Biosynthesis of Membrane Protein Glycolipid Anchors

描述（由申请人提供）：糖基磷脂酰肌醇（GPI）锚定蛋白（如叶酸受体、朊蛋白和神经细胞粘附分子（NCAM））的生物合成对正常细胞生长至关重要，并且在人类癌症和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为特征的遗传性GPI缺乏症，以及阵发性夜间血红蛋白尿（PNH），这是一种获得性溶血性疾病，由多能造血人干细胞中GPI生物合成第一步的缺陷引起。遗传废除GPI生物合成导致哺乳动物胚胎致死。GPI组装途径已被证实是原生动物和真菌疾病的治疗靶点，包括非洲昏睡病和侵袭免疫受损个体的危及生命的机会性真菌感染。本提案的目的是了解GPI蛋白生物合成的各个方面，并制定策略来操纵和控制GPI途径的总体目标。这些努力对于开发抗原生动物和抗真菌药物，以及治疗癌症和遗传性疾病至关重要，而gpi锚定蛋白在这些疾病中起着关键作用。我们对GPI蛋白生物合成的两个至关重要的步骤感兴趣：GPI锚点附着在蛋白质上和GPI脂质在内质网（ER）膜上的神秘翻转。GPI转氨酶（GPIT）是一种将GPI锚点附着在蛋白质上的酶，是一种鲜为人知的膜结合异五聚体复合物。编码三个GPI亚基的基因最近被鉴定为致癌基因，这提高了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的许多亚基的功能作用；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，这将产生与设计试剂相关的结果，这些试剂可用于选择性地阻断锥虫原虫在其哺乳动物宿主中居住时必需的GPI锚定。我们的第二个目标是鉴定GPI翻转酶，这是一种新的转运蛋白，在GPI组装过程中需要将GPI脂质中间体转运（翻转）穿过内质膜。GPI翻转是GPI组装中必不可少的步骤，也是目前唯一一个生物化学和基因定义的步骤。由于没有任何类型的内质网脂质翻转酶在功能上被鉴定出来，我们的目标是鉴定GPI翻转酶不仅有助于理解GPI组装，而且还将提供内质网脂质易位事件的基本信息。

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.