mTORC1 依赖性蛋白合成和自噬在载脂蛋白 A-I 表达的调控中解耦。

mTORC1-dependent protein synthesis and autophagy uncouple in the regulation of Apolipoprotein A-I expression.

机构信息

Laboratory of Molecular and Cellular Biology, University of Crete Medical School, Heraklion, Crete, Greece; Department of Biology, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece.

Laboratory of Molecular and Cellular Biology, University of Crete Medical School, Heraklion, Crete, Greece.

出版信息

Metabolism. 2020 Apr;105:154186. doi: 10.1016/j.metabol.2020.154186. Epub 2020 Feb 18.

DOI:10.1016/j.metabol.2020.154186

PMID:32084429

Abstract

BACKGROUND

Apolipoprotein A-I (ApoA-I) is involved in reverse cholesterol transport as a major component of HDL, but also conveys anti-thrombotic, anti-oxidative, anti-inflammatory and immune-regulatory properties that are pertinent to its protective roles in cardiovascular, inflammatory and malignant pathologies. Despite the pleiotropy in ApoA-I functions, the regulation of intracellular ApoA-I levels remains poorly explored.

METHODS

HepG2 hepatoma cells and primary mouse hepatocytes were used as in vitro models to study the impact of genetic and chemical inhibitors of autophagy and the proteasome on ApoA-I by immunoblot, immunofluorescence and electron microscopy. Different growth conditions were implemented in conjunction with mTORC inhibitors to model the influence of nutrient scarcity versus sufficiency on ApoA-I regulation. Hepatic ApoA-I expression was also evaluated in high fat diet-fed mice displaying blockade in autophagy.

RESULTS

Under nutrient-rich conditions, basal ApoA-I levels in liver cells are sustained by the balancing act of autophagy and of mTORC1-dependent de novo protein synthesis. ApoA-I proteolysis occurs through a canonical autophagic pathway involving Beclin1 and ULK1 and the receptor protein p62/SQSTM1 that targets ApoA-I to autophagosomes. However, upon aminoacid insufficiency, suppression of ApoA-I synthesis prevails, rendering mTORC1 inactivation dispensable for autophagy-mediated ApoA-I proteolysis.

CONCLUSION

These data underscore the major contribution of post-transcriptional mechanisms to ApoA-I levels which differentially involve mTORC1-dependent signaling to protein synthesis and autophagy, depending on nutrient availability. Given the established role of ApoA-I in HDL-mediated reverse cholesterol transport, this mode of ApoA-I regulation may reflect a hepatocellular response to the organismal requirement for maintenance of cholesterol and lipid reserves under conditions of nutrient scarcity.

摘要

背景

载脂蛋白 A-I（ApoA-I）作为 HDL 的主要成分参与胆固醇逆向转运，但也具有抗血栓、抗氧化、抗炎和免疫调节特性，这些特性与其在心血管、炎症和恶性病理过程中的保护作用有关。尽管 ApoA-I 具有多种功能，但细胞内 ApoA-I 水平的调节仍未得到充分探索。

方法

使用 HepG2 肝癌细胞和原代小鼠肝细胞作为体外模型，通过免疫印迹、免疫荧光和电子显微镜研究自噬和蛋白酶体的遗传和化学抑制剂对 ApoA-I 的影响。结合 mTORC 抑制剂实施不同的生长条件，以模拟营养缺乏与充足对 ApoA-I 调节的影响。还评估了高脂肪饮食喂养的小鼠中肝 ApoA-I 的表达，这些小鼠的自噬被阻断。

结果

在营养丰富的条件下，肝细胞中基础 ApoA-I 水平通过自噬和 mTORC1 依赖性从头蛋白质合成的平衡作用得以维持。ApoA-I 的蛋白水解通过涉及 Beclin1 和 ULK1 以及受体蛋白 p62/SQSTM1 的经典自噬途径发生，该途径将 ApoA-I 靶向自噬体。然而，在氨基酸不足的情况下，ApoA-I 合成的抑制占主导地位，使得 mTORC1 失活对于自噬介导的 ApoA-I 蛋白水解并非必需。

结论

这些数据强调了转录后机制对 ApoA-I 水平的主要贡献，这些机制根据营养可用性的不同，差异涉及 mTORC1 依赖性信号转导至蛋白质合成和自噬。鉴于 ApoA-I 在 HDL 介导的胆固醇逆向转运中的既定作用，这种 ApoA-I 调节模式可能反映了肝细胞对机体在营养缺乏条件下维持胆固醇和脂质储备的需求的反应。