Targeting and Regulation of O-GlcNAc Transferase at M Phase

M 期 O-GlcNAc 转移酶的靶向和调节

• 批准号: 9100880
• 负责人: Chad Eric Slawson
• 金额: $ 22.65万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9100880
• 关键词: Affinity; Amino Acids; Aneuploidy; Binding; Biological; Cell Cycle Progression; Cells; Centrosome; Complex; Coupled; Cytokinesis; Cytoplasmic Protein; Data; Development; Disease; Environment; Enzymes; Eukaryotic Cell; Goals; Grant; Growth; Holoenzymes; Hormones; Image; Kinetics; Laboratories; Lead; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Mitosis; Mitotic; Mitotic spindle; Modification; Molecular; Nuclear Proteins; Nutrient; O-GlcNAc transferase; Oligosaccharides; Outcome; Phenotype; Phosphotransferases; Post-Translational Protein Processing; Proteins; Proteomics; Recovery; Regulation; Research; Role; Serine; Signal Transduction; Solid Neoplasm; Source; Stress; Structure; Techniques; Testing; Threonine; Time; UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase; aurora B kinase; base; cancer therapy; domain mapping; extracellular; gain of function; insight; knock-down; novel; overexpression; poly-N-acetyl glucosamine; protein expression; sugar; tumor progression

O-GlcNAc is the covalent modification of proteins at serine/threonine amino acids by the sugar moiety Nacetylglucosamine. The O-GlcNAc modification is not elongated to more complex oligosaccharide structures and is found only on nuclear and cytoplasmic proteins. The modification is highly dynamic and responds to signals of the extracellular environment such hormones, stress, and nutrients. Previously, I demonstrated that 0-GlcNAc is critical for the proper progression of the cell cycle in eukaryotic cells; gain of function of either 0-GlcNAc transferase (OGT), the enzyme which adds the modification, or 0-GlcNAcase (OGA), the enzyme which removes the modification, causes mitotic exit delays. Furthermore, OGT and OGA can be found in a signaling complex with mitotic kinases such as Aurora Kinase B. The goal of this proposal is to investigate the interactions of OGT with mitotic structures such as the spindle and with mitotic proteins. We hypothesize that OGT forms complexes with various proteins during M phase progression that then targets OGT the spindle and midbody. I plan to use multiple techniques such as using FRAP (Fluorescent Recovery after Photo-bleaching) on GFP-OGT to measure the kinetics of OGT at spindle/midbody, quantitative proteomics to identify OGT targeting proteins, and finally identifying the function of Aurora Kinase B in targeting OGT to mitotic substrates and structures. Our expected contribution is significant because we expect to find insight into the regulation of OGT by mitotic targeting proteins and how these interactions are uncoupled in diseases such as cancer.

O-GlcNAc是糖基N-乙酰氨基葡萄糖对蛋白质丝氨酸/苏氨酸氨基酸的共价修饰。O-GlcNAc修饰没有延伸到更复杂的寡糖结构，只在核蛋白和细胞质蛋白上发现。这种修饰是高度动态的，并对细胞外环境的信号做出反应，如激素、压力和营养物质。以前，我证明了0-GlcNAc对于真核细胞中细胞周期的正常进行是至关重要的；获得 无论是添加修饰的O-GlcNAc转移酶(OGT)还是去除修饰的O-GlcNAcase(OGA)，都会导致有丝分裂退出延迟。此外，OGT和OGA可以在含有有丝分裂酶的信号复合体中被发现，如Aurora Kinase B。本研究的目的是研究OGT与纺锤体等有丝分裂结构以及与有丝分裂蛋白的相互作用。我们 假设OGT在M期进程中与各种蛋白质形成复合体，然后以OGT、纺锤体和中体为靶点。我计划使用多种技术，如在GFP-OGT上使用FRAP(光漂白后荧光恢复)来测量OGT在纺锤体/中体的动力学，使用定量蛋白质组学来确定OGT靶向蛋白，最后确定Aurora Kinase B在靶向OGT有丝分裂底物和结构中的功能。我们的预期贡献是巨大的，因为我们 期待深入了解有丝分裂靶向蛋白对OGT的调控，以及这些相互作用在癌症等疾病中是如何解偶联的。