二硫键对蛋白质结构和功能有重要意义，其形成是蛋白质氧化折叠的限速步骤。Peroxiredoxin 4(Prx4)利用H2O2通过氧化蛋白质二硫键异构酶(PDI)最终催化新生肽链中的二硫键形成，是一条全新的蛋白质氧化折叠通路。申请者前期工作发现Prx4与进行氧化折叠的底物蛋白直接反应引起蛋白质聚集；Prx4易被过氧化而丧失酶活力,尚不清楚这是对Prx4功能行使的一种调节形式,以上说明这一系统远比现有的线性模型复杂。拟重构Prx4介导蛋白质氧化折叠通路，在原有线性模型的基础上引入Prx4直接与底物蛋白发生全新反应的 Off-pathway及过氧化对Prx4活力的调节环路。解析这一通路重要蛋白复合物如Prx4-PDI, Prx4-Srx的晶体结构，阐释这一通路工作的结构基础。鉴定Prx4相互作用蛋白以尝试建立其与相关疾病间的初步联系。此项目有助于全面认识真核细胞内质网蛋白质氧化折叠及其生理意义。

Disulfide bonds are essential for structure and function of most secreted and membrane proteins. Disulfide bond formation is often the rate-limiting step in the oxidative protein folding. In the Peroxiredoxin 4 (Prx4) mediate oxidative protein folding pathway, Prx4 utilizes H2O2 to oxidize protein disulfide isomerase (PDI), which then introduces disulfide bonds into nascent peptides. According to our previous work, Prx4 could directly react with folding substrates causing protein aggregation, and Prx4 could be inactivated by H2O2-induced over-oxidation. All these observations suggest that the Prx4 mediated oxidative protein pathway is more complicated than the recent liner model. In this project, we are going to reconstitute the Prx4 mediated oxidative protein pathway, study interactions between components, identify the regulation mechanism by over-oxidation of Prx4.We will try to solve the crystal structures of Prx4-PDI and Prx4-Srx complexes to provide a structural basis for this pathway. Identification of Prx4's partner proteins will be also performed to give a better understanding of the roles of Prx4 in the related diseases. This project will help us to comprehensively understand the oxidative protein folding in the endoplasmic reticulum in eukaryotic cells.