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 是我的团队于 1998 年发现的。它的功能是作为显性基因 通过共激活多个核转录来调节线粒体生物合成和氧化代谢 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 特定功能，包括控制脂肪产热、肝脏的禁食反应和线粒体 骨骼肌的生物学和抗萎缩能力。激活脂肪产热的机制 防止肌肉萎缩对于糖尿病等人类代谢疾病非常重要 肥胖。初步数据说明了培养物中 PGC1α mRNA 的非常稳健且新颖的翻译控制 细胞和体内；该 mRNA 翻译由胰岛素和 IGF1 信号通过 AKT 调节， mTORC 信号传导。此外，它受到非常小的开放阅读的存在的负面调节。 框架 (uORF) 位于开始翻译规范 PGC1α1 的密码子的上游（ 典型的 PGC1α 亚型；以下简称为 PGC1α) mRNA。该 uORF 因缺失或突变而丢失 增加 PGC1α mRNA 的翻译，同时消除胰岛素/IGF1 效应。这个 uORF 编码了一个 预测的 15 个氨基酸肽在所有哺乳动物物种中都高度保守。我们将开始这些 通过使用几种使用 CRISPR 技术（现已创建）的小鼠模型进行研究，这些模型会增加或减少 通过改变这个小编码肽的起始密码子来表达这个uORF（目标1）。老鼠将会 分析对动物代谢和生理学关键方面的影响（目标 2）。这些将包括能源 通过产热脂肪、肝脏糖异生来消耗和抵抗肥胖相关的葡萄糖不耐受 和肌肉的运动耐力。由于骨骼肌及其萎缩是衰老和衰老的关键组成部分 胰岛素作用的重要目标，我们将检查肌肉选择性模型中的萎缩。机制由 通过确定是否在细胞中检查 PGC1α mRNA 的 5' UTR 和 uORF 控制翻译 uORF 通过 2 个质粒实验并通过使用分子“脚趾印”和“足迹”以顺式或反式发挥作用 分析（目标 3）。细胞提取物中 uORF 肽的存在将通过质谱法确定 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现。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来分离。最后， 目标 4 将解决胰岛素/IGF1 信号如何通过以下方式影响这种翻译控制的关键问题： 胰岛素处理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。这些数据一起将 提供重要的观点和潜在的新治疗靶点，通过线粒体 生物学、生理学和疾病过程可能在体内环境中被操纵。