Flavoproteins in Oxidative Protein Folding

描述(申请人提供)：二硫键对分泌蛋白质的天然结构和一系列生化过程的调节至关重要，然而它们在多细胞生物体中的生物合成仍然知之甚少。这一建议继续研究了一个新发现的黄素酶家族(奎斯汀-硫基氧化酶，QSOX家族)，该家族由两个硫氧还蛋白结构域和一个4-螺旋束FAD结合结构域融合而成。硫氧还蛋白和黄素结合区相互协作，确保在氧还原为过氧化氢的过程中，二硫键容易地插入到未折叠的还原蛋白质中。这项提议的三个目的是使用非重组禽类巯基氧化酶作为这一新的黄素酶家族的原型成员。蛋白质二硫键异构酶(PDI)与QSOX在氧化折叠过程中的协同作用将通过稳态和快速反应动力学进行研究。多肽和未折叠还原蛋白在QSOX上是否存在结合部位将通过动力学和光谱实验进行评估。在砷(Ill)化合物或过渡金属存在的情况下，QSOX在周转过程中的抑制将被检查，以深入了解在体内调节巯基氧化酶活性的方法。第四个目标，禽类和人类QSOX酶的异源表达，将允许突变剖析三个CxxC基序在催化中的作用。人QSOX(QSOX-1)的表达将使我们能够首次详细表征其对小分子和大分子硫醇底物的催化专一性。第五和第六个目标将这项工作扩展到该酶的较小、独立的黄素蛋白近亲：人类重组ALR(肝脏再生的增强因子)和Erv2p(酵母中与QSOX功能最接近的对应物)。

DESCRIPTION (provided by applicant): Disulfide bonds critical to the native structure of secreted proteins, and to the regulation of a range of biochemical processes, however their biosynthesis in multicellular organisms remains poorly understood. This proposal continues investigation of a newly-recognized flavoenzyme family (the Quiescin-sulfhydryl oxidase, QSOX, family) formed from the fusion of two thioredoxin domains with a 4-helix bundle FAD-binding domain. Thioredoxin and flavin-binding domains cooperate to ensure the facile insertion of disulfide bonds into unfolded reduced proteins with the reduction of oxygen to hydrogen peroxide. Three aims of this proposal use the non-recombinant avian sulfhydryl oxidase as the prototypical member of this new flavoenzyme family. The cooperation between protein disulfide isomerase (PDI) and QSOX in oxidative folding will be investigated by steady-state and rapid-reaction kinetics. The existence of a binding site on QSOX for peptides and unfolded reduced proteins will be assessed by kinetic and spectroscopic experiments. The inhibition of QSOX during turnover in the presence of arsenic(Ill) compounds or transition metals will be examined for insights into ways to modulate sulfhydryl oxidase activity in vivo. The fourth aim, heterologous expression of both avian and human QSOX enzymes, will permit a mutational dissection of the roles of the three CxxC motifs in catalysis. Expression of a human QSOX (QSOX- 1) will allow the first detailed characterization of its catalytic specificity towards small and large thiol substrates. The fifth and sixth aims extend this work to the enzyme's smaller, stand-alone, flavoprotein cousins: human recombinant ALR (augmenter of liver regeneration) and Erv2p (the closest functional counterpart of QSOX in yeast).