Targeted molecular strategies for cellular investigation of metalloproteins

金属蛋白细胞研究的靶向分子策略

• 批准号: 10688230
• 负责人: Emily L Que
• 金额: $ 31.66万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10688230
• 关键词: Affinity; Area; Binding; Biology; Cell model; Cell physiology; Cells; Chemicals; Complex; Copper; Development; Disease; Elements; Enzymes; Fluorescence Microscopy; Goals; Health; Heme; Histone Deacetylase; Homeostasis; Image; Inductively Coupled Plasma Mass Spectrometry; Investigation; Ions; Iron; Ligands; Ligation; Malignant Neoplasms; Malnutrition; Matrix Metalloproteinases; Metal Ion Binding; Metalloproteins; Metals; Micronutrients; Molecular; Molecular Target; Monitor; Organism; Pathology; Physiology; Population; Process; Protein Analysis; Proteins; Proteome; Proteomics; Roentgen Rays; Role; Site; Superoxide Dismutase; Synchrotrons; Tissues; Zinc; beta-Lactamase; cancer cell; carbonate dehydratase; combat; design; diagnostic strategy; fluorophore; insight; interest; live cell imaging; metalloenzyme; small molecule; therapeutic development; tool

Project Summary/Abstract Metals are essential micronutrients that are required for proper functioning of cells and organisms. As such, cells have evolved a complex homeostatic machinery to control the distribution and speciation of metals, also known as the metallome. Deviations from basic metallomic profiles are associated with multiple disease processes, environmental metal contamination, and nutritional deficiencies and all have detrimental effects on normal cellular function. The metallome is comprised of two main metal ion pools including the labile metal ion pool in which metals are weakly bound to cellular ligands and the tightly-bound metal ion pool in which metals are ligated to metalloproteins and other biomolecules with high affinity. Metalloproteins that constitute the tightly-bound metal ion pool represent one third of the cellular proteome and within these proteins, metals have diverse structural and catalytic functions. These metalloproteins can exist in several states including apo (metal-free), holo (metal-bound), and mismetalated depending on the cellular context. While some information is know about how the metalation state of selected metalloproteins is controlled (via metallochaperones and/or action of specific metal transporters) the details of how the labile and metalloprotein-bound metal pools interact in a cellular context as cells undergo dynamic changes is largely unknown and is a growing area of interest in the metal homeostasis field. In order to probe these interactions and characterize how the pool of metalloproteins changes in physiology and pathology, our goal is to develop a diverse chemical toolbox that will enable imaging, identification, quantification, and molecular control of metalloprotein populations in cells, tissues, and organisms. Our strategy centers on the use of precisely design molecular targeting groups that will specifically interact with the metal sites of these proteins. These targeting groups are then modified with functional tags including the following: 1) Fluorophores for live cell imaging and cell lysate protein analysis; 2) Affinity probes for proteomics studies that enable trapping of metalloproteins in their native metallation state and; 3) Photoresponsive groups for the selective control of specific metalloproteins and the development of photopharmacophores. Initial studies have focused on zinc-dependent metalloenzymes including carbonic anhydrases; however studies will be expanded to include a range of enzymes dependent on zinc (e.g. metallo- β-lactamases, histone deacetylases, matrix metalloproteinases), iron (e.g. heme and non-heme) and copper (e.g. superoxide dismutase). Tools will be applied in cellular models of metal dyshomeostasis and for profiling metalloprotein pools in cancer cells and others and will be combined with additional metallomic analysis including elemental analysis (inductively coupled plasma mass spectrometry, synchrotron-based x-ray fluorescence microscopy) and imaging of labile metal ion pools. Insights gained from the molecular tools furnished by these studies will contribute to the identification of metalloproteins of interest in cancer and other diseases and the development of therapeutic and diagnostic strategies to combat them.

项目摘要/摘要 金属是必需的微量营养素，是细胞和生物的正确功能所必需的。像这样， 细胞已经进化了一种复杂的稳态机制，以控制金属的分布和规格，也 被称为金属。与基本金属谱的偏差与多种疾病有关 过程，环境金属污染和营养缺乏，都对 正常的细胞功能。金属组完成了两个主要金属离子池，包括不稳定的金属离子 金属与细胞配体和紧密结合的金属离子池薄弱结合的池中 将金属蛋白蛋白和其他具有高亲和力的生物分子结扎。构成构成的金属蛋白 紧密结合的金属离子池代表细胞蛋白的三分之一，在这些蛋白质中，金属具有 各种结构和催化功能。这些金属蛋白可以存在于包括APO在内的几个状态 （无金属），Holo（金属结合）和不易化的，具体取决于细胞环境。而一些信息 知道如何控制选定金属蛋白的金属化状态（通过金属蛋白和/或 特定金属转运蛋白的作用）细节不稳定和金属蛋白结合金属池如何相互作用 在细胞的情况下，由于细胞的发生动态变化在很大程度上是未知的，并且是对 金属稳态场。为了探究这些相互作用并表征了库的库 金属蛋白在生理和病理学上的变化，我们的目标是开发潜水员的化学工具箱 将启用对细胞中金属蛋白群体的成像，识别，定量和分子控制， 组织和组织。我们的策略集中于使用精确设计分子靶向组的使用 特别与这些蛋白质的金属位点相互作用。然后将这些靶向组修改为 功能标签包括以下内容：1）活细胞成像和细胞裂解物蛋白分析的荧光团； 2） 蛋白质组学研究的亲和力问题，可以使金属蛋白捕获其本机金属状态 和; 3）选择性控制特定金属蛋白的光反应组和 光电行动。最初的研究集中于锌依赖性的金属酶，包括碳 赤霉酶；但是，研究将扩展到包括一系列依赖锌的酶（例如，金属 - β-内酰胺酶，组蛋白脱乙酰基酶，基质金属蛋白酶），铁（例如血红素和非血红素）和铜 （例如，超氧化物歧化酶）。工具将应用于金属dyshomeostasis的细胞模型和分析 癌细胞和其他人的金属蛋白池，将与其他金属分析结合 包括元素分析（电感耦合等离子体质谱法，基于同步加速器的X射线 荧光显微镜）和不稳定金属离子池的成像。从分子工具中获得的见解 这些研究提供的将有助于鉴定癌症和其他癌症中感兴趣的金属蛋白 疾病和理论和诊断策略的发展。