Flavoproteins in Oxidative Protein Folding

氧化蛋白折叠中的黄素蛋白

• 批准号: 8685269
• 负责人: Colin Thorpe
• 金额: $ 33.66万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8685269
• 关键词: Anabolism; Animals; Arsenicals; Binding; Biochemical Process; Biological; Biology; Blood Circulation; Capsid Proteins; Catalysis; Cell Line; Cell physiology; Chemicals; Client; Collaborations; Competence; Complement; Complex; Development; Disulfides; Enzymatic Biochemistry; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Equilibrium; Erythroid Cells; Eukaryota; Extracellular Matrix; Family; Flavins; Flavoproteins; Generations; Genes; Genomics; Grant; Growth; Growth Factor; Housing; Human; Immune; In Vitro; Investigation; Kinetics; Laboratories; Learning; Liver Regeneration; Locales; Location; Malignant Neoplasms; Malignant neoplasm of pancreas; Malignant neoplasm of prostate; Mammals; Methods; Mitochondria; Molecular; Mutagenesis; N-terminal; Organism; Oxidants; Oxidases; Oxidation-Reduction; Oxygen; Pancreas; Parasites; Pathway interactions; Peptides; Physiological; Placental Growth Factor; Plants; Play; Post-Translational Protein Processing; Prostate; Protein Disulfide Isomerase; Proteins; RNA Interference; Reaction; Regulation; Role; Structural Protein; Structure; Techniques; Thioredoxin; Thioredoxin-2; Tissues; Trypanosoma brucei brucei; Tumorigenicity; United States National Institutes of Health; Vertebrates; Work; base; bone; catalyst; chemotherapeutic agent; cofactor; cytochrome c; cytokine; design; disulfide bond; enzyme mechanism; extracellular; flexibility; high throughput screening; inhibitor/antagonist; member; mitochondrion intermembrane space; novel therapeutics; oxidation; protein folding; reconstitution; research study; 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. 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.

描述（由申请人提供）：尽管二硫键对于许多分泌蛋白的结构以及一系列生化过程的调节至关重要，但它们在多细胞生物中的生物合成仍然令人惊讶地神秘。本申请涉及几种与进化相关的 FAD 依赖性巯基氧化酶：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，肝再生增强剂 (ALR)。 QSOX 酶将二硫键直接引入未折叠的还原蛋白中，但也已被鉴定为脊椎动物的生长因子（例如骨源性生长因子、胎盘源性前列腺生长因子和红细胞刺激因子）。 QSOX1 在许多人类癌症（尤其是前列腺癌和胰腺癌）中强烈上调，并且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，并以长形式 (lfALR) 存在于线粒体膜间隙中，以短形式 (sfALR) 在多种细胞和细胞外部位发挥作用。该应用的三个具体目标中的第一个是探索两种不同的 QSOX 酶（人类 QSOX1 和来自原生动物寄生虫布氏锥虫的简单 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高通量筛选来寻找这些酶的抑制剂，并继续设计针对生物学中的 CxxC 基序的砷抑制剂。第三个目标涉及 ALR 的短形式和长形式。我们将扩展 sf- 和 lfALR 的晶体学研究，并通过快速反应技术探测 lfALR 催化的还原和氧化部分。最后，我们打算重建由 lfALR 驱动的氧化蛋白折叠途径，并在体外检查其动力学能力。总的来说，这三个目标将有助于更好地理解高等真核生物中氧化蛋白折叠的氧化还原酶学。