Surviving winter on the Qinghai-Tibetan Plateau: extensive reversible protein phosphorylation plays a dominant role in regulating hypometabolism in hibernating Nanorana parkeri

Changes in protein abundance and reversible protein phosphorylation (RPP) play important roles in regulating hypometabolism but have never been documented in overwintering frogs at high altitudes. To test the hypothesis that protein abundance and phosphorylation change in response to winter hibernation, we conducted a comprehensive and quantitative proteomic and phosphoproteomic analysis of the liver of the Xizang plateau frog, Nanorana parkeri, living on the Qinghai-Xizang (Tibet) Plateau (QTP). In total, 5 170 proteins and 5 695 phosphorylation sites in 1 938 proteins were quantified. Based on proteomic analysis, 674 differentially expressed proteins (438 up-regulated, 236 down-regulated) were screened in hibernating N. parkeri versus summer individuals. Functional enrichment analysis revealed that higher expressed proteins in winter were significantly enriched in immune-related signaling pathways, whereas lower expressed proteins were mainly involved in metabolic processes. A total of 4 251 modified sites (4 147 up-regulated, 104 down-regulated) belonging to 1 638 phosphoproteins (1 555 up-regulated, 83 down-regulated) were significantly changed in the liver. During hibernation, RPP regulated a diverse array of proteins involved in multiple functions, including metabolic enzymatic activity, ion transport, protein turnover, signal transduction, and alternative splicing. These changes contribute to enhancing protection, suppressing energy-consuming processes, and inducing metabolic depression. Moreover, the activities of phosphofructokinase, glutamate dehydrogenase, and ATPase were all significantly lower in winter compared to summer. In conclusion, our results support the hypothesis and demonstrate the importance of RPP as a regulatory mechanism when animals transition into a hypometabolic state.蛋白质丰度和可逆蛋白磷酸化的变化在调节低代谢中发挥着重要作用，但这在高海拔越冬蛙类中却从未有过报道。为了验证蛋白质丰度和蛋白质磷酸化变化以响应冬眠的假说，我们对生活在青藏高原的高山倭蛙（ Nanorana parkeri）的肝脏进行了全面的定量蛋白质组学和磷酸化蛋白质组学分析，总共定量到5170个蛋白质，对1938个蛋白质上的5695个磷酸化位点也进行了定量分析。蛋白质组学结果显示，在冬眠与活动期的个体中，共筛选出 674个（438个上调，236个下调）差异表达蛋白质。功能富集分析表明，冬季高表达的蛋白明显富集于免疫相关的信号通路，而低表达的蛋白主要参与代谢过程。肝脏中属于1638个（1555个上调，83个下调）磷酸化蛋白的4251个（4147个上调，104个下调）修饰位点发生了显著变化。大量蛋白质在冬眠期间受到可逆的磷酸化修饰，这些蛋白质参与了多种功能，包括许多代谢酶、离子泵、蛋白质周转、信号转导和可变剪切等。这些变化有助于保护机体，降低能量消耗过程，从而抑制新陈代谢。此外，与夏季相比，冬季肝脏中磷酸果糖激酶、谷氨酸脱氢酶和ATP酶的活性都明显降低。总之，我们的结果不仅支持上述假说，而且证明了当动物过渡到低代谢状态时，可逆蛋白磷酸化作为一种调节机制的重要性。.