Regulation of Translation by O-GlcNAc - Resubmission 03-05-2020

N-乙酰葡糖胺在核上的Ser（Thr）残基上的循环（O-GlcNAc酰化; OGN）， 细胞质和线粒体蛋白质作为营养传感器调节信号传导、转录， 细胞生理学异常OGN是糖尿病、癌症和阿尔茨海默病的病因学基础。OGN 几乎调控转录的各个方面，包括RNA聚合酶II、组蛋白、DNA 甲基转移酶和几乎所有的转录因子。我们和其他人最近的研究结果表明，O- GlcNAc化也调节蛋白质翻译和mRNA利用，但知之甚少。 了解营养物质和压力如何通过OGN调节蛋白质翻译不仅对我们的基础研究至关重要， 了解细胞最重要的过程之一，也是了解机制的关键 潜在的慢性衰老疾病，如糖尿病、癌症和神经退行性疾病。 我们推测，O-GlcNAc对翻译机制中蛋白质的循环调节蛋白质稳态 通过介导蛋白酶体和核糖体机制之间的通讯， 翻译速率和mRNA选择通过动态O-GlcNAc循环对许多核糖体相关蛋白。 我们提出了三个具体的目标，以促进我们对OGN在营养调控中的作用的理解。 目标1将使用最先进的质谱方法来识别新生和成熟的 核糖体相关蛋白和翻译因子被OGN修饰，我们将特别 重点放在那些似乎参与核糖体：蛋白酶体通讯。然后我们将决定 OGN在所选OGN翻译蛋白上的位点水平的功能。目标2将阐明 葡萄糖改变了翻译机器的OGN。使用活HEK 293细胞和兔网织红细胞 翻译系统，我们将通过进行RNA测序分析来确定OGN是否在mRNA选择中起作用。 多聚核糖体制剂。目的3将确定O-GlcNAc转移酶（OGT） 和O-GlcNAcase（OGA）响应于蛋白酶体抑制而快速靶向核糖体。 这些研究不仅阐明了营养物质如何调节蛋白质合成的分子机制， 但它们也是揭示糖尿病中高血糖如何异常改变蛋白质的关键 在许多组织中表达。这些研究揭示的分子机制可能会导致全新的 用于治疗慢性衰老疾病，特别是糖尿病的靶点。

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, including RNA polymerase II, histones, DNA methyltransferases, and nearly all transcription factors. Recent findings by us and others indicate that O- GlcNAcylation also regulates protein translation and mRNA utilization, but much less is known. Understanding how nutrients and stress regulate protein translation via OGN is not only critical to our basic understanding of one of the cell’s most vital processes, but also is key to understanding mechanisms underlying chronic diseases of aging, such as diabetes, cancer and neurodegeneration. We hypothesize that O-GlcNAc cycling on proteins in the translational machinery regulates proteostasis by mediating communication between the proteasome and ribosomal machinery, and that nutrients regulate translation rates and mRNA selection by dynamic O-GlcNAc cycling on many ribosome-associated proteins. We propose three specific aims to advance our understanding of OGN’s roles in nutrient regulation of translation: Aim 1 will use state-of-the-art mass spectrometric methods to identify both nascent and mature ribosome associated proteins and translation factors that are modified by OGN, and we will specifically focus on those that appear to be involved in ribosome:proteasome communication. We will then determine the functions of OGN at the site level on selected OGN translation proteins. Aim 2 will elucidate how high glucose alters the OGN of the translation machinery. Using both live HEK293 cells and a rabbit reticulocyte translation system, we will determine if OGN plays a role in mRNA selection by performing RNA seq analyses of polysome preparations. Aim 3 will determine the mechanisms by which the O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) are rapidly targeted to ribosomes in response to proteasome inhibition. These studies are not only elucidating molecular mechanisms of how nutrients regulate protein synthesis, but they also are key to revealing how hyperglycemia, as occurs in diabetes, abnormally alters protein 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.