Zhang Yichi, English Simon G, Storey Kenneth B
Institute of Biochemistry and Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, ON, K1S 5B6, Canada.
Department of Molecular Biology, Hamon Center for Regenerative Science and Medicine, Sen. Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX, 75390, USA.
Mol Biol Rep. 2018 Oct;45(5):751-761. doi: 10.1007/s11033-018-4214-8. Epub 2018 Jun 19.
Xenopus laevis, otherwise known as the African clawed frog, undergoes natural dehydration of up to 30% of its total body water during the dry season in sub-Saharan Africa. To survive under these conditions, a variety of physiological and biochemical changes take place in X. laevis. We were interested in understanding the role that the calcineurin-NFAT pathway plays during dehydration stress response in the skeletal muscles of X. laevis. Immunoblotting was performed to characterize the protein levels of NFATc1-4, calcium signalling proteins, in addition to myogenic proteins (MyoD, MyoG, myomaker). In addition, DNA-protein interaction ELISAs were used to assess the binding of NFATs to their consensus binding sequence, and to identify the effect of urea on NFAT-binding. Our results showed that NFATc1 and c4 protein levels decreased during dehydration, and there were no changes in NFATc2, c3, and calcium signalling proteins. However, MyoG and myomaker both showed increases in protein levels during dehydration, thus indicating that the late myogenic program involving myoblast differentiation, but not satellite cell activation and myoblast proliferation, could be involved in preserving the skeletal muscle of X. laevis during dehydration. In addition, we observed that urea seems to reduce NFATc3-binding to DNA during control, but not during dehydration, possibly indicating that NFATc3 is protected from the denaturing effects of urea as it accumulates during dehydration. These findings expand upon our knowledge of adaptive responses to dehydration, and they identify specific protein targets that could be used to protect the skeletal muscle from damage during stress.
非洲爪蟾，也就是非洲爪蛙，在撒哈拉以南非洲的旱季会经历全身水分高达30%的自然脱水过程。为了在这些条件下生存，非洲爪蟾会发生各种生理和生化变化。我们感兴趣的是了解钙调神经磷酸酶 - 活化T细胞核因子（calcineurin-NFAT）通路在非洲爪蟾骨骼肌脱水应激反应中所起的作用。我们进行了免疫印迹分析，以表征NFATc1 - 4、钙信号蛋白以及生肌蛋白（MyoD、MyoG、成肌素）的蛋白质水平。此外，我们使用了DNA - 蛋白质相互作用酶联免疫吸附测定法来评估NFAT与它们的共有结合序列的结合情况，并确定尿素对NFAT结合的影响。我们的结果表明，脱水过程中NFATc1和c4的蛋白质水平下降，而NFATc2、c3和钙信号蛋白没有变化。然而，MyoG和成肌素在脱水过程中蛋白质水平均有所增加，这表明涉及成肌细胞分化而非卫星细胞激活和成肌细胞增殖的晚期生肌程序可能参与了非洲爪蟾脱水期间骨骼肌的保护。此外，我们观察到，在对照期间尿素似乎会降低NFATc3与DNA的结合，但在脱水期间则不会，这可能表明随着脱水过程中尿素的积累，NFATc3受到保护，免受尿素的变性作用影响。这些发现扩展了我们对脱水适应性反应的认识，并确定了在应激期间可用于保护骨骼肌免受损伤的特定蛋白质靶点。
