Structural Analysis of the GPI Transamidase Complex

描述（由申请人提供）：糖基磷脂酰肌醇（GPI）锚定蛋白（GPI- aps）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、肾二肽酶和布鲁氏锥虫（非洲昏睡病的病原体）的变异表面糖蛋白。不能合成GPI-APs导致哺乳动物胚胎死亡。多能造血人干细胞GPI生物合成缺陷导致阵发性夜间血红蛋白尿，一种获得性溶血性疾病。真菌细胞活力需要gpi - ap，它们在由寄生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点。GPI锚定是由GPI转氨酶（GPIT）催化的，GPI转氨酶是一种位于内质网（ER）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，但它们都是GPIT活动所必需的。三种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。在这个R21申请中，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。我们最终感兴趣的是建立GPIT的结构组织，描绘其亚基的作用，并了解这种重要的酶是如何调节的。在两个特定的目标中，我们提出(1)通过电子显微镜分析酵母内源性GPIT复合物(2)表达GPIT亚基和亚复合物进行x射线晶体学研究。我们的努力有望获得GPIT的中等分辨率结构，并为未来完整建筑群的高分辨率结构铺平道路。这些研究将具有很高的影响，因为没有关于GPIT的结构信息；此外，本文获得的结果将揭示内质网中其他多亚基膜结合酶，如寡糖转移酶和信号肽酶，它们在处理广泛的内质网易位蛋白（包括用于GPI锚定的蛋白）中起着关键作用，但其功能结构在很大程度上仍是一个谜。

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.