Flavoproteins in Oxidative Protein Folding

氧化蛋白折叠中的黄素蛋白

• 批准号: 8059050
• 负责人: Colin Thorpe
• 金额: $ 10万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-15 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8059050
• 关键词: Address; Anabolism; Apoptosis; Apoptotic; Arsenicals; Binding Sites; Biochemical Phenomena; Biochemical Process; Biochemical Reaction; Cell surface; Cells; Client; Disulfides; Enzymatic Biochemistry; Enzymes; Erythroid Cells; Eukaryota; Evaluation; Extracellular Matrix; Family; Family member; Flavins; Flavoproteins; Generations; Goals; Growth; Growth Factor; Homologous Gene; Human; In Vitro; Kinetics; Laboratories; Link; Liver Regeneration; Malignant Bone Neoplasm; Malignant Neoplasms; Mammalian Cell; Mammals; Methods; Mitochondria; Nature; Neuroblastoma; Organism; Oxidases; Oxidation-Reduction; Participant; Pathway interactions; Physiological; Placental Growth Factor; Play; Protein Disulfide Isomerase; Proteins; Recombinants; Regulation; Role; Site-Directed Mutagenesis; Structure; Substrate Specificity; Suggestion; Sulfhydryl Compounds; System; Thioredoxin; Tissues; Trypanosoma brucei brucei; Work; base; bone; chemotherapeutic agent; cytokine; design; disulfide bond; enzyme mechanism; extracellular; globular protein; in vivo; inhibitor/antagonist; member; protein folding; public health relevance; research study; riboflavin-binding protein; sulfhydryl oxidase

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.

描述（由申请人提供）：尽管二硫键对许多分泌蛋白的结构和一系列生化过程的调节至关重要，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。该申请的大部分内容涉及对黄素蛋白的Quiescin-巯基氧化酶（QSOX）家族知之甚少。这些脊椎动物氧化酶将二硫键直接引入未折叠的还原蛋白质中，但也被鉴定为生长因子（例如骨源性生长因子、胎盘源性前列腺生长因子和红细胞刺激因子）。QSOX在许多癌症（最显著的是前列腺癌和骨癌）中强烈上调，并可能参与细胞外基质的重塑。本申请的六个目标中的第一个探索了使给定蛋白质成为哺乳动物QSOX的良好底物的那些因素。第二个目标是表征QSOX的蛋白质底物结合位点的性质。第三个目的是研究客户蛋白质氧化重折叠过程中蛋白质二硫键异构酶和QSOX之间的合作。第四个目标是表达，纯化和启动另外两个QSOX家族成员的第一个机制研究。人QSOX 2被认为是神经母细胞瘤细胞凋亡途径中的关键分子，值得对其酶机制和底物特异性进行研究。第二个预期的候选物是重组布氏锥虫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个硫氧还蛋白结构域，并可能显示出重要的，可利用的，酶机制的差异。第五个目标是继续设计，合成和体外评价砷基抑制剂的QSOX和相关的巯基氧化酶。最后一个目标是扩展表征较小的巯基氧化酶同系物QSOX，黄素连接的增强因子的肝再生（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，但其作为巯基氧化酶的细胞内作用、其底物特异性及其酶反应机制仍然知之甚少。总的来说，这个应用程序是针对更好地了解在高等真核生物中的二硫化物生成和异构化的氧化还原酶学。公共卫生相关性：这项研究研究了一个鲜为人知的酶家族，这些酶在蛋白质折叠、细胞外基质的形成和重塑以及肝组织的再生中发挥着不同的作用。这些蛋白质中的一些是在前列腺癌和骨癌中过度表达的组织生长因子。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。