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 in 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 protein 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-Tibet Plateau. In total, 5170 proteins were quantified and 5695 phosphorylation sites on 1938 proteins were quantified, respectively. Proteomic results showed that a total of 674 (438 up, 236 down) differentially expressed proteins were screened in hibernating N. parkeri versus summer individuals. Functional enrichment analysis revealed that highly expressed proteins in winter were significantly enriched in immune-related signaling pathways, whereas low-expressed proteins were mainly involved in metabolic processes. A total of 4251 (4147 up, 104 down) modified sites belonging to 1638 (1555 up, 83 down) phosphoproteins showed a significant change in the liver. A large set of proteins are regulated during hibernation by RPP and these proteins are involved in various functions such as many metabolic enzymes, ion pumping, protein turnover, signal transduction, and alternative splicing. These changes contribute to enhancing protection, suppressing energy-consuming processes, and thus metabolic depression. Moreover, phosphofructokinase, glutamate dehydrogenase, and ATPase activities were all significantly lower in winter compared to summer. In conclusion, our present results support the hypothesis and demonstrate the importance of RPP as a regulatory mechanism when animals transition into a hypometabolic state.