MEF2D-NR4A1-FAM134B2-mediated reticulophagy contributes to amino acid homeostasis

We recently identified FAM134B2, which is an N-terminal truncated reticulophagy receptor highly induced by starvation such as fasting of mice and treatment of mammalian cells with a starvation medium that does not contain amino acids, glucose and growth factors. However, which starvation signal mediates the induction of FAM134B2 is still obscure. In this study, we found that amino acid deficiency (AAD) could mimic the starvation condition to induce FAM134B2 expression. Unexpectedly, EIF2AK4/GCN2-mediated integrated signal response (ISR) and MTOR (mechanistic target of rapamycin kinase) signals, which constitute two major signaling pathways that respond to AAD, did not contribute to AAD-induced FAM134B2 induction. mRNA-seq and siRNA screenings identified two ISR-independent transcription factors, MEF2D (myocyte enhancer factor 2D) and NR4A1 (nuclear receptor subfamily 4 group A member 1), involved in AAD-induced FAM134B2 expression. AAD induces MEF2D, resulting in the induction of NR4A1, which in turn induces FAM134B2-mediated reticulophagy to maintain intracellular amino acid levels. In conclusion, the MEF2D-NR4A1-FAM134B2 cascade is a critical signal in amino acid homeostasis. AAD: amino acid deficiency; APOC3: apolipoprotein C3; BACH1: BTB domain and CNC homolog 1; CEBP: CCAAT enhancer binding protein; DDIT3/CHOP: DNA damage inducible transcript 3; EBSS: Earle’s Balanced Salt Solution; EIF2AK4/GCN2: eukaryotic translation initiation factor 2 alpha kinase 4; ER: endoplasmic reticulum; HisOH: histidinol; ISR: integrated stress response; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF2D: myocyte enhancer factor 2D; MTOR: mechanistic target of rapamycin kinase; NR4A1: nuclear receptor subfamily 4 group A member 1; RETREG1/FAM134B: reticulophagy regulator 1; RTN2: reticulon 2, TF: transcription factor; TFEB: transcription factor EB; ZBTB10: zinc finger and BTB domain containing 10

我们近期鉴定出FAM134B2，这是一种N端截短型网状自噬受体（reticulophagy receptor），在饥饿条件下（如小鼠禁食、使用不含氨基酸、葡萄糖及生长因子的饥饿培养基处理哺乳动物细胞）表达显著上调。然而，介导FAM134B2诱导表达的饥饿信号通路仍不明确。
本研究发现，氨基酸缺乏（amino acid deficiency, AAD）可模拟饥饿状态，诱导FAM134B2的表达。出乎意料的是，作为响应氨基酸缺乏的两大主要信号通路——EIF2AK4/GCN2介导的整合应激反应（integrated stress response, ISR）与MTOR（mechanistic target of rapamycin kinase，雷帕霉素机制性靶标激酶）信号，并未参与氨基酸缺乏诱导的FAM134B2上调。
通过mRNA测序（mRNA-seq）与小干扰RNA（small interfering RNA, siRNA）筛选，我们鉴定出两个不依赖于ISR的转录因子：MEF2D（myocyte enhancer factor 2D，肌细胞增强因子2D）与NR4A1（nuclear receptor subfamily 4 group A member 1，核受体亚家族4A组成员1），它们参与了氨基酸缺乏诱导的FAM134B2表达调控。
氨基酸缺乏可诱导MEF2D表达，进而上调NR4A1，后者又可介导FAM134B2依赖的网状自噬，以维持细胞内氨基酸水平。
综上，MEF2D-NR4A1-FAM134B2级联反应是氨基酸稳态调控中的关键信号通路。

术语对照表：
AAD：氨基酸缺乏（amino acid deficiency）
APOC3：载脂蛋白C3（apolipoprotein C3）
BACH1：BTB结构域与CNC同源蛋白1（BTB domain and CNC homolog 1）
CEBP：CCAAT增强子结合蛋白（CCAAT enhancer binding protein）
DDIT3/CHOP：DNA损伤诱导转录蛋白3（DNA damage inducible transcript 3）
EBSS：伊格尔氏平衡盐溶液（Earle’s Balanced Salt Solution）
EIF2AK4/GCN2：真核翻译起始因子2α激酶4（eukaryotic translation initiation factor 2 alpha kinase 4）
ER：内质网（endoplasmic reticulum）
HisOH：组氨醇（histidinol）
ISR：整合应激反应（integrated stress response）
MAP1LC3/LC3：微管相关蛋白1轻链3（microtubule associated protein 1 light chain 3）
MEF2D：肌细胞增强因子2D（myocyte enhancer factor 2D）
MTOR：雷帕霉素机制性靶标激酶（mechanistic target of rapamycin kinase）
NR4A1：核受体亚家族4A组成员1（nuclear receptor subfamily 4 group A member 1）
RETREG1/FAM134B：网状自噬调控因子1（reticulophagy regulator 1）
RTN2：网状蛋白2（reticulon 2）
TF：转录因子（transcription factor）
TFEB：转录因子EB（transcription factor EB）
ZBTB10：含锌指与BTB结构域蛋白10（zinc finger and BTB domain containing 10）