Nutritional Control of Transcription Factor Expression

描述(由申请人提供)：哺乳动物细胞通过一种称为氨基酸反应(AAR)的信号通路增加一系列蛋白质的表达，以应对蛋白质/氨基酸的营养限制。虽然已知氨基酸的可获得性可以调节蛋白质的表达，但这些事件发生的机制尚不清楚。利用人肝癌细胞，我们已经证明了氨基酸限制通过转录控制增加了bZIP转录因子C/EBPB的表达，而C/EBPB水平的升高反过来又诱导了氨基酸反应基因的转录。对这一应用的初步研究发现，从人类C/EBPB基因3‘到蛋白质编码序列的93个碱基片段中存在氨基酸反应元件(AARE)活性。初步实验还表明，氨基酸限制会导致C/EBPB蛋白总丰度增加，并导致Thr235上C/EBPB磷酸化的核转位增加。我们的总体假设是，对人类C/EBPB表达的转录控制和翻译后对C/EBPB功能的控制是AAR途径中的重要调控步骤。利用大鼠的蛋白质限制饮食和HepG2细胞的氨基酸限制，建议的研究的具体目的是：1)通过缺失分析、体内足迹和单核苷酸突变来确定基因组中负责C/EBPB基因氨基酸依赖的转录的A；2)确定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蛋白亚型(LAP*、LAP、LIP)之一的合成或周转是否发生变化；以及4)研究C/EBPB的磷酸化是否在调节其在氨基酸可获得性信号中所起的作用方面起重要作用。我们的长期目标是以氨基酸可获得性为模型，了解哺乳动物细胞如何通过基因表达对其营养环境做出反应。

DESCRIPTION (provided by applicant): 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.