Targeted molecular strategies for cellular investigation of metalloproteins

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，在本发明中， 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， 被称为金属层偏离基本金属组学特征与多种疾病相关 过程、环境金属污染和营养缺乏，所有这些都对人类的健康产生不利影响。 正常的细胞功能金属组由两个主要的金属离子池组成， 其中金属与细胞配体弱结合的池和其中金属与细胞配体紧密结合的金属离子池 与金属蛋白和其他生物分子以高亲和力连接。金属蛋白质构成了 紧密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多种状态存在，包括载脂蛋白 （无金属）、全金属（金属结合）和错金属化，这取决于细胞环境。虽然一些信息 已知如何控制所选金属蛋白的金属化状态（通过金属伴侣和/或 特定金属转运蛋白的作用）不稳定和金属蛋白结合的金属池如何相互作用的细节 在细胞环境中，细胞经历动态变化在很大程度上是未知的， 金属稳态场为了探测这些相互作用并描述 我们的目标是开发一个多样化的化学工具箱， 将使成像，鉴定，定量，和细胞中金属蛋白群体的分子控制成为可能， 组织和有机体。我们的策略集中在使用精确设计的分子靶向基团， 特异性地与这些蛋白质的金属位点相互作用。然后，这些目标群体被修改为 1）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，能够捕获处于其天然代谢状态的金属蛋白 和; 3）用于选择性控制特定金属蛋白和开发 植物寄生体。最初的研究集中在锌依赖性金属酶，包括碳 脱水酶;然而，研究将扩展到包括依赖于锌的一系列酶（例如金属- β-内酰胺酶、组蛋白脱乙酰酶、基质金属蛋白酶）、铁（例如血红素和非血红素）和铜 (e.g.超氧化物歧化酶）。工具将应用于金属稳态失调的细胞模型和分析 癌细胞和其他细胞中的金属蛋白库，并将与其他金属组学分析相结合 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 荧光显微术）和不稳定金属离子池的成像。从分子工具中获得的见解 这些研究提供的信息将有助于鉴定癌症和其他疾病中感兴趣的金属蛋白。 疾病和制定防治这些疾病的治疗和诊断战略。

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.