Lipid Carriers in Membrane Glycoproteins

膜糖蛋白中的脂质载体

• 批准号: 7062506
• 负责人: WILLIAM J LENNARZ
• 金额: $ 29.39万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-08-01 至 2007-09-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7062506
• 关键词: SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; X ray crystallography; active sites; affinity chromatography; endoplasmic reticulum; enzyme activity; enzyme structure; glycoprotein biosynthesis; glycoproteins; high performance liquid chromatography; immunoprecipitation; lipid transport; mass spectrometry; matrix assisted laser desorption ionization; membrane lipids; membrane proteins; molecular cloning; protein disulfide isomerase; protein disulfide reductase (glutathione); protein folding; protein metabolism; thiols

DESCRIPTION (provided by applicant): There is growing evidence that lack of proper N-glycosylation can induce serious medical problems. It is also known that secretory glycoproteins that do not fold correctly can produce metabolic disorders. The objective of this program, using yeast as a model for higher eukaryotes, is to study two yeast enzymes involved in the metabolic fate of glycoproteins. One is protein disulfide isomerase (PDI), a lumenal enzyme of the endoplasmic reticulum (ER) that mediates correct disulfide bond formation in newly synthesized glycoproteins (and proteins). The other is peptide N-glycanase (PNGase), a cytoplasmic enzyme that removes oligosaccharide chains from glycopeptides and possibly unfolded glycoproteins. A collaborative effort will be made to obtain the three dimensional structure of PDI. Two major objectives are to understand how this enzyme acts as a sulfhydryl oxidase and as a disulfide isomerase, and if it acts as a chaperone. With respect to PNGase, it has been observed that it interacts with the proteosome via only one protein, Rad 23p. Therefore, in a collaborative effort, attempts will be made to crystallize and obtain the three-dimensional structures of PNGase and of Rad23p. Knowledge of these structures will be very important because deletion analyses have established which domains of these proteins are essential for their interaction. PNGase in mice and humans contains a N-terminal extension that is required for interaction with the mouse HR23Bp, and interestingly, with a number of other proteins related to the proteosome. To understand these multiple protein interactions, three approaches will be taken. One is to isolate the proteosome and determine if mHR23Bp, mPNGase and other proteins involved in ubiquitination are in this proteosome complex. The second approach will be to determine the sequence of reactions that result in the destruction of misfolded glycoproteins by a reconstitution approach involving the individual components. The third will be deletion analysis of these various mouse proteins, especially mAMFR, (an E3 ligase and a cell surface receptor) and mS4 (a component of the 19S "lid" of the proteosome) and mPNGase. Overall the proposed studies should provide much insight into the folding of glycoproteins containing disulfide bonds and the destruction of glycoproteins that cannot achieve their native state.

描述（由申请人提供）：越来越多的证据表明，缺乏适当的 N-糖基化会导致严重的医疗问题。还已知，不正确折叠的分泌糖蛋白会产生代谢紊乱。该计划的目的是使用酵母作为高等真核生物的模型，研究两种参与糖蛋白代谢命运的酵母酶。一种是蛋白质二硫键异构酶 (PDI)，它是内质网 (ER) 的一种腔内酶，可介导新合成的糖蛋白（和蛋白质）中正确的二硫键形成。另一种是肽 N-聚糖酶 (PNGase)，一种细胞质酶，可从糖肽和可能的未折叠糖蛋白中去除寡糖链。将共同努力获得 PDI 的三维结构。两个主要目标是了解这种酶如何充当巯基氧化酶和二硫键异构酶，以及它是否充当伴侣。对于 PNGase，据观察它仅通过一种蛋白质 Rad 23p 与蛋白酶体相互作用。因此，在共同努力下，将尝试结晶并获得PNGase和Rad23p的三维结构。了解这些结构非常重要，因为缺失分析已经确定了这些蛋白质的哪些结构域对于它们的相互作用至关重要。小鼠和人类中的 PNGase 包含一个 N 末端延伸，这是与小鼠 HR23Bp 相互作用所必需的，有趣的是，它还与许多其他与蛋白酶体相关的蛋白质相互作用。为了理解这些多种蛋白质相互作用，将采取三种方法。一是分离蛋白酶体，并确定 mHR23Bp、mPNGase 和其他参与泛素化的蛋白质是否存在于该蛋白酶体复合物中。第二种方法是通过涉及各个成分的重构方法来确定导致错误折叠糖蛋白破坏的反应顺序。第三个是对这些不同的小鼠蛋白的缺失分析，特别是 mAMFR（一种 E3 连接酶和细胞表面受体）和 mS4（蛋白酶体 19S“盖子”的组成部分）和 mPNGase。总体而言，拟议的研究应该对含有二硫键的糖蛋白的折叠以及无法达到其天然状态的糖蛋白的破坏提供更多的见解。