Control of PGC1alpha Translation and Function

a.摘要 转录辅激活因子PGC 1 α是本课题组在1998年发现的。它的功能是一种支配性的 通过共激活几种核转录来调节线粒体生物发生和氧化代谢 控制线粒体基因表达的广泛程序的因素。PGC 1 α还具有重要的组织 特定的功能，包括控制脂肪产热，肝脏的禁食反应，和线粒体 生物学和抵抗骨骼肌萎缩。激活脂肪产热的机制， 预防肌肉萎缩在人类代谢疾病如糖尿病和 肥胖初步数据表明，在培养的细胞中，PGC 1 α mRNA的翻译控制非常稳健和新颖。 细胞和体内;这种mRNA翻译由胰岛素和IGF 1信号通过AKT调节， mTORC信号传导。而且，它是负调控的存在一个非常小的开放阅读 在开始翻译经典PGC 1 α1的密码子的上游（ 典型的PGC 1 α同种型;以下简称PGC 1 α）mRNA。由于缺失或突变导致的uORF缺失 增加PGC 1 α mRNA的翻译，同时消除胰岛素/IGF 1效应。这个uORF编码一个 在所有哺乳动物物种中高度保守的15个氨基酸的预测肽。我们将开始这些 使用CRISPR技术（现已创建）的几种小鼠模型进行研究， 通过改变该小编码肽的起始密码子来表达该uORF（Aim 1）。小鼠将被 分析对动物代谢和生理学关键方面的影响（目的2）。其中包括能源 通过产热脂肪和肝脏中的脂肪异生来消耗和抵抗肥胖相关的葡萄糖耐受不良 和运动耐量。由于骨骼肌及其萎缩是衰老的关键组成部分， 作为胰岛素作用的重要靶点，我们将在肌肉选择性模型中检查萎缩。机制 将通过确定细胞中PGC 1 α mRNA的5' UTR和uORF控制翻译， 通过2个质粒实验和使用分子“足印”和“足迹”，uORF以顺式或反式发挥功能 测定（目的3）。将通过质谱法测定细胞提取物中uORF肽的存在 使用合成的“重”肽作为关键的内标。此外，我们将建立一个体外 翻译系统，并确定这种调节是否可以在体外重演。关键监管组成部分 该系统将通过使用寡核苷酸的已建立的亲和层析方法分离。最后， 目的4将解决胰岛素/IGF 1信号传导如何影响这种翻译控制的关键问题， 胰岛素处理的细胞中的定量磷蛋白质谱。磷酸化蛋白质组学分析也将 应用于通过上述亲和方法分离的组分。这些数据将 提供了至关重要的观点和潜在的新的治疗靶点， 生物学、生理学和疾病过程可以在体内环境中操纵。

a. Abstract The transcriptional coactivator PGC1α was discovered by my group in 1998. It functions as a dominant regulator of mitochondrial biogenesis and oxidative metabolism by coactivating several nuclear transcription factors that control the broad program of mitochondrial gene expression. PGC1α also has important tissue specific functions, including control of adipose thermogenesis, the fasting response in liver, and mitochondrial biology and resistance to atrophy in skeletal muscle. Mechanisms that activate thermogenesis in fat and prevent atrophy in muscle are of enormous importance in human metabolic diseases such as diabetes and obesity. Preliminary data illustrates a very robust and novel translational control of PGC1α mRNA in cultured cells and in vivo; this mRNA translation is regulated by insulin and IGF1 signaling through AKT and mTORC signaling. Moreover, it is negatively regulated by the presence of a very small open-reading frame (uORF) just upstream of the codon that begins translation of the canonical PGC1α1 (the canonical PGC1α isoform; hereafter just called PGC1α) mRNA. Loss of this uORF by deletion or mutation increases the translation of PGC1α mRNA while ablating the insulin/IGF1 effect. This uORF encodes a predicted peptide of 15 amino acids that is strongly conserved in all mammalian species. We will begin these studies by using several mouse models using CRISPR technology (now created) which increase or decrease expression of this uORF by altering the start codon of this small encoded peptide (Aim 1). Mice will be analyzed for effects on key aspects of animal metabolism and physiology (Aim 2). These will include energy expenditure and resistance to obesity-linked glucose intolerance via thermogenic fat, gluconeogenesis in liver and exercise tolerance in muscle. Since skeletal muscle and its atrophy is a critical component of aging and an important target of insulin action, we will examine atrophy in the muscle-selective models. Mechanisms by which the 5' UTR and uORF control translation of PGC1α mRNA will be examined in cells by determining if the uORF functions in cis or trans via 2 plasmid experiments and through use of molecular “toeprint” and “footprint” assays (Aim 3). The presence of the uORF peptide in cell extracts will be determined by Mass Spectrometry with the use of synthetic “heavy” peptides as key internal standards. Moreover, we will set up an in vitro translation system and determine if this regulation can be recapitulated in vitro. Key regulatory components of this system will be isolated by established affinity chromatography methods using oligonucleotides. Finally, Aim 4 will address the critical question of how insulin/IGF1 signaling impacts this translational control through quantitative phosphoprotein Mass Spectrometry in insulin treated cells. Phospho-proteomic analyses will also be applied to components isolated through the affinity methods described above. Together, these data will provide crucial perspectives and potential new therapeutic targets through which mitochondrial biology, physiology and disease processes might be manipulated in in vivo settings.