Control of PGC1alpha Translation and Function

A.摘要 转录辅活化子pGc1α是我的团队在1998年发现的。它作为一种支配性的 共同激活多个核转录调控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。Pgc1α也有重要的组织 特殊功能，包括控制脂肪产热、肝脏中的空腹反应和线粒体 生物学与骨骼肌萎缩的抵抗力。激活脂肪和脂肪产热的机制 防止肌肉萎缩对人类代谢性疾病如糖尿病和 肥胖。初步数据显示，在培养的α细胞中，PGC1mRNA的翻译控制非常强大和新颖 细胞和体内；这种mRNA翻译受胰岛素和IGF1信号调节，通过AKT和 MTORC信令。此外，它还受到一个非常小的开放读数的存在的负面调节 框架(UORF)位于开始翻译规范的pGc1α1( 典型的前列环素α亚型，以下简称前列环素α。该uORF因缺失或突变而丢失 在抑制胰岛素/α效应的同时，增加PGC1IGF1mRNA的翻译。这个uORF编码一个 预测的由15个氨基酸组成的多肽，在所有哺乳动物物种中都高度保守。我们将开始这些 通过使用CRISPR技术(现已创建)的几种小鼠模型进行研究，这些模型可以增加或减少 通过改变这个小编码肽的起始密码子来表达这个uORF(目的1)。老鼠会成为 分析对动物新陈代谢和生理的关键方面的影响(目标2)。这些将包括能源 肝脏中热源性脂肪、糖异生对肥胖相关糖耐量异常的消耗和抵抗 以及肌肉中的运动耐力。由于骨骼肌及其萎缩是衰老的关键组成部分，因此 作为胰岛素作用的重要靶点，我们将研究肌肉选择性模型中的萎缩。机制，由 将在细胞中检测PGC1mRNA的5‘非编码区和uORF控制翻译，方法是确定是否有α UORF在顺式或反式中的作用通过两个质粒实验以及通过使用分子“脚印”和“足迹”来实现 化验(目标3)。细胞提取液中uORF多肽的存在将通过质谱学来确定 使用合成的“重”多肽作为关键的内部标准。此外，我们还将建立一个体外培养的 翻译系统，并确定这一调节是否可以在体外重述。关键的监管组成部分 该系统将通过已建立的使用寡核苷酸的亲和层析方法来分离。最后， 目标4将解决关键问题，即胰岛素/IGF1信号如何通过 胰岛素处理细胞中的定量磷蛋白质谱仪。磷酸蛋白质组学分析也将 应用于通过上述亲和方法隔离的组件。这些数据加在一起将 提供重要的视角和潜在的新的治疗靶点，通过线粒体 生物学、生理学和疾病过程可能在活体环境中被操纵。

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.