Flavoproteins in Oxidative Protein Folding

描述(申请人提供)：虽然二硫键对许多分泌蛋白质的结构和一系列生化过程的调节是关键的，但它们在多细胞生物体中的生物合成仍然令人惊讶地神秘。本申请涉及几种进化相关的依赖于FAD的巯基氧化酶：黄素蛋白Quiescin-巯基氧化酶(QSOX)家族的成员，以及较小的单域Erv样氧化酶的代表，肝再生增强因子(ALR)。QSOX酶将二硫键直接引入到未折叠的还原蛋白中，但也被鉴定为脊椎动物的生长因子(例如骨源性生长因子、胎盘源性躺卧生长因子和红系细胞刺激因子)。QSOX1在许多人类癌症(最明显的是前列腺癌和胰腺癌)中表达强烈上调，可能参与细胞外基质的重塑。ALR与QSOX具有相同的FAD结合结构域，在线粒体的膜间隙中以长形式(LfALR)存在，以短形式(SfALR)在多种细胞和细胞外部位发挥作用。本申请的三个具体目标中的第一个目的是探索两种不同的QSOX酶(人QSOX1和来自原生动物寄生虫布鲁氏锥虫的较简单的QSOX)催化未折叠还原蛋白底物有效氧化的分子机制。第二个目标是通过定量的高通量筛选寻找这些酶的抑制剂，并继续设计针对生物学中CxxC基序的砷抑制剂。第三个目标涉及短期和长期形式的ALR。我们将扩大我们对SF-ALR和lfALR的结晶学研究，并利用快速反应技术探索lfALR催化的还原和氧化部分。最后，我们打算重建由lfALR驱动的氧化蛋白质折叠途径，并在体外检测它们的动力学能力。总体而言，这三个目标将有助于更好地理解高等真核生物氧化蛋白质折叠的氧化还原酶学。

DESCRIPTION (provided by applicant): 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. This application deals with several evolutionarily-related FAD-dependent sulfhydryl oxidases: members of the Quiescin-sulfhydryl oxidase (QSOX) family of flavoproteins, and a representative of the smaller single-domain Erv-like oxidases, augmenter of liver regeneration (ALR). The QSOX enzymes introduce disulfide bonds directly into unfolded reduced proteins, but have also been identified as growth factors in vertebrates (e.g. bone-derived growth factor, placental-derived prostrate growth factor, and erythroid cell stimulating factor). QSOX1 is strongly up-regulated in a number of human cancers (most notably of prostrate and pancreas) and may be involved in the remodeling of the extracellular matrix. ALR shares the same FAD-binding domain as QSOX and is found in a long form (lfALR) in the intermembrane space of the mitochondrion and in a short form (sfALR) functioning in a variety of cellular and extracellular locales. The first of three specific aims of this application explores the molecular mechanism by which two diverse QSOX enzymes (human QSOX1 and the simpler QSOX from the protozoan parasite Trypanosoma brucei) catalyze the efficient oxidation of unfolded reduced protein substrates. The second aim is to search for inhibitors of these enzymes by quantitative high-throughput screening and to continue the design of arsenical inhibitors targeting CxxC motifs in biology. The third aim deals with short and long forms of ALR. We will extend our crystallographic investigations of sf- and lfALR and probe the reductive and oxidative halves of lfALR catalysis by rapid reaction techniques. Finally, we intend to reconstitute oxidative protein folding pathways driven by lfALR and examine their kinetic competence in vitro. Overall, these three aims will contribute to a better understanding of the redox-enzymology of oxidative protein folding in higher eukaryotes.