Structural Analysis of the GPI Transamidase Complex

描述(申请人提供)：糖基磷脂酰肌醇(GPI)锚定蛋白(GPI-APs)广泛存在于真核生物中。GPI-APs的例子包括叶酸受体、乙酰胆碱酯酶、肾脏二肽酶和布鲁氏锥虫的变种表面糖蛋白，后者是非洲昏睡病的病原体。不能合成GPI-AP会导致哺乳动物胚胎死亡。多能造血干细胞中GPI生物合成缺陷会导致阵发性睡眠性血红蛋白尿，这是一种获得性溶血性疾病。GPI-AP是真菌细胞存活所必需的，它们在由寄生原虫引起的锥虫病、疟疾和利什曼病等疾病中非常重要。GPI组装途径是治疗真菌和原虫疾病的药物靶点。GPI锚定是由GPI转氨酶(GPI Transamidase，GPIT)催化的，它是一种位于内质网(ER)的5亚单位膜结合复合体。催化亚基Gpi8与caspase有同源性；其他亚基的功能尚不清楚，但都是GPIT活性所必需的。其中三个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R21的应用中，我们建议使用电子显微镜和X射线结晶学来启动GPIT络合物的结构-功能研究。我们最终感兴趣的是建立GPIT的结构组织，描述其亚单位的作用，并了解这种重要的酶是如何调节的。在两个特定的目标中，我们建议(1)用电子显微镜分析酵母中内源的GPIT复合体和(2)表达GPIT亚单位和亚复合体用于X射线结晶学研究。我们的努力有望产生一个中等分辨率的GPIT结构，并为未来完整的复合体的高分辨率结构铺平道路。这些研究将产生很大的影响，因为没有关于GPIT的结构信息；此外，这里获得的结果将揭示内质网中的其他多亚单位膜结合酶，如寡糖转移酶和信号肽酶，它们在处理广泛的内质网转位蛋白方面发挥关键作用，包括目的是GPI锚定的蛋白，但其功能结构在很大程度上仍然是一个谜。

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.