Flavoproteins in Oxidative Protein Folding

描述（由申请人提供）：二硫键对分泌蛋白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中的生物合成仍然知之甚少。本研究继续研究了一个新发现的黄酶家族（quiestin -巯基氧化酶，QSOX，家族），它是由两个硫氧还蛋白结构域与一个4螺旋束fad结合结构域融合而成的。硫氧还蛋白和黄素结合域合作，确保二硫键在未展开的还原蛋白中随着氧还原为过氧化氢而容易插入。本提案的三个目的是利用非重组禽巯基氧化酶作为这个新黄酶家族的原型成员。本文将通过稳态和快速反应动力学研究蛋白二硫异构酶（PDI）和QSOX在氧化折叠中的协同作用。QSOX上是否存在肽和未折叠的还原蛋白的结合位点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作用将被检查，以了解体内调节巯基氧化酶活性的方法。第四个目标是禽和人QSOX酶的异源表达，将允许对三个CxxC基序在催化中的作用进行突变解剖。人类QSOX （QSOX- 1）的表达将允许首次详细表征其对小型和大型硫醇底物的催化特异性。第五个和第六个目标将这项工作扩展到酶的较小的，独立的黄素蛋白表兄弟：人类重组ALR（肝脏再生增强剂）和Erv2p（酵母中QSOX的最接近的功能对偶物）。

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).