Biosynthesis of Membrane Protein Glycolipid Anchors

膜蛋白糖脂锚的生物合成

• 批准号: 7938503
• 负责人: ANANT K MENON
• 金额: $ 19.8万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7938503
• 关键词: Abbreviations; Acetylcholinesterase; Acetylglucosamine; African Trypanosomiasis; Anabolism; Antifungal Agents; Benzophenones; Binding; Binding Proteins; Binding Sites; Biochemistry; Biological; Biological Assay; COOH-terminal signal transamidase; Carrier Proteins; Catalytic Domain; Chemicals; Complex; Conserved Sequence; Data; Defect; Detergents; Development; Disease; Dolichol; Elements; Embryo; Endoplasmic Reticulum; Enzymes; Ethanolamines; Event; Evolution; Face; Family suidae; GPI Membrane Anchors; Genes; Genetic; Glucosamine; Glycolipids; Glycosylphosphatidylinositol-anchor Biosynthesis Pathway; Glycosylphosphatidylinositols; Hemagglutinin; Hematopoietic; Hereditary Disease; Human; Individual; Inherited; Label; Life; Lipids; Liver; Malignant Neoplasms; Mammals; Mannose; Maps; Membrane; Membrane Proteins; Molecular; Mutagenesis; Mycoses; Neural Cell Adhesion Molecules; Normal Cell; Oncogene Proteins; Oncogenes; PI-Glycan; Parasites; Pathway interactions; Pharmaceutical Preparations; Phosphatidylinositols; Phospholipase C; Phospholipids; Play; Positioning Attribute; Prions; Procedures; Protein Biosynthesis; Proteins; Protozoa; Radiolabeled; Rattus; Reaction; Reagent; Recruitment Activity; Reporter; Research Personnel; Role; Seizures; Side; Structure; System; Testing; Triton X100; Trypanosoma; Trypanosoma brucei brucei; Venous Thrombosis; Work; base; cancer therapy; catalyst; cell growth; design; dolichyl-diphosphooligosaccharide - protein glycotransferase; fatty acid elongases; folate-binding protein; fungus; glycoprotein phospholipase D; human stem cells; innovation; interest; man; mutant; novel; phosphoethanolamine; programs; proteoliposomes; radiotracer; rat Piga protein; reconstitution; therapeutic target; transamidases

DESCRIPTION (provided by applicant): The biosynthesis of glycosylphosphatidylinositol (GPI)-anchored proteins (such as the folate receptor, prion protein, and the neural cell adhesion molecule (NCAM)) is critical for normal cell growth, and perturbed in human cancers as well as a number of genetic diseases. The latter include an inherited GPI deficiency characterized by venous thrombosis and seizures, and paroxysmal nocturnal hemoglobinuria (PNH), an acquired hemolytic disease that is caused by a defect in the first step of GPI biosynthesis in multipotent hematopoietic human stem cells. Genetic abrogation of GPI biosynthesis results in embryonic lethality in mammals. The GPI assembly pathway has been validated as a therapeutic target for protozoal and fungal diseases, including African sleeping sickness and the life-threatening opportunistic fungal infections that afflict immuno-compromised individuals. The aims of this proposal are to understand aspects of the biosynthesis of GPI-proteins with the overall objective of developing strategies to manipulate and control the GPI pathway. Such efforts are central to the development of anti-protozoal and anti-fungal drugs, as well as to the treatment of cancer and inherited diseases in which GPI-anchored proteins play a key part. We are interested in two critically important steps of GPI-protein biosynthesis: the attachment of a GPI anchor to protein and the enigmatic flip-flop of GPI lipids across the endoplasmic reticulum (ER) membrane. GPI transamidase (GPIT), the enzyme that attaches GPI anchors to protein, is a poorly understood membrane-bound hetero-pentameric complex. Genes encoding three of the GPIT subunits have been recently identified as oncogenes, raising the possibility that the GPI pathway may provide a novel target for human cancer treatment. Our aim is to define the functional role of GPIT's many subunits; we are especially interested in the oncoproteins PIG-U and GAA1 that we hypothesize to be the GPI binding elements of the complex. We also propose analyses of trypanosome GPIT that will, in the long term, yield results pertinent to the design of reagents that could be used to selectively block essential GPI anchoring in trypanosomatid protozoa while these parasites are resident in their mammalian hosts. Our second aim is to identify GPI flippase, the novel transporter that is required to translocate (flip) GPI lipid intermediates across the ER membrane during GPI assembly. GPI flipping is an obligatory step in GPI assembly and the only one that currently remains to be biochemically and genetically defined. Since no ER lipid flippase of any type has been functionally identified, our aim to identify the GPI flippase will not only contribute specifically to an understanding of GPI assembly but will also provide fundamental information on lipid translocation events in the ER in general.

描述（由申请人提供）：糖基磷脂酰肌醇（GPI）锚定蛋白（例如叶酸受体、朊病毒蛋白和神经细胞粘附分子（NCAM））的生物合成对于正常细胞生长至关重要，并且在人类癌症以及许多遗传疾病中受到干扰。后者包括以静脉血栓形成和癫痫发作为特征的遗传性 GPI 缺陷，以及阵发性睡眠性血红蛋白尿 (PNH)，这是一种获得性溶血性疾病，由多能造血干细胞中 GPI 生物合成第一步缺陷引起。 GPI 生物合成的基因消除会导致哺乳动物胚胎死亡。 GPI 组装途径已被验证为原虫和真菌疾病的治疗靶点，包括非洲昏睡病和困扰免疫功能低下个体的危及生命的机会性真菌感染。该提案的目的是了解 GPI 蛋白质生物合成的各个方面，总体目标是制定操纵和控制 GPI 途径的策略。这些努力对于抗原虫和抗真菌药物的开发以及 GPI 锚定蛋白在其中发挥关键作用的癌症和遗传性疾病的治疗至关重要。我们对 GPI 蛋白质生物合成的两个至关重要的步骤感兴趣：GPI 锚定蛋白与蛋白质的连接以及 GPI 脂质跨内质网 (ER) 膜的神秘翻转。 GPI 转酰胺酶 (GPIT) 是将 GPI 锚附着到蛋白质上的酶，是一种人们知之甚少的膜结合异五聚体复合物。编码三个 GPIT 亚基的基因最近被鉴定为癌基因，这提高了 GPI 通路可能为人类癌症治疗提供新靶点的可能性。我们的目标是定义 GPIT 许多子单元的功能作用；我们对癌蛋白 PIG-U 和 GAA1 特别感兴趣，我们假设它们是复合物的 GPI 结合元件。我们还提出了对锥虫 GPIT 的分析，从长远来看，这将产生与试剂设计相关的结果，这些试剂可用于选择性地阻断锥虫原生动物中必需的 GPI 锚定，而这些寄生虫居住在其哺乳动物宿主中。我们的第二个目标是鉴定 GPI 翻转酶，这是一种新型转运蛋白，在 GPI 组装过程中将 GPI 脂质中间体跨内质网转运（翻转）所需的。 GPI 翻转是 GPI 组装中的必要步骤，也是目前唯一有待生物化学和遗传学定义的步骤。由于尚未在功能上鉴定出任何类型的 ER 脂质翻转酶，因此我们鉴定 GPI 翻转酶的目标不仅将有助于了解 GPI 组装，而且还将提供有关 ER 中脂质易位事件的基本信息。