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.

项目概要： 天冬酰胺（N）连接的糖基化（NLG）是最常见的膜蛋白修饰， 真核生物中的分泌蛋白。然而，糖生物学的一个主要挑战是鉴定和 了解调节NLG的细胞机制和非必需基因产物。此外，虽然 该途径靶点丰富，涉及至少34种基因产物，调节该途径的药理学抑制剂， 进程不可用。我们设计并实施了一种生物发光成像策略， 在细胞水平上检测NLG位点占有率的变化。该平台可以集成到实验中， 从微孔板检测到体内成像，高通量筛选工作已经提供了两个 结构上独立的NLG抑制剂类别。第一种抑制剂NGI-1阻断寡糖基转移酶 活性，并导致这种多亚基酶复合物的保真度损失。我们计划找出 酸和NGI-1结合口袋，以便更准确地了解小分子OST的机制 抑制作用第二种抑制剂的细胞靶点尚未确定，因此我们建议 研究和鉴定NLG抑制的这种新型作用机制的方法。我们还 设计并验证了一种检测异常糖基化的荧光成像方法， 用于在单细胞水平上检测聚糖位点占据的异质性。我们建议推进这一点 为了研究调节NLG的遗传因子， 以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的调节的见解，并提供了一种技术， 定量体外和体内特定细胞中NLG的动态变化和异质性。