Biosynthesis of Membrane Protein Glycolipid Anchors

膜蛋白糖脂锚的生物合成

• 批准号: 6879017
• 负责人: ANANT K MENON
• 金额: $ 32.81万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-11-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6879017
• 关键词: Escherichia coli; acyltransferase; chemical structure function; electron microscopy; endoplasmic reticulum; enzyme complex; fluorescence resonance energy transfer; glycoproteins; glycosylphosphatidylinositols; hexosyltransferase; high performance liquid chromatography; intermolecular interaction; intracellular transport; lipid biosynthesis; mass spectrometry; membrane proteins; molecular site; nonblood lipoprotein; protein biosynthesis; protein localization; protein purification; site directed mutagenesis; tissue /cell culture; yeasts

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.

描述（由申请人提供）：本提案的目的是 争取支持继续进行我们关于 糖基磷脂酰肌醇（GPIs）和GPIs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 粘附分子和原生动物表面抗原。GPI的缺陷 造血细胞生物合成引起阵发性睡眠性血红蛋白尿症 (PNH)一种人类获得性溶血性疾病， 由于缺乏GPI锚定的红细胞上的补体激活 补体调节蛋白。GPI生物合成结果的遗传废除 在哺乳动物的胚胎致死率上。本提案的目的是了解 GPI锚生物合成的各个方面，其总体目标是 来操纵和控制GPI通路。这些努力是核心 抗原生动物和抗真菌药物的发展，以及 GPI锚定蛋白发挥关键作用的人类疾病的可能治疗方法 部分 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转酰胺酶（GPT），第一个和最后一个酶， 组装途径，并追求新的发现，早期酶的GPI 组装途径定位于与ER相关的ER膜结构域 （MAM）。GPI-GnT和GPT都是新型的多亚基复合物：GPI-GnT是 负责合成第一个GPI生物合成中间体， N-乙酰葡糖胺磷脂酰肌醇，GPT是附着在 GPI锚定蛋白质。我们将定义酵母的亚基组成 GPI-GnT复合物及其糖核苷酸和磷脂结合特性 使用光亲和标记，定点诱变，和 荧光共振能量转移我们将类似地定义人类GPT， 专注于复杂的组成，亚基相互作用所需的 复杂的组装和内质网所需的靶向基序 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， 有助于GPI生物合成的调节。我们将专注于PIG-L， 第二种酶的途径，其活动是已知的，是本地化的 妈妈我们将使用截短的和嵌合的PIG-L构建体， 分离，荧光和电子显微镜，和单细胞 荧光光漂白技术研究PIG-L的靶向 MAM和MAM内的动态特性。