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。这种应用的很大一部分涉及鲜为人知的Quiescin-巯基氧化酶(QSOX)黄素蛋白家族。这些脊椎动物氧化酶直接将二硫键引入未折叠的还原蛋白中，但也被鉴定为生长因子(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)。QSOX在许多癌症(尤其是前列腺癌和骨癌)中表达强烈上调，并可能参与细胞外基质的重塑。这项应用的六个目标中的第一个目的是探索使给定蛋白质成为哺乳动物QSOX良好底物的那些因素。第二个目标是描述QSOX与蛋白质底物结合的性质。第三个目的是研究蛋白质二硫键异构酶和QSOX在客户蛋白氧化重折叠过程中的协同作用。第四个目标是表达、纯化和启动对另外两个QSOX家族成员的机制研究。人QSOX2被认为是神经母细胞瘤细胞凋亡途径中的关键分子，值得研究其酶机制和底物特异性。第二个候选对象是重组布氏锥虫QSOX。与后生动物QSOX不同，它们的原生动物对应物基本上缺乏第二硫氧还蛋白结构域，并可能在酶机制上显示出重要的和可利用的差异。第五个目标是继续设计、合成和体外评估基于砷的QSOX抑制剂和相关的巯基氧化酶。最终目标扩展了QSOX的较小的巯基氧化酶同系物的特征，QSOX是黄素连接的肝再生增强因子(ALR)。虽然ALR在哺乳动物中具有一系列重要的细胞因子样功能，但其在细胞内的作用、其底物特异性及其酶反应机制尚不清楚。总体而言，这一应用旨在更好地理解高等真核生物中二硫键生成和异构化的氧化还原酶学。与公共健康相关：这项研究研究了一系列知之甚少的酶，它们在蛋白质折叠、细胞外基质的形成和重塑以及肝组织的再生中扮演着不同的角色。其中一些蛋白质是组织生长因子，在前列腺癌和骨癌中过度表达。更好地了解这些重要蛋白质的作用机制可能有助于化疗药物的设计。

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. Much of this application deals with the poorly-understood Quiescin-sulfhydryl oxidase (QSOX) family of flavoproteins. These vertebrate oxidases introduce disulfide bonds directly into unfolded reduced proteins but have also been identified as growth factors (e.g. bone-derived growth factor, placental-derived prostrate growth factor, and erythroid cell stimulating factor). QSOXs are strongly up-regulated in a number of cancers (most notably of prostrate and bone) and may be involved in the remodeling of the extracellular matrix. The first of six aims of this application explores those factors that make a given protein a good substrate of mammalian QSOX. The second goal is to characterize the nature of QSOX's binding site for protein substrates. A third aim investigates the cooperation between protein disulfide isomerase and QSOX during the oxidative refolding of client proteins. A fourth goal is to express, purify and initiate the first mechanistic study of two additional QSOX family members. Human QSOX2 has been described as a key player in the pathways to apoptosis in neuroblastoma cells and merits a study of enzyme mechanism and substrate specificity. A second intended candidate is recombinant Trypanosoma brucei QSOX. Unlike metazoan QSOXs, their protozoan counterparts substantially lack the second thioredoxin domain and may show important, and exploitable, differences in enzyme mechanism. A fifth aim is a continuation of the design, synthesis and in vitro evaluation of arsenical-based inhibitors of QSOX and related sulfhydryl oxidases. A final goal extends characterization of the smaller sulfhydryl oxidase homolog of QSOX, the flavin-linked augmenter of liver regeneration (ALR). While ALR has a range of important cytokine-like functions in mammals, its intracellular role as a sulfhydryl oxidase, its substrate specificity and its enzymatic reaction mechanism remain poorly understood. Overall, this application is directed towards a better understanding of the redox-enzymology of disulfide generation and isomerization in higher eukaryotes. PUBLIC HEALTH RELEVANCE: This research studies a family of poorly understood enzymes that play diverse roles in protein folding, in the formation and remodeling of the extracellular matrix, and in the regeneration of liver tissue. Some of these proteins are tissue growth factors that are over-expressed in prostrate and bone cancer. A better understanding of the mechanism of these important proteins may help in the design of chemotherapeutic agents.