Biosynthesis of Membrane Protein Glycolipid Anchors

描述(申请人提供)：糖基磷脂酰肌醇(GPI)锚定蛋白的生物合成(如叶酸受体、Prion蛋白和神经细胞黏附分子(NCAM))是正常细胞生长的关键，在人类癌症和一些遗传性疾病中受到干扰。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，由多潜能造血干细胞GPI生物合成的第一步缺陷引起。GPI生物合成的遗传取消会导致哺乳动物的胚胎死亡。GPI组装途径已被证实是治疗原生动物和真菌疾病的靶点，包括非洲昏睡病和危及生命的机会性真菌感染，这些疾病困扰着免疫系统受损的人。这项建议的目的是了解GPI-蛋白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。这些努力是开发抗原生动物和抗真菌药物的核心，也是治疗癌症和遗传性疾病的核心，在这些疾病中，GPI锚定蛋白发挥了关键作用。我们感兴趣的是GPI-蛋白质生物合成的两个关键步骤：将GPI锚点连接到蛋白质上和GPI脂类穿过内质网(ER)膜的神秘翻转。GPI转氨酶(GPI Transamidase，GPIT)是一种膜结合型异五聚体，是一种将GPI锚点连接到蛋白质上的酶，人们对它知之甚少。编码三个GPIT亚单位的基因最近被确定为癌基因，这增加了GPI途径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许多亚基的功能作用；我们特别对癌蛋白PIG-U和GAA1感兴趣，我们假设它们是复合体的GPI结合元件。我们还建议对锥虫GPIT进行分析，从长远来看，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虫中的必要GPI锚定，而这些寄生虫驻留在哺乳动物宿主中。我们的第二个目标是确定GPI Flppase，这是一种新型的转运蛋白，在GPI组装过程中需要将GPI脂质中间体转运(翻转)到ER膜上。GPI翻转是GPI组装的必经步骤，也是目前唯一有待生化和基因定义的步骤。由于尚未从功能上鉴定出任何类型的内质网脂质翻转酶，我们确定GPI翻转酶的目的不仅将有助于对GPI组装的专门了解，而且还将提供有关内质网中脂质转位事件的基本信息。

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.