Rocco-Machado Nathália, Deng Max, He Yi, Levine Rodney L
Laboratory of Biochemistry, National Heart, Lung and Blood Institute, Maryland, USA.
Laboratory of Biochemistry, National Heart, Lung and Blood Institute, Maryland, USA.
Arch Biochem Biophys. 2025 Feb;764:110268. doi: 10.1016/j.abb.2024.110268. Epub 2024 Dec 12.
Ca/calmodulin-dependent protein kinase II α (CaMKIIα) "autonomous" activation induced by Thr286 phosphorylation has a crucial role in synaptic plasticity. Previous studies showed that in Alzheimer's disease brain, CaMKIIα autophosphorylation at Thr286 is reduced while the level of cysteine-oxidized CAMKIIα is elevated. We performed tryptic mapping of the oxidized CaMKIIα and discovered the formation of a disulfide between the N-terminal Cys6 and the regulatory domain Cys280. The apparent pK values of Cys6 and Cys280 are 7.1 and 7.7, respectively, lower than the 8.5 for free Cys. The low apparent pK of Cys6 facilitates the oxidation of its thiol to the sulfenic acid at physiological pH. The thiolate of Cys280 can then attack the sulfenic acid to form a disulfide. Using an antibody against phosphorylated Thr286, we showed that disulfide formation prevents Thr286 phosphorylation. CaMKIIα autonomous activation induced by disulfide formation is much lower than the autonomous activation induced by phosphorylation. The decreased autonomous activation may contribute to the synaptic impairment of Alzheimer's disease. We also generated a CaMKIIα mutant in which Cys6 was mutated to Ser6. This mutation prevented disulfide formation and restored autonomous activation induced by phosphorylation. Our findings provide insight into the mechanistic details of CaMKIIα autonomous activation induced by disulfide formation that may contribute to the impairment of long-term potentiation in Alzheimer's disease.
苏氨酸286磷酸化诱导的钙/钙调蛋白依赖性蛋白激酶IIα（CaMKIIα）“自主”激活在突触可塑性中起关键作用。先前的研究表明，在阿尔茨海默病大脑中，苏氨酸286位点的CaMKIIα自磷酸化减少，而半胱氨酸氧化型CAMKIIα的水平升高。我们对氧化型CaMKIIα进行了胰蛋白酶图谱分析，发现N端半胱氨酸6（Cys6）和调节结构域半胱氨酸280（Cys280）之间形成了二硫键。Cys6和Cys280的表观pK值分别为7.1和7.7，低于游离半胱氨酸的8.5。Cys6较低的表观pK值有利于其硫醇在生理pH值下氧化为亚磺酸。然后Cys280的硫醇盐可以攻击亚磺酸形成二硫键。使用抗磷酸化苏氨酸286的抗体，我们发现二硫键的形成阻止了苏氨酸286的磷酸化。二硫键形成诱导的CaMKIIα自主激活远低于磷酸化诱导的自主激活。自主激活的降低可能导致阿尔茨海默病的突触损伤。我们还构建了一个Cys6突变为Ser6的CaMKIIα突变体。这种突变阻止了二硫键的形成，并恢复了磷酸化诱导的自主激活。我们的研究结果深入了解了二硫键形成诱导CaMKIIα自主激活的机制细节，这可能导致阿尔茨海默病中长时程增强的损伤。
