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.

项目总结 N-乙酰氨基葡萄糖在核上丝氨酸(Thr)残基(O-GlcN酰化；OGN)上的循环， 细胞质和线粒体蛋白质作为营养感受器调节信号， 转录和细胞生理学。OGN异常是糖尿病、癌症病因的基础 和阿尔茨海默氏症。OGN几乎调控转录的每一个方面，以响应 营养素。在阐明细胞因子功能的方法方面取得了长足的进步。 OGN.虽然我们可以增加或减少细胞中的全局OGN，但实现 对OGN功能的机械性理解是缺乏一种方法来在单个 在不影响细胞内其他数千种OGN蛋白质的情况下。 我们发现RNA聚合酶II的C-末端结构域由52个 序列的不完全重复YSPTSPS在没有被磷酸化时是严重的OGN。 CTD的OGN是转录启动所必需的，与磷酸化是相互作用的，并且 在伸长之前，糖必须被O-GlcNAcase除去。虽然已经有很多研究 关于CTD的磷酸化在转录中的作用，相比之下，还没有研究 OGN在CTD上的具体角色！ 我们建议开发一种光遗传学方法来特异性地靶向O-GlcNAc 转移酶(OGT)到特定的蛋白质。我们的计划是改造LOV2光诱导二聚体(ILID) 系统。在这个系统中，光诱导的分子缔合发生在亚秒级的时间尺度上 而黑暗中的回归可以在十分钟内发生。首先，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，其中有Tet蛋白，即羟甲基化胞嘧啶的酶 残基，但也靶向OGT到染色质。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。最后，我们将研究OGT对染色质的作用 OGN对RNA polII CTD的营养和应激反应以及细胞类型的影响 已修改站点的差异。我们将阐明OGN-CTD的相互作用组并确定 如果OGN在RNA PolII中发挥作用，则在启动子处暂停。 这些研究不仅阐明了营养物质如何调节的分子机制 转录，但它们也是揭示高血糖如何发生在糖尿病中的关键， 异常改变了许多组织中的基因表达。其中揭示的分子机制 研究可能会为治疗慢性老年性疾病带来全新的靶点， 尤其是糖尿病。