Nutritional Control of Transcription Factor Expression

哺乳动物细胞通过增加蛋白/氨基酸的表达来应对蛋白质/氨基酸的营养限制 通过一种称为氨基酸反应的信号通路获得广泛的蛋白质 (AAR)。虽然已知氨基酸的可用性可以调节蛋白质的表达，但其机制 这些事件是如何发生的还没有被很好地理解。使用人类HepG2肝癌细胞，我们有 先前的文献证明，限制氨基酸会增加bZIP转录因子的表达 C/EBPB通过转录调控，而C/EBPB水平的升高又诱导转录 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 人C/EBPB基因3‘端93个碱基片段具有氨基酸反应元件(AARE)活性 到蛋白质编码序列。初步实验还表明，氨基酸限制会导致 C/EBPb蛋白总丰度增加和C/EBPb核转位增加 在Thr235上被磷酸化。我们的全球假设是人类C/EBPB的转录控制 C/EBPB功能的表达和翻译后调控是AAR中的重要调控步骤 路径。利用大鼠蛋白质限制饮食和HepG2细胞的氨基酸限制，具体目的 建议的研究是：1)确定与氨基酸依赖有关的基因组AARs 利用缺失分析、活体足迹和单核苷酸转录C/EBPB基因 诱变；2)鉴定AARE结合蛋白并鉴定它们在AAR途径中的作用； 确定三种C/EBPB蛋白异构体之一的合成或周转是否发生变化(LAP*， LAP，LIP)；以及4)研究C/EBPB的磷酸化是否在调节作用中起重要作用 它在发出氨基酸可获得性信号方面发挥了作用。我们的长期目标是了解哺乳动物细胞是如何 以氨基酸可利用性为模型，通过基因表达对其营养环境做出反应。

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.