Receptors for clostridial neurotoxins

描述（由申请人提供）： 肉毒杆菌中毒首次被描述是在大约200年前。这种疾病是由肉毒杆菌神经毒素（BoNT）引起的，肉毒杆菌神经毒素是由肉毒梭菌的产毒菌株产生的七种相关毒素（A-G）。这些毒素是已知的最有毒的物质。它们通过进入神经元并裂解介导神经递质胞吐作用的蛋白质来发挥作用，从而导致瘫痪和死亡。BoNT被认为通过双受体机制结合到神经元表面，其中受体是由神经节苷脂和蛋白质组成的复合物。确定毒素受体和介导进入的途径，可能提供一种阻断这些毒素作用的方法。最近的证据表明，突触结合蛋白家族的成员作为BoNT/B受体的蛋白质组分。突触结合蛋白I和II表现出不同的结合和介导BoNT/B进入的能力。本提案的第一个目的是探索这些差异，目的是将突触结合蛋白的结构与其作为毒素受体的功能联系起来。其他BoNT进入细胞的确切机制尚不清楚。因此，在第二个目标中，我们将确定其他BoNT进入靶细胞的途径。我们的初步数据表明，不同的毒素通过不同的途径进入细胞。在第三个目标中，我们将通过鉴定介导BoNT/E内化的受体来进一步探索BoNT宿主识别和进入的方式。我们专注于这种血清型，因为我们已经证明了使用BoNT/E亲和基质的可行性;长期目标是鉴定其他BoNT的受体。最后，在第四个目标中，我们将开发基于FRET的传感器，其可用于在体外和活细胞中真实的监测BoNT活性。这些传感器将使高通量筛选成为可能，以识别能够拮抗毒素作用的小分子。 BoNT目前在临床上用于治疗各种肌肉张力障碍，并且还作为潜在的生物武器大规模生产。本文提出的研究将为BoNTs的分子作用机制提供新的见解，并可能提供一种新的方法来预防这些物质的中毒。

DESCRIPTION (provided by applicant): Botulism was first described almost 200 years ago. This disease is caused by the botulinum neurotoxins (BoNT), which are seven related toxins (A-G) produced by toxigenic strains of Clostridium botulinum. These toxins are the most poisonous substances known. They act by entering neurons and cleaving proteins that mediate the exocytosis of neurotransmitters, resulting in paralysis and death. BoNTs are thought to bind to the surface of neurons via a double-receptor mechanism in which the receptor is a complex composed of gangliosides and protein(s). Identification of the toxin receptors, and the pathways that mediate entry, might provide a means to block the action of these toxins. Recent evidence indicates that members of the synaptotagmin family serve as the proteins components of the BoNT/B receptor. Synaptotagmin I and II exhibit distinct abilities to bind and mediate entry of BoNT/B. The first Aim of this proposal explores these differences with the goal of relating the structure of synaptotagmin with its ability to function as a toxin receptor. The precise mechanism by which the other BoNTs gain entry into cells is not known. Therefore, in the second Aim we will identify the pathways through which other BoNTs enter target cells. Our preliminary data indicate that different toxins enter cells via distinct pathways. In the third Aim we will further explore the means of BoNT host recognition and entry by identifying the receptor(s) that mediate internalization of BoNT/E. We have focused on this serotype because we have demonstrated feasibility using a BoNT/E affinity matrix; the long term goal is to identify receptors for additional BoNTs. Finally, in the fourth Aim we will develop FRET-based sensors that can be used to monitor BoNT activity in vitro and in living cells in real time. These sensors will make it possible to carryout high throughput screening to identify small molecules that can antagonize the action of the toxins. BoNTs are currently being used clinically to treat a variety of muscle dystonias and are also produced on large scales as potential biological weapons. The studies proposed here will provide new insights into the molecular mechanism of action of the BoNTs, and may provide a novel means to prevent poisoning by these substances.