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的糖蛋白的正确折叠。(2)确定细胞如何处理不正确折叠的糖蛋白。这些目标将通过研究蛋白二硫异构酶（PDI）和肽- n -聚糖酶（PNGase）两种酶来实现。在Specific Aim 1中，我们将把酵母PDI的结构联系起来，这是我们最近与Hermann Schindelin博士合作确定的，与这种酶在催化新生蛋白质折叠和生成二硫键的功能有关。此外，我们将解决人类PDI的晶体结构，以确定其与酵母PDI差异的结构基础。我们已经证明了另一种酶，PNGase，它将错误折叠的蛋白质去糖基化，是由五种蛋白质组成的降解复合物的一部分，这些蛋白质在体外相互作用，我们还与Hermann Schindelin博士合作，对PNGase的各种结构域进行了结构分析，包括与其结合伙伴的共晶结构。特异性目的2是研究这种多蛋白复合物是否存在于体内，以及它是否在将错误折叠的糖蛋白从内质网管腔输送到细胞质溶胶中起作用，在细胞质溶胶中它们被去糖基化和降解。这些研究具有很高的医学相关性，因为有许多人类疾病涉及错误折叠的蛋白质。错误折叠（糖）蛋白的无效去除导致聚集形成，这种情况发生在阿尔茨海默病、帕金森病、亨廷顿病、克雅氏病和戈谢氏病以及许多其他退行性疾病中。越来越多的人类遗传疾病被证明是由分泌蛋白运输问题引起的，包括由囊性纤维化跨膜传导调节因子（CFTR）突变引起的囊性纤维化、肺气肿和肝硬化。1-抗胰蛋白酶缺乏与凝血因子缺陷患者血友病的关系

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.