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: 10390048
• 负责人: Emily L Que
• 金额: $ 4.81万
• 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/10390048
• 关键词: Affinity; Area; Biology; Cell model; Cell physiology; Cells; Chemicals; Complex; Copper; Development; Diagnostic; Disease; Enzymes; Fluorescence Microscopy; Goals; Health; Heme; Histone Deacetylase; Homeostasis; Image; Inductively Coupled Plasma Mass Spectrometry; Investigation; Ions; Iron; Ligands; Malignant Neoplasms; Malnutrition; Matrix Metalloproteinases; Metalloproteins; Metals; Micronutrients; Molecular; Molecular Target; Monitor; Organism; Pathology; Physiology; Population; Process; Protein Analysis; Proteins; Proteome; Proteomics; Roentgen Rays; Role; Site; Structure; Superoxide Dismutase; Synchrotrons; Tissues; Zinc; base; beta-Lactamase; cancer cell; carbonate dehydratase; combat; design; fluorophore; insight; interest; live cell imaging; metalloenzyme; microscopic imaging; 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, which 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 known 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 change 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 designed 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.

项目总结/摘要 金属是细胞和生物体正常运作所需的必需微量营养素。因此，细胞 已经进化出一种复杂的稳态机制来控制金属的分布和形态， 作为金属。偏离基本金属组学特征与多种疾病过程有关， 环境金属污染和营养缺乏，这些都对正常的 细胞功能金属组由两个主要的金属离子池组成，包括在金属组中的不稳定金属离子池。 其中金属与细胞配体弱结合，而其中金属 以高亲和力连接到金属蛋白和其他生物分子上。金属蛋白质构成了紧密的- 结合的金属离子池代表了细胞蛋白质组的三分之一，在这些蛋白质中，金属具有不同的 结构和催化功能。这些金属蛋白可以以几种状态存在，包括apo（无金属）， 全息（金属结合），和错金属化，这取决于细胞环境。虽然我们知道一些关于 如何控制所选金属蛋白的金属化状态（通过金属伴侣和/或特异性 金属转运蛋白）的细节，如何不稳定和金属蛋白结合的金属池相互作用，在细胞的情况下， 由于细胞经历动态变化在很大程度上是未知的， 领域为了探索这些相互作用，并描述金属蛋白库在生理学中如何变化， 和病理学，我们的目标是开发一个多样化的化学工具箱，使成像，识别， 细胞、组织和生物体中金属蛋白群体的定量和分子控制。我们的战略 其核心是使用精确设计的分子靶向基团，这些基团将特异性地与金属相互作用， 这些蛋白质的位置。然后用功能标签修饰这些靶向基团，所述功能标签包括： 用于活细胞成像和细胞裂解物蛋白质分析的荧光团; 2）用于蛋白质组学研究的亲和探针， 使金属蛋白能够被捕获在它们的天然金属化状态; 3）光响应基团， 特异性金属蛋白的选择性控制和生物活性载体的开发。初步研究表明， 集中在锌依赖性金属酶，包括碳酸酐酶;然而，研究将扩大 包括一系列依赖于锌的酶（例如，金属-β-内酰胺酶、组蛋白脱乙酰酶、基质金属酶）， 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 应用于金属稳态异常的细胞模型和用于分析癌细胞中的金属蛋白库， 其他，并将结合额外的金属分析，包括元素分析（归纳 耦合等离子体质谱法、基于同步加速器的X射线荧光显微术）和不稳定的 金属离子池。从这些研究提供的分子工具中获得的见解将有助于 癌症和其它疾病中感兴趣的金属蛋白的鉴定以及治疗和 诊断策略，以打击他们。