LIPID CARRIERS IN MEMBRANE GLYCOPROTEIN SYNTHESIS

大多数分泌蛋白和膜蛋白是N-糖基化的，这种修饰通常对其稳定性和功能有深远的影响。事实上，很明显，在许多人类疾病中，蛋白质糖基化被改变。 虽然我们现在对N-糖基化的基本机制有了很好的理解，但控制新形成的N-连接糖蛋白随后折叠成其最终稳定的三维结构的因素还没有很好的理解。 此外，我们对不能正确折叠的糖蛋白如何被分解代谢只有部分了解。 我们将研究简单真核生物S.酿酒酵母，因为这种生物可以很容易地进行基因操作。 关于二硫键的形成在内质网（ER）的蛋白质折叠过程中，我们将研究的机制，存在于内质网，蛋白质二硫键异构酶（PDI）的管腔中的酶。 这种酶催化硫醇氧化形成二硫键和这些二硫键的异构化。 此外，PDI充当伴侣。 我们已经准备了一系列位点特异性的半胱氨酸到丝氨酸突变体，以及一组PDI的C-末端缺失，并将在体外和体内实验中使用这些，以更好地了解PDI促进蛋白质折叠和氧化和二硫键异构化的机制。 在新合成的糖蛋白在内质网中不能正确折叠的情况下，在高等真核生物中已经表明，这些错误折叠的蛋白质从内质网中输出到胞质溶胶中并降解;正在积极研究其catenorization的机制。 在酵母中，这种处理过程不太清楚，特别是关于糖蛋白上聚糖的命运。 最近发现的一种可溶性酶，PNGase，可以使糖蛋白去糖基化，可能在酵母的这一过程中起关键作用。因此，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在错误折叠蛋白质的催化中的可能功能。 此外，将在体外研究酶的底物特异性。 由于ER内的PDI和胞质溶胶中的PNGase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节糖蛋白折叠和catalysts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.