Understanding the mechanism of adaptor protein engagement by OGT and its functional effects on glycosylation

了解 OGT 与接头蛋白结合的机制及其对糖基化的功能影响

• 批准号: 10797591
• 负责人: Cassandra Marie Joiner
• 金额: $ 6.46万
• 依托单位: ST. OLAF COLLEGE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10797591
• 关键词: Active Sites; Adaptor Signaling Protein; Alzheimer' s Disease; Binding; Binding Proteins; Binding Sites; Biological Assay; Cell Extracts; Cell Nucleus; Cell physiology; Cytoplasm; Cytoplasmic Protein; Disease; Enzymes; Eukaryota; Event; Foundations; Future; Glucose; Goals; Individual; Libraries; Link; Malignant Neoplasms; Maps; Mediating; Methods; Modification; N-terminal; Nuclear Proteins; Nutrient; O-GlcNAc transferase; Pathway interactions; Protein Glycosylation; Proteins; Research; Role; Scaffolding Protein; Scientist; Solvents; Stress; Surface; Therapeutic; Therapeutic Intervention; experimental study; glycosylation; insight; interest; mutant; novel; protein protein interaction; screening; therapeutic development; therapeutically effective; tool; unnatural amino acids

PROJECT SUMMARY/ABSTRACT The identification of O-linked beta-N-acetylglucosamine (O-GlcNAc) modified proteins in the nucleus and cytoplasm overturned the paradigm that glycosylated proteins are only found in the secretory pathway of eukaryotes. Since then, O-GlcNAc modifications, installed by the O-GlcNAc transferase (OGT) enzyme, have been identified on proteins involved in almost all cellular processes. O-GlcNAc levels rise upon increase of glucose levels, and perturbations in protein O-GlcNAcylation has been implicated in diseases caused by protein misregulation, such as cancer and Alzheimer’s disease. It has been speculated that methods to regulate O- GlcNAcylation levels on targeted substrates would be therapeutically advantageous. To date, over one thousand protein targets have been identified, however the mechanisms by which OGT chooses those substrates eludes scientists, making it challenging to develop effective therapeutic interventions. Substrate selection does not occur at the active site of OGT. Instead, OGT’s N-terminal tetratricopeptide repeat (TPR) domain has been implicated in substrate selection through two proposed mechanisms, either through 1) intrinsic interactions with substrates and/or 2) interactions with substrates mediated by adaptor protein binding that alter OGT’s enzymatic activity. The TPR domain contains 13.5 repeats that form a unique superhelix with two 100 Å long binding surfaces, the concave, lumenal surface that has been implicated in direct substrate binding and a convex, solvent-exposed surface that we hypothesize engages non-substrate protein interactors, such as adaptors. While several studies have provided insights into intrinsic substrate binding, adaptor-mediated substrate selection mechanisms are poorly understood due to the limited tools for selectively capturing non-substrate interactions. I propose experiments to identify unique adaptor binding sites along the solvent-exposed surface of OGT’s TPR domain and to develop strategies to interrogate the role of adaptor interactions in OGT substrate selection. In Aim 1, we will use a library of photoactivatable unnatural amino acid (UAA)-containing OGT constructs to covalently capture known adaptor proteins and generate a map of adaptor binding sites along the solvent-exposed surface of the TPR domain. Additionally, we will use TPR mutants and glycotransferase assays to interrogate the functional consequences of disrupting the OGT-adaptor binding interfaces on the glycosylation of individual substrates. In Aim 2, we will use the same library of UAA-containing OGT constructs to covalently capture novel TPR-surface interactors from whole cell extracts and develop a two-step screening strategy to separate adaptor proteins that alter OGT’s activity towards protein substrates from scaffolding proteins that do not alter OGT’s substrate glycosylation activity upon binding. Results from this study will provide the first comprehensive map of non- substrate binding sites along the TPR domain and identify novel adaptor proteins for future mechanistic studies. This information will enable the advancement of new strategies to selectively interrogate O-GlcNAc’s role on specific substrates for future therapeutic applications.

项目概要/摘要 细胞核中 O-连接 β-N-乙酰氨基葡萄糖 (O-GlcNAc) 修饰蛋白的鉴定 细胞质颠覆了糖基化蛋白仅存在于细胞分泌途径中的范式 从那时起，由 O-GlcNAc 转移酶 (OGT) 进行的 O-GlcNAc 修饰已经出现。 已在几乎所有细胞过程中涉及的蛋白质中发现 O-GlcNAc 水平随 增加而增加。 葡萄糖水平和蛋白质 O-GlcNAcNA 酰化的扰动与蛋白质引起的疾病有关 据推测，调节 O- 的方法可能会导致癌症和阿尔茨海默病等疾病的失调。 迄今为止，目标底物上的 GlcNAc 酰化水平超过一千。 蛋白质靶标已被确定，但 OGT 选择这些底物的机制尚不清楚 科学家们认为，开发有效的治疗干预措施具有挑战性。 相反，OGT 的 N 端四肽重复 (TPR) 结构域与 OGT 的活性位点有关。 通过两种提议的机制进行底物选择，要么通过 1) 与底物的内在相互作用 和/或 2) 由接头蛋白结合介导的与底物的相互作用，改变 OGT 的酶活性。 TPR 结构域包含 13.5 个重复，形成独特的超螺旋，具有两个 100 Å 长的结合表面， 与直接底物结合有关的凹腔表面和暴露于溶剂的凸面 我们追求的表面涉及非底物蛋白质相互作用物，例如接头，同时进行了一些研究。 提供了对内在底物结合的见解，接头介导的底物选择机制是 我建议，由于选择性捕获非基质相互作用的工具有限，人们对其了解甚少。 鉴定沿 OGT TPR 结构域溶剂暴露表面的独特接头结合位点的实验 并制定策略来探讨接头相互作用在 OGT 底物选择中的作用。 将使用包含 OGT 构建体的光活化非天然氨基酸 (UAA) 库来共价捕获 已知的接头蛋白，并生成沿溶剂暴露表面的接头结合位点图 此外，我们将使用 TPR 突变体和糖转移酶测定来探究其功能。 破坏 OGT 接头结合界面对单个底物糖基化的影响。 目标 2，我们将使用相同的包含 UAA 的 OGT 构建体库来共价捕获新型 TPR 表面 从全细胞提取物中提取相互作用蛋白，并开发两步筛选策略来分离接头蛋白 通过不改变 OGT 底物的支架蛋白改变 OGT 对蛋白质底物的活性 这项研究的结果将提供第一个全面的非-结合图谱。 沿 TPR 结构域的底物结合位点，并鉴定用于未来机制研究的新型接头蛋白。 这些信息将促进新策略的进展，以选择性地探究 O-GlcNAc 在 未来治疗应用的特定底物。