Nutritional Control of Transcription Factor Expression

哺乳动物细胞对蛋白质/氨基酸的营养限制的反应是通过增加蛋白质/氨基酸的表达， 蛋白质通过一个信号通路，这将被称为氨基酸反应的广谱 （AAR）。虽然已知氨基酸的可利用性可以调节蛋白质表达，但其机制尚不清楚。 这些事件是如何发生的还不清楚。使用人HepG 2肝癌细胞，我们有 先前有文献记载，氨基酸限制增加了bZIP转录因子的表达， C/EBPb通过转录控制表达，而C/EBPb水平升高又诱导转录 来自氨基酸反应基因。对这一应用的初步研究已经产生了新的观察结果， 在来自人C/EBPb基因3 ′端的93 bp片段中存在氨基酸应答元件（AARE）活性， 与蛋白质编码序列相关联初步实验也表明，氨基酸限制导致 C/EBPb蛋白总丰度增加，C/EBPb核转位增加 在Thr 235上磷酸化。我们的总体假设是，人类C/EBPb的转录控制 C/EBPb功能的表达和翻译后控制是AAR中重要的调控步骤 通路大鼠限蛋白饮食和HepG 2细胞氨基酸限制的研究 建议的研究是：1）确定负责氨基酸依赖性的基因组AARE 通过使用缺失分析、体内足迹法和单核苷酸多态性分析来检测C/EBPb基因的转录。 诱变; 2）鉴定AARE结合蛋白并表征它们在AAR途径中的作用; 3） 确定三种C/EBPb蛋白质同种型之一的合成或周转是否有变化（图1 *， 研究C/EBPb的磷酸化是否在调节C/EBPb的作用中起重要作用。 它在氨基酸可用性的信号中发挥作用。我们的长期目标是了解哺乳动物细胞 通过基因表达对营养环境做出反应，使用氨基酸可用性作为模型。

Mammalian cells respond to nutritional limitation of protein/amino acids by increasing the expression of a wide spectrum of proteins via a signaling pathway that will be referred to as the Amino Acid Response (AAR). Although it is known that amino acid availability can modulate protein expression, the mechanisms by which these events occur are not well understood. Using human HepG2 hepatoma cells, we have documented previously that amino acid limitation increases the expression of the bZIP transcription factor C/EBPb through transcriptional control and that, in turn, an elevated level of C/EBPb induces transcription from amino acid responsive genes. Initial studies for this application have yielded the novel observation that there is amino acid response element (AARE) activity in a 93 bp fragment from the human C/EBPb gene 3' to the protein coding sequence. Preliminary experiments have also shown that amino acid limitation causes an increase in the total abundance of C/EBPb protein and an increase in the nuclear translocation of C/EBPb phosphorylated on Thr235. Our global hypothesis is that transcriptional control of human C/EBPb expression and post-translational control of C/EBPb function represent important regulatory steps in the AAR pathway. Using protein restricted diets in rats and amino acid limitation of HepG2 cells the Specific Aims of the proposed studies are to: 1) identify the genomic AARE responsible for amino acid-dependent transcription of the C/EBPb gene by using deletion analysis, in vivo footprinting, and single nucleotide mutagenesis; 2) identify the AARE binding proteins and characterize their role in the AAR pathway; 3) determine if there are changes in synthesis or turnover of one of the three C/EBPb protein isoforms (LAP*, LAP, LIP); and 4) investigate whether or not phosphorylation of C/EBPb is important in regulating the role that it plays in signaling amino acid availability. Our long-term goal is to understand how mammalian cells respond to their nutritional environment through gene expression, using amino acid availability as the model.