ASCT2参与SARS介导的蛋氨酸供应的牛乳腺上皮细胞的β-酪蛋白合成。,Journal of Agricultural and Food Chemistry

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ASCT2参与SARS介导的蛋氨酸供应的牛乳腺上皮细胞的β-酪蛋白合成。
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2019-10-09 , DOI: 10.1021/acs.jafc.9b03833
Wenting Dai ₁ , Fengqi Zhao _{1,

2} , Jianxin Liu ₁ , Hongyun Liu ₁

Affiliation

乳腺细胞对蛋氨酸（Met）的吸收取决于相应的氨基酸（AA）转运蛋白，该转运蛋白在转运蛋白以外的乳蛋白生产中起着调节作用。我们以前的研究已经确定，丝氨酰tRNA合成酶（SARS）可能是一种新的介质，可以调节原代牛乳腺上皮细胞（BMEC）中必需的AA刺激的酪蛋白合成。但是，Met在奶牛乳蛋白生产中的调控机制仍需进一步阐明。在这里，我们旨在研究Met对BMEC中牛奶蛋白质合成的影响，并探讨其潜在机制。评估Met对AA转运蛋白，酪蛋白合成及相关信号传导途径的影响，在接受或不加AA转运蛋白抑制的情况下，用0.6 mM Met处理6 h的BMEC进行评估（ASCT2，相应抑制剂（GPNA）的中性AA转运蛋白活性。此外，SARS对细胞的影响主要是在用0.6 mM Met处理6 h的BMECs中进行评估，无论是否通过RNAi干扰抑制SARS。通过实时定量聚合酶链反应法分析AA转运蛋白和通路相关基因的基因表达，并通过western blot法测定相关蛋白的蛋白表达。结果显示，与其他浓度的Met相比，0.6 mM的Met显着增强了细胞生长和β-酪蛋白的合成。在13个氨基酸转运蛋白中，0.6 mM Met高度增加了ASCT2表达。通过添加500μMASCT2抑制剂GPNA，Met刺激的ASCT2表达和增强的乳腺细胞内Met摄取均降低。在存在0.6 mM Met的情况下，抑制ASCT2活性（通过GPNA）和抑制SARS表达（通过RNAi）均会降低β-酪蛋白的合成。此外，0.6 mM Met增加了mTOR，S6K1、4EBP1和Akt的基因表达。相反，GPNA对ASCT2的抑制作用降低了这四个基因的基因表达。总体而言，这项工作表明，ASCT2通过增强BMEC中的Met摄取和激活mTOR信号通路，参与了SARS介导的Met对β-酪蛋白合成的刺激。

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ASCT2 Is Involved in SARS-Mediated β-Casein Synthesis of Bovine Mammary Epithelial Cells with Methionine Supply.

The methionine (Met) uptake into mammary cells depends upon the corresponding amino acid (AA) transporters, which play a regulatory role in the mammary protein production beyond transport. Our previous studies have identified that seryl-tRNA synthetase (SARS) could be a novel mediator to regulate essential AA-stimulated casein synthesis in primary bovine mammary epithelial cells (BMECs). However, the regulatory mechanisms of Met in milk protein production in dairy cows remain further clarified. Here, we aimed to investigate the effects of Met on milk protein synthesis in BMECs and explore the underlying mechanism. The effects of Met on the AA transporter, casein synthesis, and the related signaling pathway were evaluated in the BMECs treated with 0.6 mM Met for 6 h combined with or without the inhibition of AA transporter (ASCT2, a neutral AA transporter) activity by the corresponding inhibitor (GPNA). Besides, the effects of SARS on the cells were mainly evaluated in the BMECs treated with 0.6 mM Met for 6 h together with or without SARS knockdown by RNAi interference. The gene expression of AA transporters and pathway-related genes were analyzed by the real-time quantitative polymerase chain reaction method, and the protein expression of related proteins were determined by the western blot assay. Results showed that 0.6 mM Met remarkably enhanced cell growth and β-casein synthesis compared to the supply of other Met concentrations. Among 13 amino acid transporters, 0.6 mM Met highly increased ASCT2 expression. This Met-stimulated ASCT2 expression and the enhanced mammary intracellular Met uptake were both decreased by the addition of 500 μM GPNA, an inhibitor of ASCT2. In the presence of 0.6 mM Met, the inhibition of ASCT2 activity (by GPNA) and SARS expression (by RNAi) both reduced β-casein synthesis. Additionally, 0.6 mM Met increased the gene expression of mTOR, S6K1, 4EBP1, and Akt; in contrast, the inhibition of ASCT2 by GPNA lowered the gene expression of these four genes. Collectively, this work suggests that ASCT2 is involved in the SARS-mediated Met stimulation of β-casein synthesis through enhancing mammary Met uptake and activating the mTOR signaling pathway in BMECs.

更新日期：2019-10-09

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