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

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 本项目的总体目标是了解OST 3/6巯基氧化还原酶在N连接蛋白糖基化和ER氧化还原稳态中的作用。巯基依赖性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，并且由称为巯基氧化还原酶的结构上不同的酶家族催化。许多ER硫醇氧化还原酶参与蛋白质折叠的特点，然而，折叠和糖基化的整体机制仍然很差的特点。内质网中的N-糖基化是真核生物控制蛋白质折叠的重要过程和关键步骤。OST 3/6蛋白是丰富的ER膜连接的硫氧还蛋白折叠硫醇氧化还原酶，参与寡糖基转移酶复合物中N-连接蛋白糖基化的氧化还原控制。OST 3/6缺陷与严重的蛋白质糖基化不足和ER应激相关。人OST 3/6样蛋白N33的纯合缺失与转移性前列腺癌相关，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，有两个已知的人类N33基因自然敲除的病例与非综合征性精神发育迟滞有关。在本研究中，我们将系统地研究OST 3/6蛋白的生物学功能。将检查OST 3/6缺陷对蛋白糖基化和ER应激效率的影响。利用巯基介导的底物捕获法和全基因表达分析鉴定OST 3/6蛋白的可能靶点。OST 3/6在二硫键形成中的作用将在一系列硫醇氧化还原酶测定中得到解决。这些实验将在酿酒酵母和哺乳动物细胞中进行。我们还想开发OST 3/6敲除小鼠模型。这些模型将为更好地理解OST 3/6蛋白缺陷对癌症和脑疾病的后果提供工具，并将有助于分析OST 3/6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. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.