Biosynthesis of N-linked Glycoproteins: Two Fates - Folding or Degradation

描述(由申请人提供)：本提案提出的长期目标是：(1)更好地了解细胞如何确保新合成糖蛋白的正确折叠。(2)确定细胞如何处理错误折叠的糖蛋白。这些目标将通过研究两种酶来实现，即蛋白质二硫键异构酶(PDI)和多肽-N-葡聚糖酶(PNGase)。在具体目标1中，我们将把我们最近与Hermann Schindelin博士合作确定的酵母PDI的结构与该酶在新生蛋白质中催化折叠和产生二硫键的功能联系起来。此外，我们还将解开人PDI的晶体结构，以确定其与酵母PDI不同的结构基础。我们已经证明了另一种酶PNGase，它可以去糖基化错误折叠的蛋白质，是由五种在体外相互作用的蛋白质组成的降解复合体的一部分，我们还与Hermann Schindelin博士合作对PNGase的各个结构域进行了结构分析，包括与其一些结合伙伴的共晶结构。具体目的2是研究这种多蛋白复合体是否在体内存在，以及它是否在将错误折叠的糖蛋白从内质网的管腔传送到胞浆中发挥功能，在那里它们被去糖化和降解。这些研究具有很高的医学意义，因为有许多人类疾病涉及错误折叠的蛋白质。去除错误折叠(糖蛋白)的效率低下会导致聚集体形成，这会发生在阿尔茨海默氏症、帕金森氏症、亨廷顿病、克雅氏病、高谢病和许多其他退行性疾病中。越来越多的人类遗传病是由分泌性蛋白运输问题引起的，包括囊性纤维化跨膜传导调节因子(CFTR)突变引起的囊性纤维化，β1抗胰蛋白酶缺乏引起的肺气肿和肝硬变，以及凝血因子缺陷患者的血友病。

DESCRIPTION (provided by applicant): The long-term objectives are presented in this proposal are: (1) To gain a better understanding of how cells ensure the correct folding of newly synthesized glycoproteins. (2) To determine how cells dispose of incorrectly folded glycoproteins. These objectives will be accomplished by studying two enzymes, protein disulfide isomerase (PDI) and peptide-N-glycanase (PNGase). In Specific Aim 1 we will relate the structure of yeast PDI, which we have recently determined in collaboration with Dr. Hermann Schindelin to the function of this enzyme in catalyzing the folding and generation of disulfide bonds in nascent proteins. In addition, we will solve the crystal structure of human PDI in order to determine the structural basis for its differences from yeast PDI. We have demonstrated that the other enzyme, PNGase, which deglycosylates misfolded proteins is part of a degradation complex that consists of five proteins that interact in vitro, and we have also collaborated with Dr. Hermann Schindelin in the structural analyses of various domains of PNGase including cocrystal structures with some of its binding partners. Specific Aim 2 is to investigate whether this multi-protein complex exists in vivo and if it functions in routing misfolded glycoproteins out of the lumen of the ER into the cytosol, where they are deglycosylated and degraded. These studies are of high medical relevance because there are numerous human disorders that involve misfolded proteins. Inefficient removal of misfolded (glyco) proteins results in aggregate formation, which occurs in Alzheimer's, Parkinson's, Huntington's, Creutzfeldt-Jakob, and Gaucher's disease, and many other degenerative disorders. An increasing number of human genetic diseases turn out to be caused by secretory protein trafficking problems including cystic fibrosis caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), emphysema and cirrhosis due to ?1-antitrypsin deficiency, and hemophilia in patients with defects in coagulation factors.