The structure and function of eukaryotic protein glycosylation enzymes

真核蛋白质糖基化酶的结构和功能

• 批准号: 10412104
• 负责人: Huilin Li
• 金额: $ 42.59万
• 依托单位: VAN ANDEL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-18 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10412104
• 关键词: 3-Dimensional; Antigens; Architecture; Asparagine; Binding; Binding Proteins; Biological Assay; CA-125 Antigen; CA-19-9 Antigen; Carbohydrates; Catalytic Domain; Cellular biology; Classification; Co-Translational Protein Processing; Complex; Cryoelectron Microscopy; Crystallization; Development; Diagnosis; Enzymes; Foundations; Glycoproteins; Glycosides; Goals; Gold; Homologous Gene; Human; In Vitro; Integral Membrane Protein; Knowledge; Link; Malignant neoplasm of ovary; Malignant neoplasm of pancreas; Maps; Membrane; Membrane Proteins; Microsomes; Modeling; Modification; Multiprotein Complexes; Mutagenesis; Negative Staining; Normal Cell; Oligosaccharides; Peptides; Play; Polysaccharides; Positioning Attribute; Preparation; Protein Biosynthesis; Protein Glycosylation; Proteins; Reaction; Receptor Protein-Tyrosine Kinases; Research; Resolution; Ribosomes; Role; Sampling; Side; Structure; Transferase; Transmembrane Domain; Triplet Multiple Birth; Tumor Markers; Work; X-Ray Crystallography; Yeasts; antitumor agent; base; cancer cell; cancer diagnosis; cancer therapy; design; flexibility; glycosylation; improved; in vitro activity; in vivo; inhibitor; insight; mimetics; model building; neoplastic cell; polypeptide; prevent; progression marker; recruit; secretory protein; senescence; sugar; tumor diagnosis; tumor progression; tumorigenesis

Project Summary Asparagine (N-linked) glycosylation is a ubiquitous modification of eukaryotic secretory and membrane proteins. N-glycosylation is the most common type of glycosylation, with 90% of glycoproteins being N- glycosylated. The N-glycans are initially added to a triplet sequence N-X-S/T by a multi-protein transmembrane complex called oligosaccharyl transferase (OST). Most of the protein N-glycosylation occurs while a protein is being synthesized by the ribosome and being transported through the translocon. Hence, OST physically interacts and forms a super-complex with the ribosome and translocon. The mechanisms of protein synthesis and nascent peptide translocation are better understood thanks to available structures of ribosomes and translocons. However, our understanding of eukaryotic protein N-glycosylation is very limited due to the lack of high-resolution OST structures. Our goal is to bridge this major knowledge gap by characterizing the structure and function of the eight-protein OST complex of yeast. A recent study demonstrated that inhibiting human OST induces senescence in receptor tyrosine kinase–driven tumor cells, suggesting that OST may be a target for the development of anti-tumor agents. Furthermore, because transformation of a normal cell to a cancer cell is usually accompanied by N-glycan branching and extension, several N-glycans have been widely used as tumor markers: for example, carbohydrate antigen (CA) CA19-9 for detecting pancreatic cancer, and CA125, which is considered the gold standard marker for diagnosing ovarian cancer. We propose a comprehensive structure and function study of the yeast OST complex, with a combined approach of cryo-EM, X-ray crystallography, structure-based mutagenesis and cell biology, and in vitro activity assays. Our work has important implications in tumorigenesis and cancer diagnosis and treatment.

项目摘要 天冬酰胺（N连接）糖基化是真核秘密和膜的无处不在的修饰 蛋白质。 N-糖基化是最常见的糖基化类型，其中90％的糖蛋白是N- 糖基化。最初通过多蛋白跨膜添加N-聚糖N-X-S/T中 复合物称为寡核酸转移酶（OST）。大多数蛋白质N-糖基化发生在蛋白质是 由核糖体合成并通过易位运输。因此，身体上 与核糖体和易位的相互作用并形成超复合物。蛋白质合成的机制 和新生的意大利辣香肠易位可以更好地理解，感谢您对核糖体的可用结构和 易位。但是，由于缺乏缺乏 高分辨率OST结构。我们的目标是通过表征结构来弥合这一主要知识差距 八蛋白OST酵母菌络合物的功能。最近的一项研究表明，抑制人类 OST在受体酪氨酸激酶驱动的肿瘤细胞中诱导感受，这表明OST可能是一个靶标 为了开发抗肿瘤剂。此外，因为将正常细胞转化为癌症 细胞通常伴随着N-聚糖分支和延伸，几个N-聚糖已被广泛使用 作为肿瘤标记：例如，用于检测胰腺癌的碳水化​​合物抗原（CA）CA19-9，和 CA125，被认为是诊断卵巢癌的黄金标准标记。我们提出了一个 酵母OST复合物的综合结构和功能研究，具有冷冻EM的综合方法 X射线晶体学，基于结构的诱变和细胞生物学以及体外活性评估。我们的工作有 肿瘤发生，癌症诊断和治疗的重要意义。