Mechanisms of Copper Transport and Catalysis

铜的传输和催化机制

• 批准号: 10397998
• 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/10397998
• 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; 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; macrophage; novel; pathogen; peptide hormone; peptidylglycine alpha-amidating monooxygenase; programs

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 铜是真核细胞所必需的微量元素，作为酶促氧化还原辅因子。在高 浓度的金属是有毒的，复杂的稳态机制已经发展，以确保 维持在正确的生理水平。原核生物如革兰氏阴性病原体具有 减少了对金属的需求，并显示出对铜的敏感性， 哺乳动物细胞由于这个原因，巨噬细胞利用致病性铜毒性作为它们的一部分。 防御入侵者，而后者通过上调其输出者来对抗宿主防御 机械.我们的研究主要集中在铜的运输和催化机制， 分成三个独立但重叠的项目项目1研究了 单核单加氧酶，例如肽基甘氨酸单加氧酶（PHM）， 能够将关键的翻译后酰胺安装到肽激素中。我们已经确定了三个 重要的未回答的问题-（一）电子转移的途径（二）化学形态的 反应中间体和（iii）底物触发机制，并设计了一种方法 审问每个人项目2的重点是通过研究致病铜输出的机制， CusCBAF的E.杆菌在这里，我们使用一种新的标记方法来跟踪金属离子的通量， 它们通过泵移动。我们建立在最近的结果，成功地确定了作用， CuS F和CuS B在金属化CuS A中的作用，铜从CuS F转移到CuS B的速率，以及CuS F和CuS B在金属化CuS A中的同一性。 CusF-CusB复合物作为共享的配体中间体。未回答的问题包括 CusF到CusA金属交换的机制，金属结合残基在选择性中的作用， 效率，以及新型His 2 Phe Cu（I）/Ag（I）结合位点在CusS调节剂中的功能。项目3 通过研究新发现的铜的结构/功能，将我们的计划带入新的领域- 依赖性激酶MEK 1/2和ULK 1/2参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了Pi在开发工具集方面的记录，这些工具集可以询问 铜的化学性质，但特别是与氧反应的Cu（I）状态，并且是 哪种铜在细胞中被运输。这项工作的两个方面特别有用： 利用X射线吸收和发射的Cu（I）配位结构的金属定向探针 光谱和（ii）利用硒代甲硫氨酸取代偶联的标记策略的发展 到Se K EXAFS。这项研究的预期成果是更好地理解生物学如何 利用铜的基本化学性质来实现功能，认识到功能需要 选择性金属化的要求、催化的结构决定因素和 由强大的细菌输出泵诱导的致病性毒力。