Proteomic Profile of Carbonylated Proteins Screen the Regulation of Calmodulin-Dependent Protein Kinases-AMPK-Beclin1 in Aerobic Exercise-Induced Autophagy in Middle-Aged Rat Hippocampus
Proteomic Profile of Carbonylated Proteins Screen the Regulation of Calmodulin-Dependent Protein Kinases-AMPK-Beclin1 in Aerobic Exercise-Induced Autophagy in Middle-Aged Rat Hippocampus
复制标题
羰基化蛋白的蛋白质组学谱筛选钙调蛋白依赖性蛋白激酶-AMPK-Beclin1在中年大鼠海马有氧运动诱导的自噬中的调节作用
DOI：
10.1159/000500742
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发表时间：
2019-10-01
期刊：
影响因子：
3.5
通讯作者：
Yin, Dazhong
中科院分区：
文献类型：
--
作者：
Liu, Wenfeng;Xia, Yan;Yin, Dazhong
背景：羰基化是蛋白质的氧化修饰，是氧化应激的标志。毒性羰基化蛋白的积累可能是海马老化或神经退行性变的发病机制之一。大量的证据表明，规律的有氧运动可能会减轻羰基化蛋白质的功能障碍，但运动的适应机制尚不清楚。目的：本研究采用蛋白质组学方法探讨海马羰基应激机制，以及钙调素依赖性蛋白激酶（CAMK）-腺苷酸活化蛋白激酶（AMPK）-Beclin 1信号通路在有氧运动延缓衰老或改善功能中的作用。研究方法：24只13月龄健康雄性SD大鼠（平均体重693.21 ± 68.85 g），随机分为中年安静对照组（M-SED，n = 12）和中年有氧运动跑组（M-EX，n = 12）。M-EX组参加常规有氧运动-跑步机跑步-运动强度在10周内从最大耗氧量（V stecO 2 max）的50-55%逐渐增加到65-70%。通过亲和素磁珠和电喷雾四极杆飞行时间质谱（ESI-Q-TOF-MS）分析氧化修饰的靶蛋白。蛋白质印迹法用于检测分子靶点。结果：规律性有氧运动可恢复中年大鼠的闭会期间习惯性行为，挽救海马的形态结构。ESI-Q-TOF-MS筛选出仅在M-SED中发现的56个羰基化蛋白和仅在M-EX中发现的16个羰基化蛋白，表明有氧运动降低羰基应激。有趣的是，Ca 2 +/CAMK II α（CAMK II α）仅在4-羟基壬烯醛加合物的氧化修饰位点的M-SED基团中发生羰基化，而定期有氧运动可减轻CAMK II α羰基化。有规律的有氧运动显著增加CAMK Ⅱ α和AMPKα1的表达和磷酸化活性水平。它还上调海马中Beclin 1和微管相关蛋白1-轻链3的表达。结论：CAMK Ⅱ α羰基化可能是海马衰老的潜在生物标志物。此外，规律有氧运动通过激活CAMK-AMPK-Beclin 1信号通路诱导的自噬可能通过减轻蛋白质羰基化（羰基应激）来减轻海马神经变性或病理变化。
Background: Carbonylation is an oxidative modification of the proteins and a marker of oxidative stress. The accumulation of toxic carbonylated proteins might be one of the onsets of pathogenesis in hippocampal aging or neurodegeneration. Enormous evidence indicates that regular aerobic exercise might alleviate the dysfunction of carbonylated proteins, but the adaptational mechanisms in response to exercise are unclear. Objective: This study explored the carbonyl stress mechanism in the hippocampus using proteomics and the role of calmodulin-dependent protein kinases (CAMK)-AMP-activated protein kinase (AMPK)-Beclin1 signaling pathways in alleviating aging or improving function with regular aerobic exercise. Methods: Twenty-four healthy 13-month-old male Sprague-Dawley rats (average 693.21 ± 68.85 g) were randomly divided into middle-aged sedentary control group (M-SED, n = 12) and middle-aged aerobic exercise runner group (M-EX, n = 12). The M-EX group participated in regular aerobic exercise – treadmill running – with exercise intensity increasing gradually from 50–55% to 65–70% of maximum oxygen consumption (V˙O2max) over 10 weeks. The targeted proteins of oxidative modification were profiled by avidin magnetic beads and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS). Western blots were used to test for molecular targets. Results: Regular aerobic exercise restores the intersessional habituation and rescues the hippocampus morphological structure in middle-aged rats. ESI-Q-TOF-MS screened 56 carbonylated proteins only found in M-SED and 16 carbonylated proteins only found in M-EX, indicating aerobic exercise decreased carbonyl stress. Intriguingly, Ca2+/CAMK II alpha (CAMKIIα) was carbonylated only in the M-SED group at the oxidative modification site of 4-hydroxynonenal adducts, while regular aerobic exercise alleviated CAMKIIα carbonylation. Regular aerobic exercise significantly increased the expression and phosphorylated, active levels of CAMKIIα and AMPKα1. It also upregulated the expression of Beclin1 and microtubule-associated protein1-light chain 3 in the hippocampus. Conclusion: Quantification of CAMKIIα carbonylation may be a potential biomarker of the hippocampal senescence. Additionally, regular aerobic exercise-induced autophagy via the activation of CAMK-AMPK-Beclin1 signaling pathway may mitigate the hippocampal neurodegeneration or pathological changes by alleviating protein carbonylation (carbonyl stress).