LIPID CARRIERS IN MEMBRANE GLYCOPROTEIN SYNTHESIS

大多数分泌蛋白和膜蛋白都是N-糖基化的，这种修饰往往会对它们的稳定性和功能产生深远的影响。事实上，很明显，在一些人类疾病中，蛋白质的糖基化发生了改变。虽然我们现在对N-糖基化的基本机制有了很好的了解，但控制新形成的N-连接糖蛋白随后折叠成最终稳定的三维结构的因素还不是很清楚。此外，我们对不能正确折叠的糖蛋白是如何分解代谢的只有部分了解。我们将在简单的真核生物酿酒酵母中研究糖蛋白的二硫键形成和折叠，以及未折叠糖蛋白的分解代谢，因为这种生物很容易被遗传操纵。关于内质网(ER)中蛋白质折叠过程中二硫键的形成，我们将研究内质网管腔中存在的一种酶--蛋白质二硫键异构酶(PDI)的机制。这种酶既催化硫醇氧化形成二硫键，又催化这些二硫键异构化。此外，PDI还充当监护人。我们已经准备了一系列丝氨酸突变的半胱氨酸位点，以及一组PDI的C端缺失，并将在体外和体内实验中使用这些来更好地了解PDI促进蛋白质折叠的机制以及在氧化和二硫键异构化中的功能。在新合成的糖蛋白在内质网中不能正确折叠的情况下，在高等真核生物中，这些错误折叠的蛋白被从内质网输出到胞浆中并降解；它们分解代谢的机制正在积极研究中。在酵母中，这种处理过程不太清楚，特别是关于糖蛋白上的多糖的命运。最近发现的一种可溶性酶，PNGase，它能使糖蛋白脱糖，可能在酵母的这一过程中发挥关键作用。因此，我们将对酵母PNGase进行克隆和测序。然后，在一系列体内实验中，将研究PNGase在错误折叠蛋白分解代谢中的可能功能。此外，该酶还将在体外进行底物专一性研究。由于内质网中的PDI和胞浆中的PNGase都与未折叠的蛋白质相互作用，因此对这两种酶的清楚理解将为调控糖蛋白折叠和分解代谢的因素提供新的见解。

Most secretory and membrane proteins are N-glycosylated and this modification often has profound effects on their stability and function. In fact, it is clear that in a number of human disorders protein glycosylation is altered. Although we now have a good understanding of the basic mechanism of N-glycosylation, the factors that control the subsequent folding of the newly formed N-linked glycoproteins into their final, stable three dimensional structure are not well understood. In addition, we only have a partial picture of how glycoproteins that do not fold correctly are catabolized. We will study disulfide bond formation and folding of glycoproteins, as well as the catabolism of unfolded glycoproteins, in the simple eukaryote, S. cerevisiae, because of the ease in which this organism can be genetically manipulated. With respect to disulfide bond formation during protein folding in the endoplasmic reticulum (ER), we will study the mechanism of an enzyme present in the lumen of the ER, protein disulfide isomerase (PDI). This enzyme catalyzes both the oxidation of thiols to form disulfide bonds and the isomerization of these disulfide bonds. In addition, PDI serves as a chaperone. We have prepared a collection of site specific cysteine to serine mutants, as well as a set of C-terminal deletions of PDI, and will use these in in vitro and in vivo experiments to better understand the mechanism by which PDI facilitates protein folding and functions in oxidation and disulfide bond isomerization. In the case of newly synthesized glycoproteins that do not fold correctly in the ER, it has been shown in higher eukaryotes that these misfolded proteins are exported out of the ER into the cytosol and degraded; the mechanism for their catabolism is being actively studied. In yeast this disposal process is less well understood, especially with respect to the fate of glycans on glycoproteins. A recently discovered soluble enzyme, PNGase, that deglycosylates glycoproteins may play a key role in this process in yeast. Therefore yeast PNGase will be cloned and sequenced. Then, in a series of in vivo experiments the possible function of PNGase in the catabolism of malfolded proteins will be investigated. In addition, the enzyme will be studied in vitro with respect to substrate specificity. Since both PDI inside the ER and PNGase in the cytosol interact with unfolded proteins, a clear understanding of both of these enzymes will provide new insights into factors regulating glycoprotein folding and catabolism.