Structural Analysis of the GPI Transamidase Complex

GPI 转酰胺酶复合物的结构分析

基本信息

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
项目类别：
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

项目摘要

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）锚定的蛋白质（GPI-APS）在真核生物中无处不在。 GPIAPS的例子包括叶酸受体，乙酰胆碱酯酶，肾二肽酶和Brucei锥虫的变异表面糖蛋白，这是非洲睡眠病的致病药物。无法合成GPIAP会导致哺乳动物的胚胎致死性。多能造血人干细胞中的GPI生物合成有缺陷会导致阵发性夜间血红蛋白尿，这是一种产生的溶血性疾病。真菌细胞生存能力需要GPIAP，它们在由寄生虫原生动物引起的锥虫病，疟疾和利什曼病等疾病中很重要。 GPI组装途径是真菌和原生动物疾病的药物靶标。 GPI锚定是由GPI跨成果酶（GPIT）催化的，这是一种位于内质网（ER）中的5-亚基膜结合复合物。催化亚基GPI8与caspase共享同源性；其他亚基的功能作用尚不清楚，但GPIT活性都是必需的。在某些癌症中，三个非催化亚基过表达，表明GPIT与肿瘤发生之间有联系。在此R21应用中，我们建议使用电子显微镜和X射线晶体学对GPIT复合物进行结构功能研究。我们最终有兴趣建立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酶。在两个具体的目标中，我们建议（1）通过电子显微镜分析酵母的内源性GPIT复合物，（2）表示X射线晶体学研究的Express GPIT亚基和亚复合物。期望我们的努力产生GPIT的中等分辨率结构，并为完整复合物的未来高分辨率结构铺平道路。由于GPIT没有结构信息，因此这些研究将产生很大的影响。此外，此处获得的结果将阐明ER中的其他多重亚基膜结合酶，例如寡素胆汁胆管酶和信号肽酶，这些酶在处理广泛的ER转换蛋白方面起着至关重要的作用。公共卫生相关性：诸如叶酸受体和乙酰胆碱酯酶等糖基磷脂酰肌醇（GPI）锚定的蛋白质无处不在。它们也是由真菌病原体和寄生原生动物引起的疾病的关键参与者。 GPI跨成像酶是负责合成GPI锚定蛋白的酶，是一种复杂的结构，由五个组成的成分组成，这些成分嵌入生物膜中。我们有兴趣使用现代结构生物学的工具来了解该酶的结构，以了解其工作原理的第一步。