Reconstitution of leucine-mediated autophagy via the mTORC1-Barkor pathway in vitro
Reconstitution of leucine-mediated autophagy via the mTORC1-Barkor pathway in vitro
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DOI：
10.4161/auto.8.2.18563
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发表时间：
2012-02
期刊：
影响因子：
13.3
通讯作者：
Xianghua Yan;Qiming Sun;J. Ji;Yaqin Zhu;Zhengfei Liu;Q. Zhong
中科院分区：
文献类型：
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作者：
Xianghua Yan;Qiming Sun;J. Ji;Yaqin Zhu;Zhengfei Liu;Q. Zhong
补充支链氨基酸，特别是亮氨酸，通过调节蛋白质的合成和降解，是改善营养不良的关键。新出现的证据表明，亮氨酸缺乏导致的蛋白质分解与自噬有关。在这项研究中，我们的目标是建立一种无细胞的方法，重述亮氨酸介导的体外自噬，并分析其生化要求。我们发现，在无细胞实验中，自噬小体结合蛋白Barkor/Atg14(L)的膜结合被富营养介质中的胞浆抑制，这种抑制是通过营养剥夺来释放的。我们还发现，雷帕霉素可以有效地逆转营养丰富的胞浆的抑制，这表明mTORC1在这个无细胞系统中的自噬抑制中发挥了重要作用。此外，我们还证明，在培养细胞中补充亮氨酸可以阻止Barkor斑点的形成和自噬活性。因此，我们建立了一种新的无细胞实验方法，以mTORC1依赖的方式概括了亮氨酸介导的自噬抑制；该实验将有助于我们剖析氨基酸在自噬和相关人类代谢性疾病中的调节。
Supplementation of branched chain amino acids, especially leucine, is critical to improve malnutrition by regulating protein synthesis and degradation. Emerging evidence has linked leucine deprivation induced protein breakdown to autophagy. In this study, we aimed to establish a cell-free assay recapitulating leucine-mediated autophagy in vitro and dissect its biochemical requirement. We found that in a cell-free assay, membrane association of Barkor/Atg14(L), a specific autophagosome-binding protein, is suppressed by cytosol from nutrient-rich medium and such suppression is released by nutrient deprivation. We also showed that rapamycin could efficiently reverse the suppression of nutrient rich cytosol, suggesting an essential role of mTORC1 in autophagy inhibition in this cell-free system. Furthermore, we demonstrated that leucine supplementation in the cultured cells blocks Barkor puncta formation and autophagy activity. Hence, we establish a novel cell-free assay recapitulating leucine-mediated autophagy inhibition in an mTORC1-dependent manner; this assay will help us to dissect the regulation of amino acids in autophagy and related human metabolic diseases.