Flavoproteins in Oxidative Protein Folding

虽然二硫键对许多分泌蛋白的结构和对蛋白质的调节是至关重要的， 在一系列生物化学过程中，它们在多细胞生物中的生物合成仍然令人惊讶 很神秘虽然氧化蛋白质折叠几乎完全被认为是从细胞内的 从这个角度来看，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。本申请涉及一种细胞外活性的FAD依赖性 容易引入二硫化物的巯基氧化酶（人Quiescin-sulfhydryl oxidase 1; QSOX 1 直接结合到未折叠的还原蛋白质中。QSOX 1在许多人中高度上调， 癌症（最明显的是乳腺癌、胰腺癌和前列腺癌），其中它促进粘附和侵袭， 转化细胞蛋白质二硫键异构酶（PDI）家族的成员也在外面部具有活性 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 细胞目前的工作是针对了解活动的QSOX 1和PDI的关键 外表面/细胞外硫醇/二硫化物（SH/SS）转化。三个具体目标之一 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， 固定和活细胞，并采用常规和超分辨率荧光显微镜。 第二个目的是探索在人成纤维细胞中敲低QSOX 1水平对细胞凋亡的影响。 SH/SS平衡从质膜表面进入细胞外基质。全面 对QSOX 1在细胞外空间中的生理底物的评估将告知以下作用： 氧化酶在粘附和侵袭中的作用。第三个目的是使用一个新的方法来解决PDI的外面部作用。 氧化还原成像和异构酶底物的蛋白质组学鉴定的组合。新 将开发敏感的检测方法，以评估单功能和多功能 砷和硝基苯乙烯衍生物作为外面部PDI的抑制剂。总的来说，这三个目标将 有助于更好地理解SH/SS转化的氧化还原酶学， 在哺乳动物细胞行为中的重要作用。

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.