Dissecting and Reconstructing the Dolichol Pathway

描述（由申请人提供）：该计划的目标是研究组成多萜醇途径的生物合成酶的综合作用。具体而言，研究将集中在催化前七个反应的酶，这些反应发生在内质网（ER）膜的胞质表面上，并导致焦磷酸多萜醇-GlcNAc 2 Man 5的生物合成。N-连接糖基化是所有真核生物中必不可少的过程，导致新生糖蛋白生物合成的步骤在整个真核生物界是高度保守的。尽管该途径在真核细胞中的重要性，但对组成酶的结构、机制和综合功能知之甚少。本项目的研究将集中在酵母S.啤酒。了解酵母酶的进展将直接使哺乳动物同源物的鉴定，并提供深入了解人类和其他哺乳动物的多萜醇途径。 该研究方案的具体目标如下： 1.鉴定、生物化学表征和开发用于催化多萜醇途径中七个连续转化序列的酶的同源或异源表达系统。将采用生物化学方法鉴定序列中“缺失”的酶。 2.发展实验方法以研究底物如何沿着多萜醇途径中酶的“装配线”转移。在体内和体外的方法，用于评估的途径和作用的多萜结合底物将被提交。 3.研究多萜醇合成途径中催化多萜醇合成的酶的底物特异性。将评价糖基供体和受体底物的专属性。具体地说，我们感兴趣的是评估多萜醇途径酶是否可以将糖直接转移到糖肽受体。这样的发现对于制备定制的糖蛋白产物将是有用的。 4.对该途径中的靶点进行结构分析。这一具体目标将与格鲁吉亚大学的Prestegard教授合作进行核磁共振分析，与波士顿大学医学院的Karen艾伦教授合作进行X射线分析。

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.