ROLES OF OST3/6 THIOL OXIDOREDUCTASES IN N-LINKED PROTEIN GLYCOS/REDOX HOMEOSTAS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 该项目的总体目标是提供对OST 3/6巯基氧化还原酶在Nlinked 蛋白质糖基化和ER氧化还原稳态。硫醇依赖的氧化还原过程涉及 氧化应激防御、信号转导和蛋白质折叠、修饰和调节， 被称为硫醇氧化还原酶的结构不同的酶家族催化。大量ER硫醇 参与蛋白质折叠的氧化还原酶已经被表征;然而， 并且糖基化的特征仍然很差。ER中的N-连接蛋白质糖基化是一个重要的 这是真核生物蛋白质折叠过程中的一个关键步骤。OST 3/6蛋白在ER中含量丰富 参与N-连接蛋白氧化还原控制的膜连接硫氧还蛋白-折叠巯基氧化还原酶 寡糖基转移酶复合物中的糖基化。OST 3/6缺陷与严重的蛋白质 糖基化不足和ER应激。人OST 3/6样蛋白N33的纯合缺失与 转移性前列腺癌及其等位基因缺失与人结肠直肠癌和胰腺癌相关。 这一观察结果表明N33可能具有肿瘤抑制功能。此外，还有两个已知的案例 人类N33基因的自然敲除与非综合征性精神发育迟滞有关。在 本研究将系统地研究OST 3/6蛋白的生物学功能。的影响 OST 3/6缺陷将在蛋白糖基化和ER应激的效率方面进行检查。可能 利用巯基介导的底物捕获方法和全局基因， 表情分析OST 3/6在二硫键形成中的作用将在一系列硫醇中得到解决。 氧化还原酶测定。这些实验将在酿酒酵母和哺乳动物中进行 细胞我们还想开发OST 3/6敲除小鼠模型。这些模型将提供工具， 更好地了解OST 3/6蛋白缺乏对癌症和脑部疾病的影响， 对OST 3/6的生物学功能分析具有重要意义。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall goal of this project is to provide understanding of the roles of OST3/6 thiol oxidoreductases in Nlinked protein glycosylation and ER redox homeostasis. Thiol-dependent redox processes are involved in oxidative stress defense, signal transduction, and protein folding, modification and regulation, and are catalyzed by structurally distinct families of enzymes known as thiol oxidoreductases. Numerous ER thiol oxidoreductases involved in protein folding have been characterized; however, the overall machinery of folding and glycosylation remains poorly characterized. The N-linked protein glycosylation in the ER is an essential process and a key step in the control of protein folding in eukaryotes. OST3/6 proteins are abundant ER membrane-linked thioredoxin-fold thiol oxidoreductases involved in the redox control of N-linked protein glycosylation in the oligosaccharyltransferase complex. OST3/6 deficiency is associated with severe protein underglycosylation and ER stress. Homozygous deletion of human OST3/6 like protein, N33, correlates with metastatic prostate cancer and its allelic deletion is associated with human colorectal and pancreatic cancers. This observation suggests a possible tumor suppressor function of N33. In addition, there are two known cases of a natural knockout of N33 in humans which are associated with nonsyndromic mental retardation. In the proposed study, we will systematically characterize the biological function of OST3/6 proteins. The effect of OST3/6 deficiency will be examined with regard to efficiency of protein glycosylation and ER stress. Possible targets of OST3/6 proteins will be identified using thiol-mediated substrate-trapping method and global gene expression analysis. The OST3/6 roles in disulfide bond formation will be addressed in series of thiol oxidoreductase assays. These experiments will be carried in yeast Saccharomyces cerevisiae and mammalian cells. We also would like to develop OST3/6 knockout mouse models. These models will provide tools in a better understanding of the consequences of OST3/6 protein deficiency on cancer and brain disorders and will be useful in the analysis of OST3/6 biological function.