Structural Analysis of the GPI Transamidase Complex

GPI 转酰胺酶复合物的结构分析

• 批准号: 8196655
• 负责人: ANANT K MENON
• 金额: $ 25.35万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8196655
• 关键词: Abbreviations; Acetylcholinesterase; African Trypanosomiasis; Alkaline Phosphatase; Amino Acids; Anabolism; Architecture; Area; Binding; Biochemistry; Biological; Biological Assay; Biology; COOH-terminal signal transamidase; Canis familiaris; Caspase; Catalytic Domain; Cell Adhesion Molecules; Cell Survival; Cell surface; Cellular biology; Cleaved cell; Complex; Cysteine Protease; Defect; Disease; Drug Delivery Systems; Electron Microscopy; Embryo; Endoplasmic Reticulum; Enzymes; Epitopes; Eukaryota; Family; Future; GPI Membrane Anchors; Glycosylphosphatidylinositols; Glypican; Hematopoietic; Human; Laboratories; Learning; Leishmaniasis; Light; Link; Location; Malaria; Malignant Neoplasms; Mammals; Mannose; Maps; Membrane; Membrane Glycoproteins; Membrane Proteins; Multienzyme Complexes; NCAM1 gene; PI-Glycan; Pathway interactions; Peptide Signal Sequences; Phosphatidylinositols; Play; Prions; Process; Protein Subunits; Proteins; Protozoa; Recruitment Activity; Reporting; Resolution; Roentgen Rays; Role; Structure; Trypanosoma brucei brucei; Trypanosomiasis; Universities; Variant; Work; X-Ray Crystallography; Yeasts; design; dolichyl-diphosphooligosaccharide - protein glycotransferase; enzyme structure; experience; folate-binding protein; glycosylation; human stem cells; inhibitor/antagonist; insight; interest; man; membrane dipeptidase; pathogen; phosphoethanolamine; protein complex; protein structure; rat Piga protein; reconstitution; signal peptidase; structural biology; tool; transamidases; tumorigenesis

DESCRIPTION (provided by applicant): Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are ubiquitous in eukaryotes. Examples of GPI-APs include folate receptor, acetylcholinesterase, renal dipeptidase and the variant surface glycoproteins of Trypanosoma brucei, the causative agent of African sleeping sickness. Inability to synthesize GPI-APs results in embryonic lethality in mammals. Defective GPI biosynthesis in multipotent hematopoietic human stem cells causes paroxysmal nocturnal hemoglobinuria, an acquired hemolytic disease. GPI-APs are needed for fungal cell viability and they are important in diseases such as trypanosomiasis, malaria and leishmaniasis that are caused by parasitic protozoa. The GPI assembly pathway is a drug target for fungal and protozoal diseases. GPI anchoring is catalyzed by GPI transamidase (GPIT), a 5-subunit membrane- bound complex located in the endoplasmic reticulum (ER). The catalytic subunit, Gpi8, shares homology with caspases; the functional role of the other subunits is unclear, but all are required for GPIT activity. Three of the non-catalytic subunits are over-expressed in certain cancers, indicating a link between GPIT and oncogenesis. In this R21 application we propose to initiate structure-function studies of the GPIT complex using electron microscopy and X-ray crystallography. We are ultimately interested in establishing the structural organization of GPIT, delineating the role of its subunits, and understanding how this important enzyme is regulated. In two specific aims we propose to (1) analyze the endogenous GPIT complex from yeast by electron microscopy and (2) express GPIT subunits and sub-complexes for X-ray crystallographic studies. Our efforts are expected to yield a medium-resolution structure of GPIT and pave the way for a future high-resolution structure of the intact complex. These studies will have high impact as there is no structural information on GPIT; also, results obtained here will shed light on other multi-subunit membrane bound enzymes in the ER such as oligosaccharyltransferase and signal peptidase that play a critical role in processing a wide range of ER-translocated proteins, including proteins destined for GPI anchoring, but whose functional architecture remains largely a mystery. PUBLIC HEALTH RELEVANCE: Glycosylphosphatidylinositol (GPI)-anchored proteins such as folate receptor and acetylcholinesterase are ubiquitous in eukaryotes. They are also key players in diseases caused by fungal pathogens and parasitic protozoa. GPI transamidase, the enzyme responsible for synthesizing GPI-anchored proteins, is a complex structure consisting of five components that are embedded in a biological membrane. We are interested in using applying the tools of modern structural biology to learn about the structure of this enzyme as a first step to understanding how it works.

描述（由申请人提供）：糖基磷脂酰肌醇（GPI）锚定蛋白（GPI-AP）在真核生物中普遍存在。 GPI-AP 的例子包括叶酸受体、乙酰胆碱酯酶、肾二肽酶和布氏锥虫（非洲昏睡病的病原体）的变异表面糖蛋白。无法合成 GPI-AP 会导致哺乳动物胚胎死亡。人类多能造血干细胞中的 GPI 生物合成缺陷会导致阵发性睡眠性血红蛋白尿，这是一种获得性溶血性疾病。 GPI-AP 是真菌细胞活力所必需的，它们对于由寄生原生动物引起的锥虫病、疟疾和利什曼病等疾病很重要。 GPI 组装途径是真菌和原虫疾病的药物靶点。 GPI 锚定由 GPI 转酰胺酶 (GPIT) 催化，GPIT 是一种位于内质网 (ER) 中的 5 亚基膜结合复合物。催化亚基 Gpi8 与半胱天冬酶具有同源性；其他亚基的功能作用尚不清楚，但所有亚基都是 GPIT 活动所必需的。三个非催化亚基在某些癌症中过度表达，表明 GPIT 与肿瘤发生之间存在联系。 在此 R21 应用中，我们建议使用电子显微镜和 X 射线晶体学启动 GPIT 复合物的结构功能研究。我们最终感兴趣的是建立 GPIT 的结构组织，描述其亚基的作用，并了解如何调节这种重要的酶。在两个具体目标中，我们建议 (1) 通过电子显微镜分析酵母的内源 GPIT 复合物，以及 (2) 表达 GPIT 亚基和子复合物用于 X 射线晶体学研究。我们的努力预计将产生 GPIT 的中等分辨率结构，并为未来完整复合体的高分辨率结构铺平道路。 由于 GPIT 没有结构信息，这些研究将产生很大影响；此外，这里获得的结果将揭示 ER 中的其他多亚基膜结合酶，例如寡糖转移酶和信号肽酶，它们在处理各种 ER 易位蛋白（包括注定用于 GPI 锚定的蛋白）中发挥关键作用，但其功能架构在很大程度上仍然是一个谜。 公共卫生相关性：糖基磷脂酰肌醇 (GPI) 锚定蛋白，如叶酸受体和乙酰胆碱酯酶，在真核生物中普遍存在。它们也是真菌病原体和寄生原生动物引起的疾病的关键参与者。 GPI 转酰胺酶是负责合成 GPI 锚定蛋白的酶，是一种复杂的结构，由嵌入生物膜的五种成分组成。我们有兴趣应用现代结构生物学工具来了解这种酶的结构，作为了解其工作原理的第一步。