Nutrient Regulation of Cell Physiology by O-GlcNAcylation

O-GlcNAc 酰化对细胞生理学的营养调节

基本信息

批准号：
10668984
负责人：
GERALD Warren HART
金额：
$ 28.72万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
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/10668984
关键词：
Affect Aging Alzheimer&apos 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 Darkness 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 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 metabolomics novel optogenetics peptide O-linked N-acetylglucosamine-beta-N-acetylglucosaminidase promoter recruit response scaffold sensor sugar tool transcriptomics

项目摘要

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.

项目概要 N-乙酰氨基葡萄糖在核上 Ser(Thr) 残基上的循环（O-GlcNAcylation；OGN），细胞质和线粒体蛋白作为营养传感器来调节信号传导，转录和细胞生理学。 OGN 异常是糖尿病、癌症的病因和阿尔茨海默病。 OGN 调节转录的几乎每个方面营养素。在开发阐明其功能的方法方面已经取得了长足的进步奥格恩。虽然我们可以增加或减少细胞中的整体 OGN，但最大的障碍是对 OGN 功能的机械理解缺乏一种方法来改变 OGN 的单一功能蛋白质，而不影响细胞内其他数千个 OGN 蛋白质。我们发现RNA聚合酶II的C端结构域由52个组成序列的不完美重复 YSPTSPS 在未磷酸化时严重 OGN。 CTD 的 OGN 是转录起始所必需的，与磷酸化作用相反，并且在延伸之前，糖必须被 O-GlcNAcase 去除。虽然已经有很多研究 CTD磷酸化在转录中的作用，相比之下，还没有研究 OGN在CTD上的具体作用！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。我们的计划是采用 LOV2 光诱导二聚体 (iLID) 系统。在该系统中，光诱导的分子缔合发生在亚秒级的时间尺度上黑暗中的逆转可在十分钟内发生。最初，我们将使用 iLID 来调查 OGN 在胰岛素信号通路中的作用。 OGT 通常通过以下方式靶向其底物：辅助蛋白，其中包括 TET 蛋白、羟甲基化胞嘧啶的酶残基，而且还将 OGT 靶向染色质。我们将进一步研究TET蛋白的作用 OGT 对染色质的作用，特别是对 Pol II 的 CTD 的作用。最后，我们将研究以下角色： OGN 对 RNA pol II 营养和应激反应性以及细胞类型的 CTD 修改站点的差异。我们将阐明 OGN-CTD 的相互作用组，并确定如果 OGN 在 RNA pol II 在启动子处暂停中发挥作用。这些研究不仅阐明了营养物质调节的分子机制转录，但它们也是揭示高血糖（如糖尿病中发生的）如何发生的关键异常改变许多组织中的基因表达。这些揭示的分子机制研究可能会产生治疗慢性衰老疾病的全新靶标，特别是糖尿病。