ATF4 介导的 REDD1 和 sestrin2 的上调抑制了延长亮氨酸剥夺期间的 mTORC1 活性。
ATF4-Mediated Upregulation of REDD1 and Sestrin2 Suppresses mTORC1 Activity during Prolonged Leucine Deprivation.
机构信息
Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, PA, USA.
出版信息
J Nutr. 2020 May 1;150(5):1022-1030. doi: 10.1093/jn/nxz309.
BACKGROUND
The protein kinase target of rapamycin (mTOR) in complex 1 (mTORC1) is activated by amino acids and in turn upregulates anabolic processes. Under nutrient-deficient conditions, e.g., amino acid insufficiency, mTORC1 activity is suppressed and autophagy is activated. Intralysosomal amino acids generated by autophagy reactivate mTORC1. However, sustained mTORC1 activation during periods of nutrient insufficiency would likely be detrimental to cellular homeostasis. Thus, mechanisms must exist to prevent amino acids released by autophagy from reactivating the kinase.
OBJECTIVE
The objective of the present study was to test whether mTORC1 activity is inhibited during prolonged leucine deprivation through ATF4-dependent upregulation of the mTORC1 suppressors regulated in development and DNA damage response 1 (REDD1) and Sestrin2.
METHODS
Mice (8 wk old; C57Bl/6 × 129SvEV) were food deprived (FD) overnight and one-half were refed the next morning. Mouse embryo fibroblasts (MEFs) deficient in ATF4, REDD1, and/or Sestrin2 were deprived of leucine for 0-16 h. mTORC1 activity and ATF4, REDD1, and Sestrin2 expression were assessed in liver and cell lysates.
RESULTS
Refeeding FD mice resulted in activation of mTORC1 in association with suppressed expression of both REDD1 and Sestrin2 in the liver. In cells in culture, mTORC1 exhibited a triphasic response to leucine deprivation, with an initial suppression followed by a transient reactivation from 2 to 4 h and a subsequent resuppression after 8 h. Resuppression occurred concomitantly with upregulated expression of ATF4, REDD1, and Sestrin2. However, in cells lacking ATF4, neither REDD1 nor Sestrin2 expression was upregulated by leucine deprivation, and resuppression of mTORC1 was absent. Moreover, in cells lacking either REDD1 or Sestrin2, mTORC1 resuppression was attenuated, and in cells lacking both proteins resuppression was further blunted.
CONCLUSIONS
The results suggest that leucine deprivation upregulates expression of both REDD1 and Sestrin2 in an ATF4-dependent manner, and that upregulated expression of both proteins is involved in resuppression of mTORC1 during prolonged leucine deprivation.
背景
雷帕霉素靶蛋白（mTOR）在复合物 1（mTORC1）中的蛋白激酶受到氨基酸的激活，进而上调合成代谢过程。在营养缺乏的情况下，例如氨基酸不足，mTORC1 的活性受到抑制，自噬被激活。自噬产生的溶酶体内氨基酸重新激活 mTORC1。然而，在营养不足的时期持续激活 mTORC1 可能对细胞内稳态有害。因此，必须存在机制来防止自噬释放的氨基酸重新激活激酶。
目的
本研究旨在通过 ATF4 依赖性上调发育和 DNA 损伤反应 1（REDD1）和 Sestrin2 来检测延长亮氨酸剥夺期间 mTORC1 活性是否受到抑制。
方法
将 8 周龄（C57Bl/6×129SvEV）小鼠禁食过夜，其中一半于次日早上重新喂食。缺乏 ATF4、REDD1 和/或 Sestrin2 的小鼠胚胎成纤维细胞（MEFs）在亮氨酸缺乏 0-16 小时。在肝和细胞裂解物中评估 mTORC1 活性和 ATF4、REDD1 和 Sestrin2 的表达。
结果
重新喂食 FD 小鼠导致 mTORC1 的激活，同时肝中 REDD1 和 Sestrin2 的表达受到抑制。在培养的细胞中，mTORC1 对亮氨酸缺乏表现出三相反应，最初抑制，然后在 2 至 4 小时短暂再激活，然后在 8 小时后再次抑制。再抑制伴随着 ATF4、REDD1 和 Sestrin2 的上调表达。然而，在缺乏 ATF4 的细胞中，亮氨酸缺乏既不会上调 REDD1 也不会上调 Sestrin2 的表达，mTORC1 的再抑制也不存在。此外，在缺乏 REDD1 或 Sestrin2 的细胞中，mTORC1 的再抑制减弱，而在缺乏这两种蛋白质的细胞中，再抑制进一步减弱。
结论
结果表明，亮氨酸剥夺以 ATF4 依赖的方式上调 REDD1 和 Sestrin2 的表达，而两种蛋白质的上调表达都参与了延长亮氨酸剥夺期间 mTORC1 的再抑制。
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