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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 本项目的总体目标是提供对OST3/6硫醇氧化还原酶在Nlink中的作用的理解 蛋白质糖基化和内质网氧化还原动态平衡。硫醇依赖的氧化还原过程参与了 氧化应激防御、信号转导和蛋白质折叠、修饰和调节，以及 由结构不同的酶家族催化，称为硫醇氧化还原酶。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，折叠的整体机制 而且糖基化的特征仍然很差。内质网中N-连接蛋白的糖基化是必不可少的 这一过程是真核生物蛋白质折叠控制的关键步骤。OST3/6蛋白富含内质网 膜连接硫氧还蛋白-折叠硫醇氧化还原酶参与N-连接蛋白的氧化还原控制 寡糖转移酶复合体中的糖基化。OST3/6缺乏与严重的蛋白质有关 糖基化不足和内质网应激。人OST3/6样蛋白N33纯合缺失与 转移性前列腺癌及其等位基因缺失与人类结直肠癌和胰腺癌相关。 这一观察结果提示N33可能具有肿瘤抑制作用。此外，还有两个已知的案例 N33基因在人类中的自然敲除，与非综合征性精神发育迟滞有关。在 在提出的研究中，我们将系统地表征OST3/6蛋白的生物学功能。的影响 OST3/6缺乏症将从蛋白质糖基化和内质网应激的效率方面进行检查。可能的 将利用硫醇介导的底物捕捉法和全局基因识别OST3/6蛋白的靶点 表情分析。OST3/6在二硫键形成中的作用将在硫醇系列中讨论 氧化还原酶检测。这些实验将在酿酒酵母和哺乳动物身上进行。 细胞。我们还想开发OST3/6基因敲除小鼠模型。这些模型将在 更好地了解OST3/6蛋白缺乏对癌症和脑疾病的影响，并将 可用于分析OST3/6的生物学功能。

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.