Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario K1S 5B6, Canada.
Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, College of Life Sciences, Hebei Normal University, Shijiazhuang, Hebei 050024, China.
Comp Biochem Physiol Part D Genomics Proteomics. 2019 Jun;30:1-13. doi: 10.1016/j.cbd.2019.01.009. Epub 2019 Jan 25.
Vertebrate freeze tolerance requires multiple adaptations underpinned by specialized biochemistry. Freezing of extracellular water leads to intracellular dehydration as pure water is incorporated into growing ice crystals and also results in the cessation of blood supply to tissues, creating an anoxic cellular environment. Hence, the freeze tolerant wood frog, Rana sylvatica, must endure both dehydration and anoxia stresses in addition to freezing. The metabolic responses to freezing, dehydration and anoxia involve both protein/enzyme adaptations and the production of metabolites with metabolic or osmotic functions, particularly glucose and urea. The present study uses a phosphoproteome analysis to examine the differential phosphorylation of metabolic enzymes involved in the production of these two metabolites in liver in response to freezing, anoxia, or dehydration exposures. Our results show stress-specific responses in the abundance of phosphopeptides retrieved from nine glycolytic enzymes and three urea cycle enzymes in liver of wood frogs exposed to 24 h freezing, 24 h anoxia, or dehydration to 40% of total body water loss, as compared with 5 °C acclimated controls. Data show changes in the abundance of phosphopeptides belonging to glycogen phosphorylase (GP) and phosphofructokinase 2 (PFK2) that were consistent with differential phosphorylation control of glycogenolysis and a metabolic block at PFK1 that can facilitate glucose synthesis as the cryoprotectant during freezing. Anoxia-exposed animals showed similar changes in GP phosphorylation but no changes to PFK2; changes that would facilitate mobilization of glycogen as a fermentative fuel for anaerobic glycolysis. Urea is commonly produced as a compatible osmolyte in response to amphibian dehydration. Selected urea cycle enzymes showed small changes in phosphopeptide abundance in response to dehydration, but during freezing differential phosphorylation occurred that may facilitate this ATP expensive process when energy resources are sparse. These results add to the growing body of literature demonstrating the importance and efficiency of reversible protein phosphorylation as a regulatory mechanism allowing animals to rapidly respond to environmental stress.
脊椎动物的抗冻性需要多种适应，这些适应由专门的生物化学支撑。细胞外水的冻结导致细胞内脱水，因为纯水被纳入不断生长的冰晶中，同时也导致组织的血液供应停止，从而形成缺氧的细胞环境。因此，除了冻结之外，抗冻的林蛙（Rana sylvatica）还必须忍受脱水和缺氧的压力。对冻结、脱水和缺氧的代谢反应既涉及蛋白质/酶的适应，也涉及具有代谢或渗透功能的代谢物的产生，特别是葡萄糖和尿素。本研究使用磷酸化蛋白质组分析来检查在暴露于 24 小时冻结、24 小时缺氧或脱水至总失水量的 40%的林蛙肝脏中，与 5°C 适应对照相比，这两种代谢物产生中涉及的代谢酶的差异磷酸化。我们的研究结果显示，在暴露于 24 小时冻结、24 小时缺氧或脱水至总失水量的 40%的林蛙肝脏中，与 5°C 适应对照相比，9 种糖酵解酶和 3 种尿素循环酶中回收的磷酸肽的丰度存在应激特异性反应。数据显示，糖原磷酸化酶（GP）和磷酸果糖激酶 2（PFK2）的磷酸肽丰度发生变化，这与糖原分解的差异磷酸化控制一致，并且在 PFK1 处存在代谢阻滞，可促进作为冻结时的冷冻保护剂的葡萄糖合成。缺氧暴露的动物表现出 GP 磷酸化的相似变化，但 PFK2 没有变化；这些变化将有利于糖原作为无氧糖酵解的发酵燃料的动员。在两栖动物脱水时，尿素通常作为一种相容的渗透物产生。选定的尿素循环酶的磷酸肽丰度在脱水时发生微小变化，但在冻结时发生差异磷酸化，这可能有利于当能量资源稀缺时这个昂贵的 ATP 过程。这些结果增加了越来越多的文献，证明可逆蛋白磷酸化作为一种调节机制的重要性和效率，使动物能够快速应对环境压力。
