中是致命的，即使是呼吸和其他支持护理， 抗毒素给药，多达5％的患者死亡。虽然自然发生的肉毒杆菌很少见 由于其威胁是潜在的生物恐怖武器，因此被归类为A级A类别A类别。令人惊讶的是 BONT也广泛用于医学中，以治疗100多个神经肌肉疾病和美学 目的，现在已超过20亿美元的行业，并且正在增长。 BONT在免疫学上分裂 分为7种血清型，进一步细分为亚型。今天，有100秒的Bont变体已经 通过测序工作确定，但在蛋白质水平上只有很少的研究。值得注意的是 在所有这些BONT变体中，目前仅在医疗领域使用两种，并研究了福利 使用其他变体很少见。我们的RO1项目“ BONT/A子类型的分析”已确定第一个 BONT/A血清型中的亚型具有不同的生物学特性，包括细胞进入动力学， 作用持续时间，效力和免疫学变化。这个更新项目建议扩展这些数据 为了在分子和结构水平上确定这些独特特性的基础机制。 具体而言，该项目将研究BONT/A3的作用持续时间较短的机制， BONT/A2的速度更快，更有效的细胞进入，BONT/A4的效力降低了1,000倍。 我们的协作努力在这方面是独一无二的，因为我们能够结合有关的详细机械研究 基于细胞和体内研究的子域在Holotoxin水平上进行研究。 Barbieri实验室将使用 结构建模，以指导BONT功能领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/佩莱特实验室 是否会利用这些数据在其本机宿主中创建靶向的Holotoxin构建体并进行详细说明 在包括人类细胞模型在内的各种细胞模型中进行了研究。最后，基于这些数据 研究，将在小鼠中研究某些Holotoxin构建体，以确定病理学和药理学 结构改变的后果。使用这种方法，我们能够研究大量 氨基酸取代并选择更努力和成本密集的构造的特定改动 重组全毒素。通过在进行体内研究之前利用多个细胞模型，我们能够 减少所需动物的数量，并使用特定于人类的模型。最后，结合 体外，基于细胞和体内研究将为观察到的机制提供新的见解 这些毒素的病理和药理特性。