Targeted molecular strategies for cellular investigation of metalloproteins

项目摘要/摘要 金属是细胞和生物体正常运作所必需的微量营养素。因此，细胞 已经进化出一种复杂的动态平衡机制来控制金属的分布和形态，也被称为 就像金属片一样。偏离基本金属组特征与多种疾病过程相关， 环境金属污染和营养不足，这些都对正常 细胞功能。金属组由两个主要的金属离子池组成，其中包括 哪些金属与细胞配基结合较弱，以及金属在其中紧密结合的金属离子池 与金属蛋白和其他生物分子高亲和力连接。金属蛋白构成了紧密的- 结合的金属离子池占细胞蛋白质组的三分之一，在这些蛋白质中，金属有不同的 结构和催化功能。这些金属蛋白可以几种状态存在，包括apo(无金属)， HOLO(金属结合)，以及根据细胞环境的不同而非金属化。虽然有些信息是已知的 所选金属蛋白的金属化状态是如何控制的(通过金属配位体和/或特定的 金属转运体)不稳定的和金属蛋白结合的金属池如何在细胞环境中相互作用的细节 细胞经历动态变化在很大程度上是未知的，并且是金属动态平衡的一个日益感兴趣的领域 菲尔德。为了探索这些相互作用并描述金属蛋白库如何在生理上发生变化 和病理学，我们的目标是开发一种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种群的量化和分子控制。我们的战略 重点是使用精确设计的分子靶向基团，这些基团将专门与金属相互作用 这些蛋白质的位置。然后使用包括以下内容的功能标签对这些目标群体进行修改：1) 用于活细胞成像和细胞裂解蛋白分析的荧光团；2)用于蛋白质组学研究的亲和探针 使金属蛋白能够以其天然的金属化状态被捕获；3)光响应基团 特定金属蛋白的选择性控制和光药效团的发展。初步研究表明 侧重于依赖锌的金属酶，包括碳酸氢酶；然而，研究将会扩大 包括一系列依赖锌的酶(例如金属β-内酰胺酶、组蛋白脱乙酰酶、基质 铁(如亚铁血红素和非亚铁血红素)和铜(如超氧化物歧化酶)。工具将是 应用于金属代谢紊乱的细胞模型，并用于描绘癌细胞和 其他的，并将与其他金属分析相结合，包括元素分析(归纳 耦合等离子体质谱、同步加速器X射线荧光显微镜)和不稳定物质的成像 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 癌症和其他疾病相关金属蛋白的鉴定及其治疗和治疗的进展 对抗它们的诊断策略。

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, 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.