Flavoproteins in Oxidative Protein Folding

虽然二硫键对许多分泌蛋白质的结构和调控都是至关重要的 在一系列生化过程中，它们在多细胞生物体中的生物合成仍然令人惊讶 很神秘。虽然氧化蛋白质折叠被认为几乎完全来自细胞内 展望，二硫键的产生、异构化和还原也发生在细胞外 哺乳动物细胞的表面。这个应用程序处理细胞外活跃的FAD依赖 容易引入二硫化物的巯基氧化酶(人Quiescin-巯基氧化酶1；QSOX1) 直接结合成未折叠的还原蛋白质。QSOX1在许多人类中高度上调 癌症(最明显的是乳腺癌、胰腺癌和前列腺癌)，促进黏附和侵袭 转化的细胞。蛋白二硫键异构酶(PDI)家族的成员也很活跃 它们支持病毒融合、血小板聚集和一系列癌症的侵袭性表型 细胞。本工作旨在了解QSOX1和PDI在KEY中的活动 表面/胞外硫醇/二硫化物(SH/SS)转化。三个具体目标中的第一个 建议开发灵敏的新荧光显微镜工具来评估表面SH/SS 在许多哺乳动物细胞类型中保持平衡。该目标结合了双色比率成像使用 包括固定细胞和活细胞，并使用常规和超分辨率荧光显微镜。 第二个目的是探讨下调人成纤维细胞中QSOX1的水平对细胞周期的影响。 SH/SS从质膜表面平衡进入细胞外基质。一个全面的 对细胞外间隙中QSOX1的生理底物的评估将有助于了解QSOX1的作用 黏附和侵袭中的氧化酶。第三个目标是使用一个 氧化还原成像与异构酶底物蛋白质组学鉴定相结合。新的 将开发灵敏的分析方法来评估单功能和多功能的有效性 砷和硝基苯衍生物作为面外PDI的抑制剂。总体而言，这三个目标将 有助于更好地理解所起作用的SH/SS转化的氧化还原酶学 在哺乳动物细胞行为中的关键作用。

Although disulfide bonds are critical to the structure of many secreted proteins, and to the regulation of a range of biochemical processes, their biosynthesis in multicellular organisms remains surprisingly cryptic. While oxidative protein folding has been regarded almost exclusively from an intracellular perspective, disulfide bond generation, isomerization and reduction also occur at the extracellular surface of mammalian cells. This application deals with an extracellularly-active FAD-dependent sulfhydryl oxidase (human Quiescin-sulfhydryl oxidase 1; QSOX1) that facilely introduces disulfide bonds directly into unfolded reduced proteins. QSOX1 is highly up-regulated in a number of human cancers (most notably of breast, pancreas and prostate) where it promotes adhesion and invasion of transformed cells. Members of the protein disulfide isomerase (PDI) family are also active exofacially where they support viral fusion, platelet aggregation, and the invasive phenotype of a range of cancer cells. The present work is directed towards understanding the activities of QSOX1 and PDI in key exofacial/extracellular thiol/disulfide (SH/SS) transformations. The first of three specific aims proposes the development of sensitive new fluorescence microscopy tools to evaluate surface SH/SS balance in a number of mammalian cell types. This aim combines two-color ratiometric imaging using both fixed and live cells and employs conventional and super-resolution fluorescence microscopies. The second aim explores the effects of knockdown of QSOX1 levels in human fibroblast cells on the SH/SS balance from the plasma membrane surface into the extracellular matrix. A comprehensive evaluation of the physiological substrates of QSOX1 in the extracellular space will inform the roles of the oxidase in adhesion and invasion. The third aim addresses the exofacial roles of PDI using a combination of redox imaging and the proteomic identification of isomerase substrates. New sensitive assays will be developed to evaluate the effectiveness of mono- and multifunctional arsenical and nitrostyrene derivatives as inhibitors of exofacial PDI. Overall, these three aims will contribute to a better understanding of the redox-enzymology of SH/SS transformations that play critical roles in mammalian cell behavior.