Sun H, Gao J, Ferrington D A, Biesiada H, Williams T D, Squier T C
Department of Molecular Biosciences, Mass Spectrometry Laboratory, University of Kansas, Lawrence 66045-2106, USA.
Biochemistry. 1999 Jan 5;38(1):105-12. doi: 10.1021/bi981295k.
We have investigated the ability of methionine sulfoxide reductase (MsrA) to maintain optimal calmodulin (CaM) function through the repair of oxidized methionines, which have been shown to accumulate within CaM in senescent brain [Gao, J., Yin, D. H., Yao, Y., Williams, T. D., and Squier, T. C. (1998) Biochemistry 37, 9536-9548]. Oxidatively modified calmodulin (CaMox) isolated from senescent brain or obtained by in vitro oxidation was incubated with MsrA. This treatment restores the functional ability of CaMox to activate the plasma membrane (PM) Ca-ATPase, confirming that (i) the decreased ability of CaM isolated from senescent animals to activate the PM Ca-ATPase results solely from methionine sulfoxide formation and (ii) MsrA can repair methionine sulfoxides within cytosolic proteins. We have used electrospray ionization mass spectrometry to investigate the extent and rates of methionine sulfoxide repair within CaMox. Upon exhaustive repair by MsrA, there remains a distribution of methionine sulfoxides within functionally reactivated CaMox, which varies from three to eight methionine sulfoxides. The rates of repair of methionine sulfoxides within individual tryptic fragments of CaMox vary by a factor of 2, where methionine sulfoxides located within hydrophobic sequences are repaired in preference to methionines that are more solvent accessible within the native structure. However, no single methionine sulfoxide is completely repaired in all CaM oxiforms. Decreases in the alpha-helical content and a disruption of the tertiary structure of CaM have previously been shown to result from methionine oxidation. Repair of selected methionine sulfoxides in CaMox by MsrA results in a partial refolding of the secondary structure, suggesting that MsrA repairs methionine sulfoxides within unfolded sequences until native-like structure and function are re-attained. The ability of CaMox isolated from senescent brain to fully activate the PM Ca-ATPase following repair by MsrA suggests the specific activity of MsrA is insufficient to maintain CaM function in aging brain. These results are discussed in terms of the possible regulatory role MsrA may play in the modulation of CaM function and calcium homeostasis under conditions of oxidative stress.
我们研究了甲硫氨酸亚砜还原酶（MsrA）通过修复氧化甲硫氨酸来维持钙调蛋白（CaM）最佳功能的能力，氧化甲硫氨酸已被证明会在衰老大脑的CaM中积累[高杰、尹东辉、姚瑶、威廉姆斯·T·D和斯奎尔·T·C（1998年）《生物化学》37卷，9536 - 9548页]。将从衰老大脑中分离或通过体外氧化获得的氧化型钙调蛋白（CaMox）与MsrA一起孵育。这种处理恢复了CaMox激活质膜（PM）Ca - ATP酶的功能能力，证实了（i）从衰老动物分离的CaM激活PM Ca - ATP酶的能力下降完全是由甲硫氨酸亚砜的形成导致的，以及（ii）MsrA可以修复胞质蛋白中的甲硫氨酸亚砜。我们使用电喷雾电离质谱法研究了CaMox中甲硫氨酸亚砜修复的程度和速率。在MsrA彻底修复后，功能重新激活的CaMox中仍存在甲硫氨酸亚砜的分布，其数量从三个到八个不等。CaMox各个胰蛋白酶片段中甲硫氨酸亚砜的修复速率相差2倍，其中位于疏水序列中的甲硫氨酸亚砜比天然结构中更易接近溶剂的甲硫氨酸优先被修复。然而，在所有CaM氧化形式中，没有单个甲硫氨酸亚砜被完全修复。先前已证明甲硫氨酸氧化会导致CaM的α - 螺旋含量降低和三级结构破坏。MsrA对CaMox中选定甲硫氨酸亚砜的修复导致二级结构部分重新折叠，这表明MsrA会修复未折叠序列中的甲硫氨酸亚砜，直到重新获得类似天然的结构和功能。从衰老大脑中分离的CaMox在被MsrA修复后完全激活PM Ca - ATP酶的能力表明，MsrA的比活性不足以在衰老大脑中维持CaM功能。我们从MsrA在氧化应激条件下可能对CaM功能和钙稳态调节中所起的作用方面讨论了这些结果。
