Dissecting and Reconstructing the Dolichol Pathway

解剖和重建 Dolichol 通路

• 批准号: 6772245
• 负责人: Barbara Imperiali
• 金额: $ 27.55万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6772245
• 关键词: Saccharomyces cerevisiae; X ray crystallography; dolichol; enzyme mechanism; enzyme structure; enzyme substrate; fungal proteins; glycopeptides; nuclear magnetic resonance spectroscopy; structural biology

DESCRIPTION (provided by applicant): The goal of this program is to investigate the integrated action of the biosynthetic enzymes that comprise the dolichol pathway. Specifically, research will focus on the enzymes that catalyze the first seven reactions that take place on the cytosolic face of the endoplasmic reticulum (ER) membrane and lead to the biosynthesis of dolichol pyrophosphate-GIcNAc2Man5. N-linked glycosylation is an essential process in all eukaryotes, and the steps leading to the biosynthesis of nascent glycoproteins are highly conserved throughout the eukaryotic kingdom. Despite the significance of this pathway in eukaryotic cells, little is known about the structure, mechanism, and integrated function of the constituent enzymes. Studies in this program will focus on the enzymes comprising the dolichol pathway in the yeast S. cerevisiae. Progress in understanding the yeast enzymes will directly enable identification of mammalian homologs and provide insight into the dolichol pathway in humans and other mammals. The specific aims of this research program are as follows: 1.To identify, biochemically characterize, and develop homologous or heterologous expression systems for the enzymes that catalyze the sequence of seven contiguous transformations in the dolichol pathway. A biochemical approach will be adopted for the identification of the "missing" enzymes in the sequence. 2.To develop experimental approaches to investigate how substrates are transferred along the "assembly line" of enzymes in the dolichol pathway. Both in vivo and in vitro methods for evaluating the pathway and the role of the dolichol-bound substrates will be presented. 3.To investigate the substrate specificity of the enzymes catalyzing the early steps in the dolichol pathway. Specificity for both glycosyl donor and acceptor substrates will be evaluated. Specifically we are interested in evaluating whether the dolichol pathway enzymes can transfer saccharide directly to a glycopeptide acceptor. Such a finding would be of utility for the preparation of tailored glycoprotein products. 4.To carry out structural analysis of targets in the pathway. This specific aim will be carried out in collaboration with Prof. Prestegard at the University of Georgia for NMR analysis and Prof. Karen Allen in the at Boston University Medical School for X-ray analysis.

描述（由申请人提供）：该计划的目标是研究构成多醇途径的生物合成酶的综合作用。具体来说，研究将集中于催化内质网 (ER) 膜胞质表面上发生的前七个反应的酶，并导致多萜醇焦磷酸 -GlcNAc2Man5 的生物合成。 N-连接糖基化是所有真核生物中的一个重要过程，导致新生糖蛋白生物合成的步骤在整个真核王国中高度保守。尽管该途径在真核细胞中具有重要意义，但人们对组成酶的结构、机制和整合功能知之甚少。该计划的研究将重点关注酿酒酵母中构成多醇途径的酶。了解酵母酶的进展将直接实现哺乳动物同源物的鉴定，并深入了解人类和其他哺乳动物的多醇途径。 本研究计划的具体目标如下： 1.鉴定、生化表征并开发催化多醇途径中七个连续转化序列的酶的同源或异源表达系统。将采用生化方法来鉴定序列中“缺失”的酶。 2.开发实验方法来研究底物如何沿着多醇途径中酶的“装配线”转移。将介绍评估多醇结合底物的途径和作用的体内和体外方法。 3.研究催化多醇途径早期步骤的酶的底物特异性。将评估糖基供体和受体底物的特异性。具体来说，我们感兴趣的是评估多醇途径酶是否可以将糖直接转移到糖肽受体。这一发现对于制备定制糖蛋白产品将是有用的。 4.对通路中的靶点进行结构分析。这一具体目标将与乔治亚大学的 Prestegard 教授（负责 NMR 分析）和波士顿大学医学院的 Karen Allen 教授（负责 X 射线分析）合作实现。