Biosynthesis of Membrane Protein Glycolipid Anchors

描述(由申请人提供)：本提案的目的是 为我们继续进行关于组装的研究获得支持 糖基磷脂酰肌醇(GPI)和GPI锚定蛋白。后者 包括功能上重要蛋白质，例如细胞表面受体、细胞 黏附分子和原生动物表面抗原。GPI的不足之处 造血细胞的生物合成引起阵发性睡眠性血红蛋白尿 (PNH)，一种人类获得性溶血性疾病，其特征是异常 GPI锚定缺陷对红细胞补体的激活作用 补充调节蛋白。GPI生物合成结果的遗传消除 在哺乳动物的胚胎致死中。这项提议的目的是为了理解 GPI锚生物合成的各个方面，总体目标是贡献 操纵和控制GPI途径的努力。这样的努力是核心 对抗原生动物和抗真菌药物的开发，以及对 GPI锚定蛋白起关键作用的人类疾病的可能治疗 一部份。 我们的具体目标是分析GPI N-乙酰-氨基葡萄糖转移酶 (GPI-GnT)和GPI转氨酶(GPT)，这两种酶是植物体内的第一种和最后一种酶 组装途径，并追寻GPI早期酶的新发现 组装途径定位于线粒体相关的内质网膜结构域 (妈妈)。GPI-GnT和GPT都是新的多亚单位复合体：GPI-GnT是 负责合成第一个GPI生物合成中间体， N-乙酰氨基葡萄糖磷脂酰肌醇，GPT是连接 GPI锚定在蛋白质上。我们将定义酵母的亚基组成 GPI-GnT复合体及其糖核苷酸和磷脂结合特性的研究 使用光亲和标记、定点突变和 荧光共振能量转移。我们将类似地定义人类GPT， 重点关注复杂的、亚单位相互作用的组成 内质网所需的复杂组装和靶向基序 本地化。MAM结构域代表了ER中不寻常的异质性 通过隔离某些GPI生物合成酶，可以 有助于调节GPI的生物合成。我们将重点关注猪-L， 该途径的第二种酶，其活性已知定位于 妈妈。我们将使用截断和嵌合的PIG-L结构，亚细胞 分离、荧光和电子显微镜，以及单细胞 荧光光漂白技术研究猪L对肿瘤细胞的靶向性 并描述其在MAM中的动态特性。

DESCRIPTION (provided by applicant): The objective of this proposal is to obtain support for the continuation of our studies on the assembly of glycosylphosphatidylinositols (GPIs) and GPI-anchored proteins. The latter include functionally important proteins such as cell surface receptors, cell adhesion molecules and protozoal surface antigens. A deficiency in GPI biosynthesis in hematopoietic cells causes paroxysmal nocturnal hemoglobinuria (PNH), an acquired hemolytic disease in humans characterized by abnormal activation of complement on erythrocytes due to a deficiency of GPI-anchored complement regulatory proteins. Genetic abrogation of GPI biosynthesis results in embryonic lethality in mammals. The aims of this proposal are to understand aspects of GPI anchor biosynthesis with the overall objective of contributing to efforts to manipulate and control the GPI pathway. Such efforts are central to the development of anti-protozoal and anti-fungal drugs, as well to the possible treatment of human diseases in which GPI-anchored proteins play a key part. Our specific aims are to analyze GPI N-acetyl-glucosaminyl-transferase (GPI-GnT) and GPI transamidase (GPT), the first and last enzymes of the assembly pathway, and to pursue the novel finding that early enzymes of the GPI assembly pathway are localized to a mitochondria-associated ER membrane domain (MAM). Both GPI-GnT and GPT are novel, multi-subunit complexes: GPI-GnT is responsible for the synthesis of the first GPI biosynthetic intermediate, N-acetylglucosaminylphosphatidylinositol, and GPT is the enzyme that attaches GPI anchors to proteins. We will define the subunit composition of the yeast GPI-GnT complex and characterize its sugar nucleotide and phospholipid binding components using photoaffinity labeling, site-directed mutagenesis, and fluorescence resonance energy transfer. We will similarly define human GPT, focusing on the composition of the complex, subunit interactions required for complex assembly and the targeting motifs needed for endoplasmic reticulum localization. The MAM domain represents an unusual heterogeneity within the ER that, through sequestration of certain of the GPI biosynthetic enzymes, may contribute to the regulation of GPI biosynthesis. We will focus on PIG-L, the second enzyme of the pathway, whose activity is known to be localized to the MAM. We will use truncated and chimeric PIG-L constructs, subcellular fractionation, fluorescence and electron microscopy, and single cell fluorescence photobleaching techniques to study the targeting of PIG-L to the MAM and characterize its dynamics within the MAM.