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: 10513912
• 负责人: Cassandra Marie Joiner
• 金额: $ 32.13万
• 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/10513912
• 关键词: Active Sites; Adaptor Signaling Protein; Alzheimer' s Disease; Binding; Binding Proteins; Binding Sites; Biological Assay; Biology; Cell Extracts; Cell Nucleus; Cell physiology; Consensus Sequence; Cytoplasm; Cytoplasmic Protein; Development; Disease; Down-Regulation; Enzymes; Eukaryota; Event; Foundations; Future; Glucose; Goals; Hela Cells; Individual; Libraries; Link; Location; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Metabolic Diseases; Methods; Modification; N-terminal; Neurodegenerative Disorders; Nuclear Proteins; Nutrient; O-GlcNAc transferase; Pathway interactions; Pattern; Post-Translational Protein Processing; Protein Glycosylation; Proteins; Proteomics; Research; Role; Scaffolding Protein; Scientist; Serine; Site; Solvents; Stress; Structure; Surface; Therapeutic; Therapeutic Intervention; Threonine; Up-Regulation; experimental study; glycosylation; glycosyltransferase; insight; interest; mimetics; mutant; novel; protein protein interaction; screening; therapeutic development; therapeutic target; 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.

项目总结/摘要 细胞核中O-连接的β-N-乙酰葡糖胺（O-GlcNAc）修饰蛋白的鉴定， 细胞质推翻了糖基化蛋白质只存在于细胞分泌途径的范式。 真核生物从那时起，由O-GlcNAc转移酶（OGT）安装的O-GlcNAc修饰已经被发现。 在几乎所有细胞过程中的蛋白质上都有发现。O-GlcNAc水平在增加 葡萄糖水平和蛋白质O-GlcNAc酰化的扰动与蛋白质引起的疾病有关。 失调，如癌症和阿尔茨海默病。据推测，调节O- 靶向底物上的GlcNAc化水平将是治疗上有利的。迄今为止， 已经确定了蛋白质靶点，但是OGT选择这些底物的机制尚不清楚。 科学家，这使得开发有效的治疗干预措施具有挑战性。不进行底物选择 在OGT的活性位点。相反，OGT的N-末端三肽重复（TPR）结构域已被牵连 在底物选择中，通过两种提出的机制，或者通过1）与底物的内在相互作用 和/或2）通过改变OGT的酶活性的衔接蛋白结合介导的与底物的相互作用。 TPR结构域包含13.5个重复序列，形成一个独特的超螺旋，具有两个100 μ m长的结合表面， 凹的、内腔表面，其与直接的基质结合有关， 表面，我们假设从事非底物蛋白质相互作用，如衔接子。虽然一些研究 提供了对内在底物结合的见解，衔接子介导的底物选择机制， 由于用于选择性捕获非底物相互作用的工具有限，对其了解甚少。我提议 沿着OGT的TPR结构域的溶剂暴露表面沿着鉴定独特衔接子结合位点的实验 并制定策略，询问适配器相互作用的OGT底物选择的作用。目标1： 将使用含有光活化非天然氨基酸（UAA）的OGT构建体的文库来共价捕获 已知的接头蛋白，并产生接头结合位点的图谱，所述接头结合位点沿着所述接头蛋白的溶剂暴露表面。 TPR结构域。此外，我们将使用TPR突变体和糖基转移酶测定来询问功能性 破坏个别底物糖基化上的OGT-接头结合界面的后果。在 目的2，我们将使用相同的含UAA的OGT构建体库共价捕获新的TPR-表面， 并开发了一种两步筛选策略来分离接头蛋白， 从不改变OGT底物的支架蛋白改变OGT对蛋白底物的活性 糖基化活性。这项研究的结果将提供第一个全面的地图， 底物结合位点沿着TPR结构域，并确定新的衔接蛋白，为今后的机制研究。 该信息将使得能够推进新的策略以选择性地询问O-GlcNAc在以下方面的作用： 用于未来治疗应用的特定底物。