Regulation of N-linked glycosylation in Mammalian Cells

哺乳动物细胞中 N 连接糖基化的调控

• 批准号: 9495850
• 负责人: Joseph N. Contessa
• 金额: $ 33.5万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9495850
• 关键词: Address; Amino Acids; Asparagine; Binding; Binding Sites; Biological Assay; Biology; CRISPR/Cas technology; Catalytic Domain; Cell Line; Cells; Chemicals; Coupled; Data; Detection; Development; Eukaryota; Eukaryotic Cell; Genetic; Genome; Glycobiology; Glycoproteins; Heterogeneity; Image; In Vitro; Investigation; Knock-out; Knowledge; Label; Libraries; Link; Mammalian Cell; Measurable; Measures; Membrane Proteins; Methodology; Methods; Modeling; Monitor; Multienzyme Complexes; Mus; Mutagenesis; Organ; Pathway interactions; Peptides; Pharmacology; Polysaccharides; Population; Post-Translational Protein Processing; Process; Proteins; Regulation; Reporter; Research; Series; Site; Small Molecule Chemical Library; Structure-Activity Relationship; Sulfonamides; System; Techniques; Tissues; Transgenic Mice; Work; base; cellular targeting; design; experimental study; fluorescence imaging; gene product; genetic approach; glycosylation; high throughput screening; imaging approach; in vivo; in vivo Model; in vivo imaging; inhibitor/antagonist; insight; molecular imaging; novel; purine analog; screening; screening program; secretory protein; small molecule; small molecule inhibitor; small molecule libraries

PROJECT SUMMARY: Asparagine (N) linked glycosylation (NLG) is the most common protein modification of membrane and secretory proteins in eukaryotes. However, A major challenge in glycobiology has been to identify and understand the cellular mechanisms and non-essential gene products that regulate NLG. Furthermore, though this pathway is target rich and involves at least 34 gene products, pharmacologic inhibitors that regulate this process have not been available. We have designed and implemented a bioluminescent imaging strategy to detect changes in NLG site occupancy at the cellular level. This platform can be integrated in experiments that range from microplate detection to in vivo imaging, and high throughput screening efforts have delivered two structurally independent classes of NLG inhibitors. The first inhibitor, NGI-1, blocks oligosaccharyltransferase activity and causes loss of fidelity for this multi-subunit enzyme complex. We plan to identify the specific amino acids and NGI-1 binding pocket in order to more precisely understand the mechanism of small molecule OST inhibition. The cellular target for the second inhibitor has yet to be defined and we therefore propose methodologies to investigate and identify this novel mechanism of action for NLG inhibition. We have also designed and validated a fluorescent imaging approach which detects abnormal glycosylation and that can be used to detect heterogeneity of glycan site occupancy at the single cell level. We propose to advance this technique to both in vitro and in vivo models in order to both investigate genetic factors that regulate NLG and to examine glycosylation differences in mouse organs and tissues. Together this work will provide new insights into the regulation of NLG by genetic factors or small molecule inhibitors, and provide a technique for quantifying dynamic changes and the heterogeneity of NLG in specific cells both in vitro and in vivo.

项目摘要： 天冬酰胺（N）连接的糖基化（NLG）是膜和最常见的蛋白质修饰 真核生物中的分泌蛋白。但是，糖生物学的主要挑战是识别和 了解调节NLG的细胞机制和非必需基因产物。此外， 该途径具有富含靶标的靶标，并且至少涉及34种基因产物，即调节这一点的药理学抑制剂 过程尚未可用。我们已经设计并实施了生物发光的成像策略 检测细胞水平NLG位点占用率的变化。该平台可以集成到实验中 范围从微板检测到体内成像，高吞吐量筛选工作已经提供了两个 NLG抑制剂的结构独立类别。第一个抑制剂NGI-1阻断了寡糖胆胆素延缓酶 活性并导致这种多生酶复合物的富裕度丧失。我们计划确定特定的氨基 酸和NGI-1结合袋，以便更精确地了解小分子OST的机制 抑制。第二抑制剂的细胞靶标尚未定义，因此我们提出 研究和确定这种新型NLG抑制作用机理的方法。我们也有 设计并验证了一种检测异常糖基化的荧光成像方法，可以是 用于检测单细胞水平的聚糖位点占用率的异质性。我们建议进步 在体外和体内模型的技术，以研究调节NLG和 检查小鼠器官和组织中的糖基化差异。这项工作将提供新的 通过遗传因素或小分子抑制剂对NLG调节的洞察力，并提供了一种技术 量化特定细胞在体外和体内的动态变化和NLG的异质性。