Supply of methionine and arginine alters phosphorylation of mechanistic target of rapamycin (mTOR), circadian clock proteins, and α-s1-casein abundance in bovine mammary epithelial cells
Supply of methionine and arginine alters phosphorylation of mechanistic target of rapamycin (mTOR), circadian clock proteins, and α-s1-casein abundance in bovine mammary epithelial cells
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蛋氨酸和精氨酸的供应改变牛乳腺上皮细胞中雷帕霉素（mTOR）机械靶标、生物钟蛋白和α-s1-酪蛋白丰度的磷酸化
DOI：
10.1039/c9fo02379h
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发表时间：
2020-01-01
期刊：
影响因子：
6.1
通讯作者：
Loor, Juan J.
中科院分区：
文献类型：
--
作者：
Hu, Liangyu;Chen, Yifei;Loor, Juan J.
甲硫氨酸（Met）和精氨酸（Arg）通过改变雷帕霉素复合物1（mTORC 1）信号通路的机制靶点活性来调节酪蛋白丰度。在非反刍动物中，生物钟网络对蛋白质合成的调节（部分通过mTORC 1的活性）的潜在作用已经得到强调。本研究的主要目的是确定反刍动物乳腺细胞中mTORC 1相关上游靶点、昼夜节律钟蛋白和蛋白激酶AMP激活催化亚基α（AMPK）的mRNA、蛋白丰度和磷酸化状态的变化，这些变化与α-s1-酪蛋白（CSN 1 S1）丰度相关，以响应单独或联合提供更多的Met和Arg。将原代牛乳腺上皮细胞（BMEC）以2 x 2处理安排与对照培养基（理想氨基酸谱，IPAA）或补充有增加的Met（incMet）、Arg（incArg）或两者（incMet + incArg）的培养基一起孵育12小时。对数据进行分析，测试Met和Arg的主效应及其相互作用。在已知为mTORC 1靶点的7种氨基酸（AA）转运蛋白中，增加Arg供应下调SLC 1A 5、SLC 3A 2、SLC 7A 1和SLC 7A 5，而增加Met供应上调SLC 7A 1。当精氨酸和蛋氨酸单独供应增加时，胞质精氨酸传感器（CASTOR 1）的mRNA丰度较低。当Arg供应增加时，p-TSC 2（TSC复合物亚基2）更大，而p-AKT（AKT丝氨酸/苏氨酸激酶1）：总（t）AKT和p-AMPK：tAMPK的磷酸化比率降低。尽管如此，p-mTOR：tmTOR的比率在incArg的情况下几乎加倍，但这种应答并没有阻止CSN 1 S1丰度的降低。周期昼夜节律调节因子1（PER 1）蛋白的丰度几乎翻了一番，但只有incMet + incArg导致更大的时钟昼夜节律调节因子（CLOCK）蛋白丰度。总的来说，数据表明，更多的Met和Arg的供应可能会影响CSN 1 S1合成BMEC通过mTORC 1，昼夜节律钟和AMPK途径的变化。在反刍动物乳蛋白合成的背景下，确定细胞内能量，总AA供应和这些途径之间的机械关系值得进一步研究。
Methionine (Met) and arginine (Arg) regulate casein protein abundance through alterations in activity of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. A potential role for the circadian clock network on the regulation of protein synthesis, partly via activity of mTORC1, has been highlighted in non-ruminants. The main objective of the study was to determine in ruminant mammary cells alterations in mRNA, protein abundance and phosphorylation status of mTORC1-related upstream targets, circadian clock proteins, and protein kinase AMP-activated catalytic subunit alpha (AMPK) in relation to alpha-s1-casein protein (CSN1S1) abundance in response to greater supply of Met and Arg alone or in combination. Primary bovine mammary epithelial cells (BMEC) were incubated for 12 h in a 2 x 2 arrangement of treatments with control media (ideal profile of amino acids, IPAA), or media supplemented with increased Met (incMet), Arg (incArg), or both (incMet + incArg). Data were analyzed testing the main effects of Met and Arg and their interaction. Among 7 amino acid (AA) transporters known to be mTORC1 targets, increasing supply of Arg downregulated SLC1A5, SLC3A2, SLC7A1, and SLC7A5, while increasing supply of Met upregulated SLC7A1. mRNA abundance of the cytosolic Arg sensor (CASTOR1) was lower when supply of Arg and Met alone increased. p-TSC2 (TSC complex subunit 2) was greater when the Arg supply was increased, while the phosphoralation ratio of p-AKT (AKT serine/threonine kinase 1):total (t) AKT and p-AMPK:tAMPK were lower. In spite of this, the ratio of p-mTOR:tmTOR nearly doubled with incArg but such response did not prevent a decrease in CSN1S1 abundance. The abundance of period circadian regulator 1 (PER1) protein nearly doubled with all treatments, but only incMet + incArg led to greater clock circadian regulator (CLOCK) protein abundance. Overall, data suggest that a greater supply of Met and Arg could influence CSN1S1 synthesis of BMEC through changes in the mTORC1, circadian clock, and AMPK pathways. Identifying mechanistic relationships between intracellular energy, total AA supply, and these pathways in the context of milk protein synthesis in ruminants merits further research.