Targeting and Regulation of O-GlcNAc Transferase at M Phase

M 期 O-GlcNAc 转移酶的靶向和调节

基本信息

批准号：
8534220
负责人：
Chad Eric Slawson
金额：
$ 21.86万
依托单位：
UNIVERSITY OF KANSAS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8534220
关键词：
Acetylglucosamine 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 novel poly-N-acetyl glucosamine protein expression sugar tumor progression

项目摘要

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 是糖部分 N 乙酰葡糖胺对丝氨酸/苏氨酸氨基酸进行的共价修饰。 O-GlcNAc 修饰不会延长为更复杂的寡糖结构，并且仅存在于核蛋白和细胞质蛋白上。这种修饰是高度动态的，并对细胞外环境的信号（如激素、压力和营养物质）做出反应。之前，我证明了 0-GlcNAc 对于真核细胞细胞周期的正常进展至关重要；的功能增益 0-GlcNAc 转移酶 (OGT)（添加修饰的酶）或 0-GlcNAcase (OGA)（去除修饰的酶）都会导致有丝分裂退出延迟。此外，OGT 和 OGA 可以在具有有丝分裂激酶（例如极光激酶 B）的信号传导复合物中找到。该提案的目的是研究 OGT 与有丝分裂结构（例如纺锤体）以及与有丝分裂蛋白的相互作用。我们假设 OGT 在 M 期进展期间与各种蛋白质形成复合物，然后将 OGT 靶向纺锤体和中体。我计划使用多种技术，例如在 GFP-OGT 上使用 FRAP（光漂白后荧光恢复）来测量 OGT 在纺锤体/中间体的动力学，定量蛋白质组学来识别 OGT 靶向蛋白，最后识别 Aurora 激酶 B 的功能将 OGT 靶向有丝分裂底物和结构。我们的预期贡献是巨大的，因为我们期望深入了解有丝分裂靶向蛋白对 OGT 的调节，以及这些相互作用如何在癌症等疾病中解偶联。