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 O连锁β-N-乙酰葡萄糖胺（O-GLCNAC）在细胞核中修饰的蛋白质的鉴定 细胞质推翻了糖基化蛋白仅在秘密途径中发现的范式 真核生物。从那时起，由O-GlCNAC转移酶（OGT）酶安装的O-GLCNAC修饰具有 在几乎所有细胞过程的蛋白质上都鉴定出来。 O-GLCNAC水平增加 葡萄糖水平和蛋白O-Glcnacylation的扰动已在蛋白质引起的疾病中浸渍 癌症和阿尔茨海默氏病等正直。已经推测，调节O-的方法 靶向底物上的Glcnacylation水平将具有热优势。迄今为止，超过一千 已经确定了蛋白质靶标，但是OGT选择这些底物的机制 科学家们，挑战开发有效的治疗干预措施。底物选择不会发生 在OGT的主动地点。相反，OGT的N末端四肽重复（TPR）域已被暗示 通过两种提出的机制选择底物，要么通过1）与底物的内在相互作用 和/或2）与改变OGT酶促活性的衔接蛋白结合介导的底物的相互作用。 TPR结构域包含13.5重复，形成一个独特的超螺旋体，具有两个100Å长绑定表面， 凹入的液体表面，在直接底物结合和凸面，溶剂暴露 我们假设的表面与非覆盖蛋白相互作用者（例如衔接子）参与。而几次研究 已经提供了有关固有底物结合的见解，适应器介导的底物选择机制是 由于有限的工具可选择性地捕获非底层相互作用，因此理解不足。我建议 实验沿OGT的TPR域的溶液暴露表面识别独特的适配器结合位点 并制定策略来询问适配器相互作用在OGT底物选择中的作用。在AIM 1中，我们 将使用光活化不自然的氨基酸（UAA）的OGT构建库来共价捕获 已知的衔接蛋白，并沿着溶液暴露的表面生成适配器结合位点的图 TPR域。此外，我们将使用TPR突变体和糖转移酶测定法来询问功能 破坏OGT适应器结合界面对单个底物糖基化的后果。在 AIM 2，我们将使用相同的含UAA的OGT构造库来共价捕获新型TPR-Surface 来自全细胞提取物的相互作用者，并制定了两步筛选策略，以分离衔接蛋白 改变了OGT从不会改变OGT底物的脚手架蛋白质对蛋白质底物的活性 结合后糖基化活性。这项研究的结果将提供第一个非 - 沿TPR结构域的底物结合位点，并确定新型的衔接蛋白，以进行未来的机械研究。 这些信息将使新策略的进步有选择地询问O-GLCNAC在 未来治疗应用的特定底物。