Nutrient Regulation of Cell Physiology by O-GlcNAcylation

O-GlcNAc 酰化对细胞生理学的营养调节

• 批准号: 10261390
• 负责人: GERALD Warren HART
• 金额: $ 28.71万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-10 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10261390
• 关键词: Affect; Aging; Alzheimer' s Disease; Binding; Binding Proteins; Biological Process; C-terminal; CRISPR/Cas technology; Cell Nucleus; Cell membrane; Cell physiology; Cells; Cellular Stress; Chemicals; Chromatin; Chronic Disease; Code; Complex; Cytoplasm; Cytoplasmic Protein; Cytosine; DNA; DNA Polymerase II; Development; Diabetes Mellitus; Disease; Eating; Enzymes; Etiology; Gene Expression; Genes; Genetic; Genetic Transcription; Glucose; Hela Cells; Human; Hyperglycemia; In Vitro; Insulin; Insulin Signaling Pathway; Kinetics; Light; Malignant Neoplasms; Messenger RNA; Metabolism; Methods; Mitochondrial Proteins; Modification; Molecular; Mus; Nuclear; Nuclear Proteins; Nutrient; O-GlcNAc transferase; Phosphorylation; Play; Post-Translational Protein Processing; Process; Proteins; RNA; RNA Polymerase II; Regulation; Role; Signal Transduction; Site; Site-Directed Mutagenesis; Stress; Surface; System; TAF1 gene; TATA-Box Binding Protein; Technology; Therapeutic; Time; Tissues; Transcription Elongation; Transcription Initiation; Work; aptamer; cell type; diabetic rat; dimer; improved; insulin signaling; link protein; novel; optogenetics; peptide O-linked N-acetylglucosamine-beta-N-acetylglucosaminidase; promoter; recruit; response; scaffold; sensor; sugar; tool; transcriptomics

PROJECT SUMMARY The cycling of N-acetylglucosamine on Ser(Thr) residues (O-GlcNAcylation; OGN) on nuclear, cytoplasmic and mitochondrial proteins serves as a nutrient sensor to regulate signaling, transcription, and cellular physiology. Abnormal OGN underlies the etiology of diabetes, cancer and Alzheimer's disease. OGN regulates nearly every aspect of transcription in response to nutrients. Great strides have been made in developing methods that elucidate the functions of OGN. While we can increase or decrease global OGN in cells, the greatest impediment toward a mechanistic understanding of OGN's functions is the lack of a method to alter OGN on a single protein without affecting the other thousands of OGN proteins within a cell. We discovered that the C-terminal domain of RNA polymerase II, which consists of 52 imperfect repeats of the sequence, YSPTSPS, is heavily OGN when it is not phosphorylated. OGN of the CTD is required for transcription initiation, is reciprocal with phosphorylation, and the sugar must be removed by O-GlcNAcase prior to elongation. While there have been many studies of the role of phosphorylation of the CTD in transcription, in contrast, there have been no studies of the specific roles of OGN on the CTD! We propose to develop an optogenetic approach to specifically target the O-GlcNAc transferase (OGT) to specific proteins. Our plan is to adapt the LOV2 light-inducible dimer (iLID) system. In this system, light-induced molecular association occurs on the sub-second time scale and reversion in the dark can occur within ten minutes. Initially, we will use iLID to investigate the roles of OGN in the insulin signaling pathway. OGT is normally targeted to its substrates by accessory proteins, among which are TET proteins, enzymes that hydroxymethylate cytosine residues, but also target OGT to chromatin. We will further investigate the roles of TET proteins in OGT actions on chromatin, particularly on the CTD of Pol II. Finally, we will study the roles of OGN on the CTD of RNA pol II in terms of its nutrient and stress responsiveness, and cell type differences in sites modified. We will elucidate the interactome of OGN-CTD and we will determine if OGN plays a role in RNA pol II pausing at promoters. These studies are not only elucidating molecular mechanisms of how nutrients regulate transcription, but they also are key to revealing how hyperglycemia, as occurs in diabetes, abnormally alters gene expression in many tissues. Molecular mechanisms revealed in these studies will likely lead to totally novel targets for the treatment of chronic diseases of aging, particularly diabetes.

项目摘要 N-乙酰葡萄糖在Ser（THR）残基（O-Glcnacylation; OGN）上的循环循环， 细胞质和线粒体蛋白是一种营养传感器，以调节信号传导， 转录和细胞生理。异常OGN是糖尿病病因，癌症的病因 和阿尔茨海默氏病。 OGN响应于转录的几乎各个方面 营养。在开发阐明功能的方法方面已经取得了长足的进步 OGN。虽然我们可以增加或减少细胞中的全局OGN，但最大的障碍是 对OGN功能的机械理解是缺乏改变单个OGN的方法 蛋白质不影响细胞内其他成千上万的OGN蛋白。 我们发现RNA聚合酶II的C末端结构域由52组成 序列不完美的重复YSPTSPS，当未磷酸化时会大量OGN。 CTD的OGN是转录启动所必需的，与磷酸化是相互的，并且 在伸长之前，必须通过O-Glcnacase除去糖。虽然有很多研究 相反，CTD在转录中的磷酸化作用，没有研究 OGN在CTD上的特定角色！ 我们建议开发一种光遗传学方法来专门针对O-GLCNAC 转移酶（OGT）到特定蛋白质。我们的计划是适应LOV2光诱导二聚体（ILID） 系统。在该系统中，光诱导的分子关联发生在亚秒时尺度上 黑暗中的恢复可以在十分钟之内发生。最初，我们将使用ILID调查 OGN在胰岛素信号通路中的作用。 OGT通常通过 辅助蛋白，其中包括TET蛋白，羟基甲酯胞嘧啶的酶 残留物，但也靶向OGT染色质。我们将进一步研究TET蛋白的作用 在OGT对染色质的作用中，特别是对POL II的CTD。最后，我们将研究 在RNA Pol II的CTD上，就其营养和压力反应性和细胞类型而言 修改站点的差异。我们将阐明OGN-CTD的相互作用组，我们将确定 如果OGN在RNA POL II在启动子中的暂停中起作用。 这些研究不仅阐明了营养如何调节的分子机制 转录，但它们也是揭示糖尿病中高血糖的关键， 异常改变许多组织中的基因表达。这些分子机制在其中揭示了 研究可能会导致完全新颖的目标来治疗慢性疾病的衰老， 特别是糖尿病。