天然药物功能蛋白质学科组（赖仞）知识库

生存适应是自然界中每一个生物面临的最基本问题。要在残酷自然界中获得生存的权利，都必须参与生存资源的残酷竞争。生存的压力推动进化，使得不同物种形成了复杂而多样的生物性状参与到其捕食、防御及竞争等生存适应的具体过程中。毒液系统就是这些生物性状之一。动物界中有大量的产毒动物，从简单的刺胞动物海葵，软体动物芋螺，环节动物水蛭，节肢动物蜘蛛、蝎子、蜈蚣、胡蜂，一直到脊椎动物的产毒蛙类、蛇类，甚至哺乳动物吸血蝙蝠、鸭嘴兽和灵长类动物蜂猴均会使用毒液进行生存适应。毒液系统优化了他们的生存适应过程，使得物种的生存竞争不再完全依靠体重、速度以及智力等因素。种间防御以及种内竞争是生存适应中的重要组成部分，但关于毒液如何参与其中还有很多疑惑。例如：1）毒液如何介导高效而持续的种间防御；2）产毒动物是否耐受自身的毒液；3）毒液如何调控适度的种内竞争；4）毒液介导的捕食、防御及竞争等生存适应过程有何机制异同等等。为了初步解决这些问题，我们开展了两部分研究工作：第一部分，我们选择金环胡蜂(Vespa mandarinia)作为种间防御的研究物种，探索了其毒液中参与种间防御的多肽毒素及其作用机制；第二部分，我们选择少棘蜈蚣(Scolopendra subspinipes mutilans)作为种内竞争的研究物种，探索了其毒液中参与种内竞争的多肽毒素及其作用机制。我们希望这两部分研究能部分揭示金环胡蜂进行种间防御以及少棘蜈蚣进行种内竞争的分子机制，同时利用这些分子机制作为研究模式，可以为更多产毒动物的相关研究提供思路。胡蜂毒液具有典型的防御特性，是研究种间防御的良好模型。在第一部分研究工作中，我们探讨了胡蜂毒素在其种间防御中发挥功能的物质基础和作用机制。首先，我们从金环胡蜂毒素中鉴定得到两种组织损伤多肽，分别为肥大细胞脱颗粒肽Mastoparan_M和趋化肽VESCP_M。这两种多肽能通过组织损伤介导疼痛、水肿以及持续的炎症反应，这是一种更加综合且持续的种间防御过程。进一步的作用机制探索发现，这两种多肽的组织损伤效果来源于他们能在不同类型细胞膜上形成孔道而对细胞膜的破坏。接着我们发现具有实际意义的一个方面，即金环胡蜂毒液中这两种多肽含量极其丰富，在胡蜂的真实叮咬中释放的浓度均达到了发挥显著功能的浓度条件。这提示Mastoparan_M和VESCP_M两种多肽毒素可能真实的参与了金环胡蜂毒液导致疼痛、水肿以及炎症反应的综合防御过程。同时，我们使用序列比对发现类似的两种多肽在胡蜂属中具有高度的一级序列保守性。利用Rosetta对他们进行进行结构模拟发现，它们具有高度类似的三维结构，这提示我们在金环胡蜂中发现的这两种多肽毒素在大胡蜂属的其他胡蜂种中可能具有类似的功能。这部分研究工作的科学意义如下：1）发现了胡蜂毒素中导致组织损伤的多肽毒素及其作用机制；2）这些多肽在胡蜂属中具有高度的保守性，类似的防御机制可能在胡蜂属中具有普适性；3）揭示了组织损伤这种长时效性的防御策略，并可以作为模式去研究其他产毒动物可能存在的组织损伤防御策略。毒液参与种内竞争的机制研究需要两个方面的基础：一是产毒动物毒液系统相对清楚的研究；二是产毒动物自身毒素作用受体（如离子通道类受体）的了解。本实验室的前期工作中已经对少棘蜈蚣的毒液种类、结构和功能进行了较为全面的研究，并已经揭示了一些少棘蜈蚣毒液参与捕食和防御的分子机制，少棘蜈蚣种内竞争机制的研究可以和其捕食和防御进行机制异同比较。同时少棘蜈蚣作为一种典型的毒液捕食性动物，对毒液存在高使用频率，其毒液系统极有可能参与了种内竞争的过程。因此我们认为少棘蜈蚣成是研究种内竞争机制的良好模型。在第二部分工作中，我们深入探讨了少棘蜈蚣毒液在其种内竞争中发挥功能的物质基础和作用机制。为了得到少棘蜈蚣自身受体信息，我们首先利用Nanopore三代测序的方法解析了少棘蜈蚣（S.s.mutilans）全基因组（第一个有毒蜈蚣基因组），并从基因组中识别出了可能参与种内竞争的离子通道类受体。通过毒素与受体的相互作用研究我们鉴定得到三种参与少棘蜈蚣种内竞争的多肽毒素（AkTx、EvTx和SsTx），这些多肽毒素通过靶向少棘蜈蚣Shal钾离子通道发挥功能。利用前期结构和功能研究比较清楚的SsTx为代表进行研究发现，SsTx通过结合Shal通道的孔区E353位氨基酸残基发挥离子通道孔道堵塞的功能而抑制通道电流产生。为了阐明少棘蜈蚣毒液靶向自身Shal通道的生理意义，我们利用SsTx对Shal通道蛋白主要分布的少棘蜈蚣神经和心血管系统进行了研究。结果表明，SsTx能提高少棘蜈蚣神经系统神经元的兴奋性，并使得少棘蜈蚣的心脏管短暂收缩，少棘蜈蚣的SsTx注射试验表明SsTx会导致其短暂运动能力下降，这和少棘蜈蚣粗毒注入其自身体内导致的反应类似，提示SsTx等毒素对Shal钾通道的抑制而介导的一系列反应可能是少棘蜈蚣进行种内竞争的分子机制。有趣的是，SsTx能够作用于哺乳动物的Shaker同源通道Kv1.1，而不能作用于少棘蜈蚣自身的Shaker通道，试验表明Shaker通道的R400位氨基酸残基介导通道对自身毒素的耐受。同时，我们的试验也未发现能作用于少棘蜈蚣自身钠通道、钙通道的多肽毒素，我们推测其耐受可能具有和Shaker类似的机制。因此，我们认为少棘蜈蚣毒素不能作用于其自身重要离子通道的现象可能是少棘蜈蚣控制其适度竞争的分子机制。这部分研究工作的科学意义有：1）揭示了少棘蜈蚣利用Shal钾离子通道进行种内竞争，同时利用Shaker等重要通道的毒素耐受控制适度种内竞争的分子机制；2）试验证明少棘蜈蚣利用毒素进行的捕食、防御及竞争具有不同的分子机制和生理效应。3）首次解析了产毒蜈蚣的基因组，为少棘蜈蚣的进一步研究提供了坚实的基础。4）揭示了产毒动物（少棘蜈蚣）使用毒素进行种内竞争的一个实例，少棘蜈蚣的这种种内竞争机制可以作为模式去研究更多的产毒动物种内竞争机制；本文对两个具有代表性的产毒动物金环胡蜂和少棘蜈蚣进行了种间防御和种内竞争的机制研究。我们在金环胡蜂中发现了利用毒素进行组织损伤这种高效而持续的种间防御策略，同时，我们在少棘蜈蚣中发现了一套利用毒素进行的适度种内竞争机制。这些结果不仅部分揭示金环胡蜂利用毒液进行种间防御以及少棘蜈蚣利用毒液进行种内竞争的分子机制，同时利用金环胡蜂和少棘蜈蚣揭示的分子机制作为研究模式，可以为更多产毒动物的相关研究提供研究思路。 

Struggle for survival is the most basic challenge faced by every living organism in nature. To survive in this cruel nature, animals must brutally compete for limited resources. The pressure for survival drives evolution, and different species form complex and diverse biological traits that are involved in the specific process of survival adaptation such as predation, defense, and competition. The venom system is one of these biological traits. A large number of venomous animals in the animal kingdom use toxins for survival , from simple sea anemone from Phylum cnidaria; conus from Mollusca; leech from Annelida; spiders, scorpions, and wasp from Arthropoda; to vertebrates such as venomous frogs, snakes and even mammals such as vampire bats, platypuses and primates such as the slow loris. Venomous animals use the venom system to optimize their process of survival adaptation so that the relationship between the competition between species is no longer completely determined by factors such as weight, speed, and intelligence.Interspecific defense and intraspecific competition are important components of survival adaptation. However, there are still many unclear questions about the involvement of toxins in the interspecific defense and intraspecific competition of venomous animals. For example, 1) How do venomous animals use venom for efficient and continuous interspecific defense; 2) Whether venomous animals can resist their own venom; 3) How toxins are used to carry out moderate intraspecific competition; 4) What are the similarities and differences in venom-mediated survival adaptation processes such as predation, defense and competition. In order to solve these problems, we carried out a two-part research: In the first part, we chose the Vespa mandarinia as the research species for interspecific defense, and explored the polypeptide toxins involved in interspecific defense in its venom and their mechanism of action. In the second part of the work, we chose the Scolopendra subspinipes mutilans as the research species for intraspecific competition, and explored the polypeptide toxins involved in intraspecific competition in its venom and their mechanism of action. We hope that these two studies can reveal the molecular mechanisms of interspecific defense and intraspecies competition by wasps and centipedes. At the same time, using these molecular mechanisms as research models can provide ideas for more studies on toxic animals.The wasp venom system has typical defensive properties and is a good model for studying interspecific defenses. In the first part of the research work, we explored the material basis and mechanism of action of wasp venom in interspecific defenses. First, we identified two tissue damaging polypeptides from the V.mandarinia toxins, the mast cell degranulation peptide Mastoparan_M and the chemotactic peptide VESCP_M. These two polypeptides mediate pain, edema, and persistent inflammatory responses through tissue damage, which is a more integrated and sustained process of interspecific defense. Further exploration of the mechanism of action revealed that the tissue damage effect of these two polypeptides was due to their ability to destroy the cell membranes through pore-formation on different types of cell membranes. Then we found that the two polypeptides in the venom of the wasp were extremely abundant, and the release concentration of the two polypeptides in the actual bite of the wasp reached the significant function concentration. This suggests that the two peptide toxins are actually involved in the defensive activity process that causes pain and other effects in the bite of the wasp. Meanwhile, we used sequence alignment to find that the two similar peptides have a high primary sequence conservation in the genus Vespa. Using Rosetta for advanced structural simulations, they have similar advanced structural features. This suggests that the two peptide toxins we found in the V.mandarinia may have similar functions in other wasp species of the genus Vespa. The scientific significance of this part of the research work is as follows: 1) The polypeptide toxin causing tissue damage in V.mandarinia and its mechanism were established; 2) These polypeptides are highly conserved in the the genus Vespa, and similar defense strategies may be conserved in the genus Vespa; 3) Reveals the long-term defense strategy of tissue damage, and similar defensive strategies may exist in other venomous animals.Research on the mechanism of venom participation in intraspecific competition requires two research foundations: one is the relatively clear study of the venom system of venomous animals; Second, the understanding of toxin receptors (such as ion channel receptors) in venomous animals. Our laboratory’s preceding work has laid a sufficient research basis for the structural function of S.s.mutilans venom components, and some molecular mechanisms of S.s.mutilans venom participation in predation and defense have been revealed. Therefore, intraspecific competition studies using S.s.mutilans can also be compared to their predation and defense processes to understand the similarities and differences between predation, defense, and competition in venomous animals. As typical predatory venomous animal, S.s.mutilans has a high frequency for using the venom system. For these reasons, we believe that S.s.mutilans is a good model for studying intraspecific competition mechanisms. In the second part of the work, we explored in-depth the material basis and mechanism of action of the S.s.mutilans venom in its intraspecific competition. In order to obtain the receptor information of the S.s.mutilans itself, we first explored the receptor information of the S.s.mutilans itself. Using the Nanopore three-generation sequencing method, we analyzed the whole genome of the S.s.mutilans. And then we identified related ion channel involved in intraspecific competition from the whole genome of S.s.mutilans. Through the interaction between toxin and receptor, three kinds of polypeptide toxins involved in intraspecific competition were identified (AkTx, EvTx, and SsTx) .and their target is S.s.mutilans Shal potassium channels. SsTx, whose structure and function were clearly studied in previous works, was selected as representative for a further mechanism study. It was found that the SsTx can bind to the E353 amino acid in the pore region of the Shal potassium channel of the S.s.mutilans causing blockage of the channel pore region and suppressing the current of the channel. To elucidate the physiological significance of centipede venom targeting its own Shal channel, We used SsTx to study the nervous and cardiovascular system of S.s.mutilans, which Shal channel is mainly distributed. The results show that SsTx can cause the excitation of the DUM neuron and the violent contraction of the cardiac tube. SsTx injection causes Short-term decline in athletic ability, and the reaction is the same as the crude venom injection in S.s.mutilans. Therefore, we believe that a series of reactions caused by the inhibition of the Shal potassium channel by SsTx and other toxins is the molecular mechanism of S.s.mutilans intraspecific competition. The interesting finding is that, SsTx can act on the Shaker homologous channel Kv1.1 in mice, but not on the Shaker channel of S.s.mutilans itself. Experiments have shown that the amino acid R400 of the Shaker channel mediates the tolerance of the channel to its own toxin. Meanwhile, we have not found any polypeptide toxin that can act on important receptors of S.s.mutilans ion channel, such as sodium channels, calcium channels. The phenomenon that the S.s.mutilans toxin cannot act on its own important ion channel may be a molecular mechanism for controlling the moderate intraspecific competition of S.s.mutilans. The scientific significance of this part of the research work is as follows: 1) It reveals that S.s.mutilans use the Shal potassium channel for intraspecific competition, while using toxin tolerance by important channels such as Shaker for controlling moderate intraspecific competition; 2) The predation, defense and competition of S.s.mutilans are survival adaptation strategies with different working molecular mechanisms and physiological effects. 3) The first analysis of the whole genome of the toxin-producing centipede (S.s.mutilans) provides a solid foundation for further research on S.s.mutilans. 4) Revealing an example of a toxin-producing animal using toxins for intraspecific competition, These intraspecific competition mechanisms of S.s.mutilans can be used as a model to study intraspecific competition mechanisms of other venomous animals..In this paper, the mechanism of interspecific defense and intraspecific competition were studied using two representative venomous animals, the V.mandarinia and S.s.mutilans. We found an efficient and persistent interspecific defense strategy in V.mandarinia based on tissue damage, and we also found a moderate intraspecific competition mechanism S.s.mutilans. Our study partially revealed the mechanisms of interspecific defense in V.mandarinia and intraspecific competition in S.s.mutilans, At the same time, these molecular mechanisms can be used as research models to provide research ideas for more venomous animals.