Control of PGC1alpha Translation and Function

PGC1alpha 翻译和功能的控制

• 批准号: 10341051
• 负责人: BRUCE M. SPIEGELMAN
• 金额: $ 58.22万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2023-07-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10341051
• 关键词: 5' Untranslated Regions; Address; Adipose tissue; Affect; Affinity; Affinity Chromatography; Aging; Amino Acids; Animals; Atrophic; Binding; Biogenesis; Biological Assay; Biology; Brain; CRISPR/Cas technology; Cell Extracts; Cell Respiration; Cells; Codon Nucleotides; Cultured Cells; Data; Defect; Diabetes Mellitus; Disease; Elements; Energy Metabolism; Exercise; Exercise Tolerance; Fasting; Fatty acid glycerol esters; Gene Expression; Genetic Models; Genetic Translation; Gluconeogenesis; Glucose Intolerance; Hormones; Human; IGF1 gene; In Vitro; Initiator Codon; Insulin; KRP protein; Link; Liver; Liver Mitochondria; Mass Spectrum Analysis; Measures; Messenger RNA; Metabolic Diseases; Metabolism; Methods; Mitochondria; Modeling; Molecular; Mus; Muscle; Mutation; Nerve Degeneration; Neuromuscular Diseases; Non-Insulin-Dependent Diabetes Mellitus; Nuclear; Obesity; Oligonucleotides; Open Reading Frames; Parkinson Disease; Pathogenesis; Peptides; Phosphoproteins; Physiology; Plasmids; Play; Post-Transcriptional Regulation; Post-Translational Protein Processing; Process; Protein Isoforms; Proteins; Proto-Oncogene Proteins c-akt; Regulation; Reporting; Resistance; Ribosomes; Series; Set protein; Signal Transduction; Skeletal Muscle; Specificity; System; Therapeutic; Thermogenesis; Tissues; Transcription Coactivator; Transfection; Translations; Untranslated Regions; Work; experimental study; genetic manipulation; human disease; improved; in vivo; mouse model; new therapeutic target; novel; novel therapeutics; phosphoproteomics; prevent; programs; response; transcription factor; translational impact

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.

一个。抽象的 转录共激活因子PGC1α在1998年被我的组发现。它起着主要作用 线粒体生物发生和氧化代谢的调节剂，通过激活几个核转录 控制线粒体基因表达的广泛程序的因素。 PGC1α还具有重要的组织 特定功能，包括控制脂肪热发生，肝脏中的禁食反应和线粒体 生物学和骨骼肌萎缩的抗性。激活脂肪中生热的机制 预防肌肉萎缩在人类代谢疾病（例如糖尿病和）中至关重要 肥胖。初步数据说明了培养的PGC1αmRNA的非常健壮和新颖的翻译控制 细胞和体内；这种mRNA翻译受胰岛素和IGF1信号的调节， MTORC信号。此外，它受到很小的开放阅读的负面调节 框架（UORF）只是密码子的上游，该密码子开始翻译的PGC1α1（ 规范PGC1α同工型； Herelafter刚刚称为PGC1α）mRNA。因删除或突变而丢失此UORF 在消融胰岛素/IGF1效应的同时，增加了PGC1αmRNA的翻译。这个uorf编码一个 预测15种氨基酸的肽，在所有哺乳动物物种中都很保存。我们将开始这些 通过使用CRISPR技术（现已创建）的几种鼠标模型进行研究，以增加或减少 通过改变该小型编码肽的起始密码子来表达该UORF（AIM 1）。老鼠会 分析了对动物代谢和生理学关键方面的影响（AIM 2）。这些将包括能量 通过热脂肪脂肪，肝脏中的糖异生的支出和抗肥胖连接的葡萄糖耐药性 和肌肉运动耐受性。由于骨骼肌及其萎缩是衰老的关键组成部分 胰岛素作用的重要靶标，我们将检查肌肉选择模型中的萎缩。机制 在细胞中将检查PGC1αmRNA的5'UTR和UORF控制翻译。 UORF在顺式或通过2个质粒实验以及通过使用分子“ toprint”和“ footprint”的函数 测定（目标3）。细胞提取物中UORF胡椒的存在将由质谱法确定 使用合成“重”肽作为关键内部标准。此外，我们将建立一个体外 翻译系统并确定该调节是否可以在体外概括。关键的监管组件 该系统将通过使用寡核苷酸的建立亲和色谱法隔离。最后， AIM 4将解决一个关键问题，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法。磷酸化的分析也将 应用于通过上述亲和力方法分离的组件。这些数据将在一起 提供关键的观点和潜在的新治疗靶标的线粒体 在体内环境中可能会操纵生物学，生理和疾病过程。