Nutritional Control of Transcription Factor Expression

描述（由申请人提供）：哺乳动物细胞对蛋白质/氨基酸的营养限制作出反应，通过被称为氨基酸反应（AAR）的信号通路增加广谱蛋白质的表达。虽然已知氨基酸可用性可以调节蛋白质表达，但这些事件发生的机制尚不清楚。利用人HepG2肝癌细胞，我们之前已经证明，氨基酸限制通过转录控制增加bZIP转录因子C/EBPb的表达，反过来，C/EBPb水平升高诱导氨基酸应答基因的转录。该应用程序的初步研究已经产生了新的观察结果，即从人类C/EBPb基因3'到蛋白质编码序列的93 bp片段中存在氨基酸反应元件（AARE）活性。初步实验还表明，氨基酸限制导致C/EBPb蛋白总丰度增加，Thr235磷酸化的C/EBPb核易位增加。我们的总体假设是，人类C/EBPb表达的转录控制和C/EBPb功能的翻译后控制是AAR途径的重要调控步骤。利用大鼠蛋白质限制饮食和HepG2细胞的氨基酸限制，拟开展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诱变确定负责C/EBPb基因氨基酸依赖性转录的基因组AARE；2)鉴定AARE结合蛋白并表征其在AAR通路中的作用；3)确定三种C/EBPb蛋白亚型（LAP*、LAP、LIP）中的一种的合成或转换是否发生变化；4)研究C/EBPb磷酸化在调节其在氨基酸可用性信号传导中的作用是否重要。我们的长期目标是了解哺乳动物细胞如何通过基因表达对其营养环境作出反应，以氨基酸可用性为模型。

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.