Department of Biology and Institute of Biochemistry, Carleton University, Ottawa, Canada.
National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.
Proteins. 2021 May;89(5):544-557. doi: 10.1002/prot.26038. Epub 2021 Jan 6.
The African clawed frog (Xenopus laevis) withstands prolonged periods of extreme whole-body dehydration that lead to impaired blood flow, global hypoxia, and ischemic stress. During dehydration, these frogs shift from oxidative metabolism to a reliance on anaerobic glycolysis. In this study, we purified the central glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to electrophoretic homogeneity and investigated structural, kinetic, subcellular localization, and post-translational modification properties between control and 30% dehydrated X. laevis liver. GAPDH from dehydrated liver displayed a 25.4% reduction in maximal velocity and a 55.7% increase in its affinity for GAP, as compared to enzyme from hydrated frogs. Under dehydration mimicking conditions (150 mM urea and 1% PEG), GAP affinity was reduced with a K value 53.8% higher than controls. Frog dehydration also induced a significant increase in serine phosphorylation, methylation, acetylation, beta-N-acetylglucosamination, and cysteine nitrosylation, post-translational modifications (PTMs). These modifications were bioinformatically predicted and experimentally validated to govern protein stability, enzymatic activity, and nuclear translocation, which increased during dehydration. These dehydration-responsive protein modifications, however, did not appear to affect enzymatic thermostability as GAPDH melting temperatures remained unchanged when tested with differential scanning fluorimetry. PTMs could promote extreme urea resistance in dehydrated GAPDH since the enzyme from dehydrated animals had a urea I of 7.3 M, while the I from the hydrated enzyme was 5.3 M. The physiological consequences of these dehydration-induced molecular modifications of GAPDH likely suppress GADPH glycolytic functions during the reduced circulation and global hypoxia experienced in dehydrated X. laevis.
非洲爪蟾（Xenopus laevis）能够承受长时间的全身极端脱水，导致血流受损、全身缺氧和缺血性应激。在脱水过程中，这些青蛙从氧化代谢转变为依赖无氧糖酵解。在这项研究中，我们将中枢糖酵解酶甘油醛-3-磷酸脱氢酶（GAPDH）纯化至电泳均一性，并研究了对照和 30%脱水的非洲爪蟾肝脏之间的结构、动力学、亚细胞定位和翻译后修饰特性。与来自水合青蛙的酶相比，脱水肝脏的 GAPDH 的最大速度降低了 25.4%，对 GAP 的亲和力增加了 55.7%。在模拟脱水条件下（150 mM 尿素和 1% PEG），GAP 的亲和力降低，K 值比对照高 53.8%。青蛙脱水还诱导丝氨酸磷酸化、甲基化、乙酰化、β-N-乙酰葡糖胺化和半胱氨酸亚硝化为显著增加，这些翻译后修饰（PTMs）。这些修饰通过生物信息学预测并通过实验验证来控制蛋白质稳定性、酶活性和核转位，这些在脱水过程中增加。然而，这些脱水反应性蛋白修饰似乎不会影响酶的热稳定性，因为用差示扫描荧光法测试时，GAPDH 的熔点没有变化。PTMs 可以促进脱水 GAPDH 对尿素的极端抗性，因为脱水动物的酶的尿素 I 为 7.3 M，而水合酶的 I 为 5.3 M。GAPDH 脱水诱导的分子修饰的生理后果可能会在脱水的非洲爪蟾中经历的循环减少和全身缺氧期间抑制 GADPH 的糖酵解功能。
