Mechanisms of Copper Transport and Catalysis

铜的传输和催化机制

• 批准号: 10610381
• 负责人: Ninian J Blackburn
• 金额: $ 61.14万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10610381
• 关键词: Amides; Binding; Binding Sites; Biology; Catalysis; Cells; Chemicals; Chemistry; Complex; Copper; Coupled; Development; Electron Transport; Ensure; Enzymes; Equilibrium; Escherichia coli; Eukaryotic Cell; Homeostasis; Host Defense; Ions; Label; Ligands; MAP2K1 gene; Macrophage; Mammalian Cell; Metals; Mixed Function Oxygenases; Mononuclear; New Territories; Outcomes Research; Oxidation-Reduction; Oxygen; Pathogenicity; Pathway interactions; Phosphotransferases; Physiological; Prokaryotic Cells; Pump; Reaction; Research; Roentgen Rays; Role; Selenomethionine; Signal Transduction; Structure; Toxic effect; Trace Elements; Virulence; absorption; cofactor; combat; design; emission spectroscopy; novel; pathogen; peptide hormone; peptidylglycine alpha-amidating monooxygenase; programs; structural determinants

PROJECT SUMMARY Copper is an essential trace element utilized by eukaryotic cells as an enzymatic redox cofactor. At high concentrations the metal is toxic, and complex homeostatic mechanisms have developed to ensure that it is maintained at the correct physiological level. Prokaryotes such as gram negative pathogens have a diminished requirement for the metal, and show sensitivity to copper at levels well tolerated by mammalian cells. For this reason, macrophages utilize pathogenic copper toxicity as part of their defense against invaders while the latter combat the host-defense by upregulating their exporter machinery. Our research is focused on the mechanisms of copper transport and catalysis and is divided into three separate but overlapping projects. Project 1 examines the reaction mechanism of mononuclear monooxygenases exemplified by peptidylglycine monooxygenase (PHM), the only enzyme capable of installing the critical post-translational amide into peptide hormones. We have identified three important unanswered questions – (i) the pathway for electron transfer (ii) the chemical speciation of reaction intermediates and (iii) the mechanism of substrate triggering, and have designed an approach for interrogating each. Project 2 focuses on the mechanism of pathogenic copper export via a study of the CusCBAF exporter of E. coli. Here we use a novel labeling approach to track the flux of metal ions as they move through the pump. We build on recent results that have successfully determined the role CusF and CusB in metalating CusA, the rates of copper transfer from CusF to CusB, and the identity of the CusF-CusB complex as a shared ligand intermediate. Unanswered questions include the mechanism of CusF to CusA metal exchange, the role of metal-binding residues in selectivity and efficiency, and the function of the novel His2Phe Cu(I)/Ag(I) binding site in the CusS regulator. Project 3 takes our program into new territory by investigating structure/function of the newly discovered copper- dependent kinases MEK1/2 and ULK1/2 involved in cell signaling and tumorogenesis. The integrated approach to all three projects leverages the Pi's track record in developing toolsets that can interrogate the chemistry of copper, but particularly the Cu(I) state which reacts with oxygen, and is the form in which copper is trafficked in the cell. Two aspects of this effort are of particular utility: (i) development of metal-directed probes of Cu(I) coordinate structure utilizing x-ray absorption and emission spectroscopies and (ii) development of labeling strategies utilizing selenomethionine substitution coupled to Se K EXAFS. The expected outcome of the research is a better understanding of how biology leverages the fundamental chemistry of copper to achieve functionality, recognizing that function requires balance between the requirements for selective metalation, the structural determinants of catalysis, and pathogenic virulence induced by robust bacterial export pumps.

项目摘要 铜是真核细胞用作酶氧化还原辅助因子的必不可少的痕量元素。在高 金属的浓度是有毒的，并且已经开发出复杂的稳态机制来确保 它保持在正确的生理水平。原核生物（例如革兰氏阴性病原体）具有 对金属的需求减少，并显示出对铜的敏感性。 哺乳动物细胞。因此，巨噬细胞利用致病铜毒性作为其一部分 防御入侵者的防御，而后者通过上调其出口商来对抗宿主防卫 机械。我们的研究侧重于铜运输和催化的机制，并分为 分为三个单独但重叠的项目。项目1考试反应机制 单核单加氧酶用肽基甘氨酸单加氧酶（PHM）示例，这是唯一的酶 能够将关键翻译后酰胺安装到肽激素中。我们已经确定了三个 重要的未解决问题 - （i）电子传输的途径（ii）化学规范 反应中间体和（iii）底物触发机理，并设计了一种方法 用于审问每个。项目2通过研究 大肠杆菌的Cuscbaf出口商。在这里，我们使用一种新颖的标签方法来跟踪金属离子的通量 他们穿过泵。我们以最新的结果为基础，这些结果成功地确定了角色 cusf和cusb在金属化CUSA中，铜从CUSF转移到CUSB的速率以及 CUSF-CUSB复合物作为共享配体中间体。未解决的问题包括 CUSF与CUSA金属交换的机制，金属结合保留在选择性和 效率以及新型HIS2Phe Cu（i）/ag（i）结合位点的功能。项目3 通过研究新发现的铜的结构/功能将我们的程序带入新领域。 依赖性激酶MEK1/2和ULK1/2参与细胞信号传导和肿瘤发生。集成 所有三个项目的方法都利用PI的往绩来开发可以询问的工具集 铜的化学反应，尤其是与氧反应的铜（i）状态，是 哪个铜被贩运在细胞中。这项工作的两个方面特别是实用性：（i）开发 使用X射线滥用和排放的Cu（i）坐标结构的金属定向问题 光谱和（ii）利用硒甲氨酸替代的标签策略的制定 到se k exafs。研究的预期结果是对生物学的更好理解 利用铜的基本化学来实现功能，认识到功能需要 选择性金属的要求，催化的结构决定者和 强大的细菌导出泵引起的致病病毒。