Lipid Carriers in Membrane Glycoproteins

描述（由申请人提供）：越来越多的证据表明，缺乏适当的N-糖基化可能导致严重的医学问题。还已知不能正确折叠的分泌性糖蛋白可产生代谢紊乱。本项目的目的是利用酵母作为高等真核生物的模型，研究参与糖蛋白代谢命运的两种酵母酶。一种是蛋白质二硫键异构酶（PDI），一种内质网（ER）的内腔酶，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），这是一种从糖肽和可能未折叠的糖蛋白中去除寡糖链的细胞质酶。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作为巯基氧化酶和二硫键异构酶，以及它是否作为伴侣。关于PNGase，已经观察到它仅通过一种蛋白质Rad 23 p与蛋白体相互作用。因此，在合作努力中，将尝试结晶并获得PNGase和Rad 23 p的三维结构。这些结构的知识将是非常重要的，因为缺失分析已经确定了这些蛋白质的哪些结构域是它们相互作用所必需的。小鼠和人类中的PNGase含有与小鼠HR 23 Bp相互作用所需的N-末端延伸，有趣的是，与许多与蛋白体相关的其他蛋白质相互作用。为了理解这些多蛋白质相互作用，将采取三种方法。一种是分离蛋白体，并确定mHR 23 Bp，mPNGase和其他参与泛素化的蛋白质是否在该蛋白体复合物中。第二种方法是通过涉及单个组分的重建方法来确定导致错误折叠的糖蛋白破坏的反应顺序。第三个将是这些各种小鼠蛋白质的缺失分析，特别是mAMFR（E3连接酶和细胞表面受体）和mS 4（蛋白体的19 S“盖子”的组分）和mPNGase。总的来说，拟议的研究应该提供更多的了解折叠的糖蛋白含有二硫键和破坏的糖蛋白，不能达到其天然状态。

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.