| 其他摘要 | 摘要 少棘蜈蚣Scolopendra subspinipes mutilans隶属于节肢动物门，多足类亚门，唇足纲，蜈蚣目，蜈蚣科，蜈蚣属。其第一对足特化为毒螯，内有囊泡状毒腺。捕食或防御时，毒液由螯枝尖端导管流出注入猎物或天敌体内。其毒素能引起昆虫的死亡与哺乳动物的剧烈疼痛和局部麻痹。目前对蜈蚣毒素的研究较为粗略，已知的信息局限在粗毒的生理功能和极少数蜈蚣物种的毒液蛋白质质谱数据上。由于蜈蚣被称为动物界的活化石，已经在自然界中生存了4亿多年，作为一类被忽视的有毒动物，其毒素分子的功能和结构进化可能有特殊之处，或许能为成为先导活性分子和分子探针发掘的巨大宝库。因此，我们采用少棘蜈蚣作为研究材料，研究其毒素分子的多样性、结构及其功能的特点，另外还利用毒素作为探针，研究毒素结合靶点的生理特性。第一章中，我们利用转录组学、蛋白质组学结合功能研究揭示了少棘蜈蚣多肽毒素的结构和功能的多样性。其中，我们发现了1个电压门控钠通道抑制剂家族，3个电压门控钾通道抑制剂家族，1个电压门控钙通道抑制剂家族和1个电压门控钙通道激动剂家族，以及一系列目前还不能确定功能的多肽毒素家族。其中一些毒素家族还具有昆虫致死活性。通过BLAST的分析，这些多肽毒素家族的成员与已知的多肽毒素基本上没有序列同源性，而且具有新型的二硫键构架方式。这些新型多肽毒素的发现具有如下的科学意义：1)证实了少棘蜈蚣毒液中存在着大量的新型小分子多肽毒素；2)这些毒素的存在极有可能与少棘蜈蚣的捕食和防御息息相关；3)这些多肽毒素家族的发现可能对先导分子和分子探针发掘提供新的素材。第二章，我们从结构和功能的角度出发，介绍1个世界上首次识别的、专门作用于Nav1.7并有可能成为疼痛治疗药物的蜈蚣多肽毒素。在本章中，我们从少棘蜈蚣毒液中发掘了一种新的蜈蚣电压门控钠通道抑制剂μ-SLPTX-Ssm6a（以下简称Ssm6a），利用膜片钳技术证明了Ssm6a是到目前为止发现的专一性最强的Nav1.7抑制剂，其在Nav1.7上的IC50为25nM，选择强度是其他电压门控钠通道亚型的至少150倍。Nav1.7在哺乳动物疼痛传递中扮演非常重要的传递者的角色，Ssm6a这些特点使得它在小鼠动物模型中表现出了超越吗啡镇痛能力的功能。Ssm6a是一个化学稳定性极强的多肽毒素，在体外试验中，Ssm6a能在血浆中保持至少一周以上的结构稳定性，对尿素和高温也有非常强的耐受性。Ssm6a的高稳定性使得其在小鼠体内的镇痛活性能保持4小时以上。更加难得的是，Ssm6a不会造成血压、心率和运动能力方面的副作用。因此，Ssm6a是一个极具镇痛药用潜力的蜈蚣多肽毒素。 第三章中，我们利用少棘蜈蚣毒素揭示了疼痛发生的分子机制辣椒素受体TRPV1通道的门控特性。我们从蜈蚣毒液中纯化鉴定到一个能引发哺乳动物疼痛反应的多肽毒素，并将其命名为RhTx。通过基因敲除和膜片钳的验证，我们发现RhTx通过专一激动TRPV1产生急性疼痛。核磁共振分析发现，RhTx拥有一个C末端结构稳定而N末端较为自由的三级结构。在此结构中，几个带负电荷的氨基酸形成了一个负电荷分子表面。毒素点突变的结果显示，正是这个负电荷分子表面是RhTx的活性结构域，而这些带负电的氨基酸则是这个毒素的关键性功能残基。我们进一步研究了关于RhTx激动TRPV1的机制，利用远红外温控技术结合膜片钳首次发现了毒素分子通过影响TRPV1的热敏感性激活通道的门控机制。随后，我们为了研究这种门控机制出现的原因进行了对TRPV1的嵌合和点突变，进而得知了RhTx结合在TRPV1的外孔区的turret结构上。我们又通过Rosetta运算RhTx和TRPV1的空间结合模型，我们计算的结果与这些结合位点信息相互佐证了这两个分子相互作用的方式。有文献报道，turret的突变会导致TRPV1对热刺激的敏感程度下降，因此RhTx结合TRPV1的turret很可能引起TRPV1热敏感性的改变。我们的工作揭示了RhTx这个引发疼痛的毒素分子，通过结合TRPV1的turret结构域，提高了TRPV1对温度的敏感性，使得TRPV1能在更低的温度下被打开。TRPV1被RhTx打开导致可兴奋性神经元的兴奋，传递痛觉信号。这部分工作的主要意义有：1)发现首个引发疼痛的蜈蚣毒素，对蜈蚣生物化学防御策略提供了首个分子机制水平上的证据；2)发现了首个2对二硫键并具有TRPV1激动剂功能的小分子多肽；3)提供了多肽毒素可以利用增加TRPV1热敏感性的方式激活TRPV1的证据；4)利用分子相互作用和结构的的证据支持了turret是TRPV1热激活门控机制的关键结构；5)通过RhTx激活TRPV1的门控方式，提供了分子利用TRPV1热激活通路与疼痛发生发展相互关联的直接证据。本文由蜈蚣毒素的发掘展开描述，对蜈蚣毒素结构和功能的多样性进行深入的研究。又利用毒素分子的高生物活性和专一性，发现了一系列具有潜在药用潜力的蜈蚣多肽毒素。最后又以蜈蚣毒素作为研究离子通道的工具，揭示了离子通道门控的特性。关键词：少棘蜈蚣，多肽毒素，Ssm6a，RhTx，离子通道，Nav1.7，疼痛，TRPV1，热激活 ; AbstractCentipede Scolopendra subspinipes mutilans L. Koch, also named Chinese Red-headed centipede, belongs to the class Chilopoda of the subphylum Myriapoda. The first pair of walking legs of them evolved into a pair of forceps. Envenomation of centipedes cause intense pain and paralysis of insects and even mammals. Despite their abundance and painful encounters with humans, the composition of centipede venom is still unclear. The current knowledge of centipede peptidic toxins only includes the activity of crude venom and mass spectrometry data. According to 400 million years evolution, centipedes are called “living fossils” and their peptidic toxins may have unique structures and functional peculiarities. Thus, the peptidic toxins from centipede venoms may construct a useful pool for the development of lead compounds and molecular probes. In these studies, we firstly reveled the diversity of structure and activities of peptidic toxins from Chinese Red-headed centipede. Furthermore, a list of peptidic toxins with potential therapeutic effect was identified by their potent and specific activities. Thirdly, the knowledge of TRPV1gating mechanism was improved by revealing the interaction between centipede toxin and ion channel.In Chapter 1, our work demonstrated the structural and functional diversity of centipede peptidic toxins by transcriptomic and proteomic analysis. Particularly, a voltage-gated sodium channels (VGSCs) inhibitor family, three voltage-gated potassium channels (VGPCs) inhibitor families, a voltage-gated calcium channels (VGCCs) inhibitor family and a VGCCs inhibitor family were identified. These peptidic toxins from centipede venom share no primary sequence identity with other known peptide by BLAST analysis. Furthermore, these peptidic toxins have novel scaffold of disulfide bridges. Some of these toxin families have insecticidal bioactivity. In addition, the bio-function of several peptidic toxin families are still unclear. The scientific importance of the discovery of these novel peptidic peptides: 1) Abundant novel peptidic toxins are packed in the venom of centipede. 2) These toxins containing substances with a wide variety of pharmacological activities may be used to interfere with the physiology of the prey or predator. 3) The discovery of novel centipede toxins may provide a new source of novel bioactive molecules with a possibility of developing potential lead compounds and biological probes.We identified a centipede toxin with potential therapeutic effect in Chapter 2. According to the potency and selectivity of peptidic toxins, this centipede toxins may be useful therapeutic. We isolated and identified a novel VGSCs inhibitor, μ-SLPTX-Ssm6a (Ssm6a), from centipede venom. By using electrophysiological analysis, Ssm6a is the most subtype-selective inhibitor against Nav1.7 reported to date. Ssm6a potently inhibits Nav1.7 with an IC50 of ～25 nM. Ssm6a has more than 150-fold selectivity for Nav1.7 over all other human Nav subtypes. Nav1.7 acts a crucial role in mammal pain signaling pathway and the inhibition of Nav1.7 by Ssm6a causes an excellent analgesic effect exceeding morphine in vivo. In addition, Ssm6a is a stable peptidic toxins in blood and urea in vitro. These characters provide Ssm6a a high persistence in animal models, which lasts the analgesic effect for more than 4 hours. Our findings suggest that Ssm6a is a powerful potential therapeutic with potent analgesic effect. In Chapter 3, we will describe our study of probing ion channel by using centipede toxin as tool. We have isolated a novel peptide toxin from the Chinese red-headed centipede. The toxin, named here RhTx, elicited strong pain behaviors when injected in mice, like the raw venom. The elicited behaviors were distinct from those mediated by inflammation but exhibited close resemblance to those associated with capsaicin injection. When tested in knock-out mice lacking the capsaicin receptor TRPV1 gene, both RhTx and capsaicin were ineffective in producing pain behaviors. Using HEK293 cells overexpressing TRPV1, we confirmed that the channel is indeed an RhTx target. Unlike most animal toxins that act as an inhibitor, RhTx is an extremely potent activator. NMR spectroscopy analysis revealed that RhTx is a compact protein with two pairs of di-sulfide bonds and a flexible N-terminus. All charged residues are found in the C-terminal half of the peptide sequence. In 3D structure these residues are clearly visible from one side of the molecule. To identify the channel-binding surface of RhTx, we synthesized mutant toxins that contained an alanine at each of the 23 non-cysteine positions. Intriguingly, four out of the five identified residues are charged while the fifth one is polar. Chimeras containing the pore region of TRPV3 exhibited disrupted toxin sensitivity, while they remained capsaicin-sensitive because the capsaicin-binding site was intact. Specifically, replacing the pore turret or pore helix had a major impact on toxin sensitivity, while replacing the ion selectivity filter and its posterior loop did not have an obvious effect. These results suggest that RhTx binds to the charge-rich outer pore region where it may directly interact with the pore turret and pore helix, two adjacent structural elements known to be critical for activation gating of TRPV1. Further testing of point mutations identified D602 in the turret, Y632 and T634 in the pore helix, and L461 in the S1-S2 linker to be critical for RhTx sensitivity. Rosetta-based molecular docking to the turret-truncated TRPV1 cryo-EM structure and a model channel with intact core domains agreed with results from functional tests, indicating direct interactions of RhTx with the pore helix and pore turret through potential electrostatic and hydrophobic interactions. At 100 nM (5 times below EC50), RhTx already lowered the activation threshold temperature by 6 °C, making the body temperatures of mouse and human at an activating level. We further found that lowering the experimental temperature could prohibit RhTx-induced activation. This is noteworthy because the same operation did not prevent capsaicin activation, which works through a separate pathway. It showed that the action of RhTx requires a transition of the heat pathway. RhTx is unique in that it is a quick and potent activator of a mammalian nociceptor by apparently targeting the heat activation machinery. The resultant burning pain has clear defensive value. RhTx adopts a different channel-binding strategy from the structurally distinct DkTx, a 75-amino acid large peptide toxin.In this thesis, we introduce our work from three aspects. First of all, the discovery of multiple peptidic toxins provide adequate material for further studies. Secondly, some of toxins with therapeutic effects from centipede venom were well studied. Thirdly, peptidic toxins can also be used as powerful probes for investigating the protein-protein interaction and gating mechanism of ion channels. |
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