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.

项目摘要/摘要 核内O-GlcNAc修饰蛋白的鉴定 细胞质颠覆了糖基化蛋白只存在于细胞分泌途径的理论。 真核生物。从那时起，由O-GlcNAc转移酶(OGT)安装的O-GlcNAc修饰已经 在几乎所有细胞过程中涉及的蛋白质上都被鉴定出来。O-GlcNAc水平随 血糖水平和蛋白质O-GlcN酰化的紊乱与蛋白质引起的疾病有关 调控不当，如癌症和阿尔茨海默氏症。据推测，调节O-的方法可能是 靶向底物上的GlcN酰化水平在治疗上是有利的。到目前为止，已有1000多人 蛋白质靶标已经确定，然而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。未发生底物选择 在OGT的活动地点。相反，OGT的N端四肽重复(TPR)结构域已被牵连 在底物选择中，通过两种建议的机制，通过1)与底物的内在相互作用 和/或2)通过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，形成一个独特的超螺旋，具有两个100？长的结合表面，即 凹面，管腔表面，与直接基材结合有关，凸起，暴露在溶剂中 我们假设的表面参与了非底物蛋白质相互作用，如接头。虽然有几项研究 提供了对内在底物结合的洞察，适配器介导的底物选择机制是 由于选择性捕捉非底物相互作用的工具有限，人们对此知之甚少。我建议 沿着OGT TPR结构域的溶剂暴露表面识别独特的接头结合位点的实验 并开发策略来询问适配器相互作用在OGT底物选择中的作用。在目标1中，我们 将使用包含光活化非天然氨基酸(UAA)的OGT构建体库来共价捕获 已知的适配器蛋白，并沿着暴露于溶剂的表面生成适配器结合点图 TPR域。此外，我们将使用TPR突变体和糖转移酶分析来询问功能 破坏OGT-适配器结合界面对个别底物糖基化的影响。在……里面 目标2，我们将使用相同的含有UAA的OGT构建库来共价捕获新的TPR表面 从整个细胞中提取相互作用蛋白，并开发一种两步筛选策略来分离 从不改变OGT底物的支架蛋白改变OGT对蛋白质底物的活性 结合时的糖基化活性。这项研究的结果将提供第一个全面的非 TPR结构域上的底物结合位点，并为未来的机制研究鉴定新的接头蛋白。 这一信息将使新的战略得以推进，以选择性地询问O-GlcNAc在 用于未来治疗应用的特定底物。