Parotoid gland secretion of bufonid toads is a rich source of toxic molecules that are used against predators, parasites and pathogens. Bufadienolides and biogenic amines are the principal compounds responsible for toxicity of parotoid secretion. Many toxicological and pharmacological analyses of parotoid secretions have been performed, but little is known about the processes related to poison production and secretion. Therefore, our aim was to investigate protein content in parotoids of the common toad, Bufo bufo, to understand the processes that regulate synthesis and excretion of toxins as well as functioning of parotoid macroglands. Applying a proteomic approach we identified 162 proteins in the extract from toad’s parotoids that were classified into 11 categories of biological functions. One-third (34.6%) of the identified molecules, including acyl-CoA-binding protein, actin, catalase, calmodulin, and enolases, were involved in cell metabolism. We found many proteins related to cell division and cell cycle regulation (12.0%; e.g. histone and tubulin), cell structure maintenance (8.4%; e.g. thymosin beta-4, tubulin), intra- and extracellular transport (8.4%), cell aging and apoptosis (7.3%; e.g. catalase and pyruvate kinase) as well as immune (7.0%; e.g. interleukin-24 and UV excision repair protein) and stress (6.3%; including heat shock proteins, peroxiredoxin-6 and superoxide dismutase) response. We also identified two proteins, phosphomevalonate kinase and isopentenyl-diphosphate delta-isomerase 1, that are involved in synthesis of cholesterol which is a precursor for bufadienolides biosynthesis. STRING protein-protein interaction network predicted for identified proteins showed that most proteins are related to metabolic processes, particularly glycolysis, stress response and DNA repair and replication. The results of GO enrichment and KEGG analyses are also consistent with these findings. This finding indicates that cholesterol may be synthesized in parotoids, and not only in the liver from which is then transferred through the bloodstream to the parotoid macroglands. Presence of proteins that regulate cell cycle, cell division, aging and apoptosis may indicate a high epithelial cell turnover in parotoids. Proteins protecting skin cells from DNA damage may help to minimize the harmful effects of UV radiation. Thus, our work extends our knowledge with new and important functions of parotoids, major glands involved in the bufonid chemical defence.

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来自蟾蜍腮腺的蛋白质：它们在腺体功能和化学防御中发挥作用吗？

蟾蜍的腮腺分泌物富含有毒分子，可用于对抗捕食者、寄生虫和病原体。蟾蜍二烯内酯和生物胺是造成腮腺分泌物毒性的主要化合物。已经对腮腺分泌物进行了许多毒理学和药理学分析，但对毒物产生和分泌的相关过程知之甚少。因此，我们的目标是研究普通蟾蜍 Bufo bufo 的腮腺中的蛋白质含量，以了解调节毒素合成和排泄的过程以及腮腺大腺体的功能。应用蛋白质组学方法，我们在蟾蜍腮腺提取物中鉴定了 162 种蛋白质，这些蛋白质被分为 11 类生物学功能。三分之一 (34.6%) 的已识别分子，包括酰基辅酶 A 结合蛋白、肌动蛋白、过氧化氢酶、钙调蛋白和烯醇化酶，参与细胞代谢。我们发现了许多与细胞分裂和细胞周期调节（12.0%；例如组蛋白和微管蛋白）、细胞结构维持（8.4%；例如胸腺素 beta-4、微管蛋白）、细胞内和细胞外运输 (8.4%)、细胞衰老相关的蛋白质和细胞凋亡（7.3%；例如过氧化氢酶和丙酮酸激酶）以及免疫（7.0%；例如白细胞介素 24 和紫外线切除修复蛋白）和应激反应（6.3%；包括热休克蛋白、过氧化物酶 6 和超氧化物歧化酶）反应。我们还鉴定了两种蛋白质，即磷酸甲羟戊酸激酶和异戊烯二磷酸 δ-异构酶 1，它们参与胆固醇的合成，而胆固醇是蟾蜍二烯内酯生物合成的前体。为已识别蛋白质预测的 STRING 蛋白质-蛋白质相互作用网络表明，大多数蛋白质与代谢过程有关，尤其是糖酵解、应激反应以及 DNA 修复和复制。GO 富集和 KEGG 分析的结果也与这些发现一致。这一发现表明，胆固醇可能在腮腺中合成，而不仅仅是在肝脏中合成，然后通过血液从肝脏转移到腮腺大腺体。调节细胞周期、细胞分裂、衰老和细胞凋亡的蛋白质的存在可能表明腮腺上皮细胞更新率高。保护皮肤细胞免受 DNA 损伤的蛋白质可能有助于最大限度地减少紫外线辐射的有害影响。因此，我们的工作通过腮腺的新的重要功能扩展了我们的知识，