Biosynthesis of Membrane Protein Glycolipid Anchors

膜蛋白糖脂锚的生物合成

• 批准号: 6724854
• 负责人: ANANT K MENON
• 金额: $ 13.33万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 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 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6724854
• 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）：该提案的目的是 为我们继续进行组装研究获得支持 糖基磷脂酰肌醇 (GPI) 和 GPI 锚定蛋白。后者 包括功能重要的蛋白质，例如细胞表面受体、细胞 粘附分子和原虫表面抗原。 GPI 缺乏 造血细胞的生物合成导致阵发性夜间血红蛋白尿 (PNH)，一种人类获得性溶血病，其特征为异常 由于 GPI 锚定缺陷而激活红细胞上的补体 补充调节蛋白。 GPI 生物合成结果的基因废除 哺乳动物胚胎致死率。该提案的目的是了解 GPI 锚生物合成的各个方面，总体目标是贡献 操纵和控制 GPI 通路的努力。这些努力是核心 抗原虫和抗真菌药物的开发，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 GPI 早期酶的新发现 组装途径定位于线粒体相关的 ER 膜结构域 （MAM）。 GPI-GnT 和 GPT 都是新颖的多亚基复合物：GPI-GnT 是 负责合成第一个GPI生物合成中间体， N-乙酰氨基葡萄糖磷脂酰肌醇，GPT 是附着酶 GPI 锚定于蛋白质。我们将定义酵母的亚基组成 GPI-GnT 复合物并表征其糖核苷酸和磷脂结合 使用光亲和标记、定点诱变的成分，以及 荧光共振能量转移。我们将类似地定义人类 GPT， 重点关注复杂的组成，亚基相互作用所需 内质网所需的复杂组装和靶向基序 本土化。 MAM 结构域代表 ER 内不寻常的异质性 通过隔离某些 GPI 生物合成酶，可能 有助于 GPI 生物合成的调节。我们将重点关注 PIG-L， 该途径的第二种酶，已知其活性位于 妈妈。我们将使用截短的嵌合 PIG-L 构建体、亚细胞 分级分离、荧光和电子显微镜以及单细胞 荧光光漂白技术研究 PIG-L 的靶向性 MAM 并描述其在 MAM 内的动态特征。