Fluorescent Cellular Zinc Sensors. What Are They Imaging?

描述（由申请人提供）：在蛋白质组中可能有多达2800个锌蛋白，是非血红素铁蛋白或铜蛋白数量的10倍以上。除了其在酶催化和蛋白质折叠和稳定性的关键功能，锌2+在正常发育和生长，在癌症，免疫反应，神经突触功能等，在许多这些活动中，锌2+的运输似乎参与了重要的作用。为了观察“游离的”或“可接近的”Zn 2+的细胞分布及其由生理和病理刺激引起的扰动，越来越多的注意力已经被给予使用在Zn 2+存在下经历其荧光性质变化的传感器。最常用的传感器TSQ（N-（6-甲氧基-8-喹啉基）-对甲苯磺酰胺）及其近亲Zinquin揭示了细胞内“可螯合的”Zn 2+的高度不对称分布和响应于诸如一氧化氮供体的试剂的荧光增强。一般认为TSQ和锌喹结合来自“游离”或适度结合的金属离子池的Zn 2+以形成荧光Zn（TSQ）2或Zn（锌喹）2。相反，初步结果与这些传感器通过形成传感器-锌-蛋白质三元复合物而变得荧光的假设完全一致。此外，使用TPEN（N，N，N '，N'-四（2-吡啶基甲基）-乙二胺）（一种用于淬灭Zn传感器荧光的细胞渗透性Zn 2+螯合剂）和一氧化氮（一种增加细胞内锌荧光的试剂）的模型研究表明，它们的一些作用也可能涉及传感器-Zn-蛋白质加合物化学。这些发现提出了关于TSQ，Zinquin和其他Zn 2+传感器的问题，“什么是成像？“该提案的总体目标是用解决这个问题所需的体内和体外补充方法来解决这个问题。具体目标是：1.建立一组基本特性，表征TSQ和Zinquin的细胞内成像。2.为了分离和鉴定TSQ结合的单个蛋白质，纯化TSQ-Zn-蛋白质加合物。3.明确TPEN与TSQ和Zinquin处理的细胞反应的细胞和分子特征。4.研究特定目的1和2在其他细胞类型和条件下的结果的一般性。5.使用TSQ、Zinquin和其他传感器以及锌蛋白进行模型研究。6.检验传感器-锌-蛋白加合物在其他传感器的细胞锌离子成像中发挥重要作用的假设。主要的新工具，将在这项研究中采用的是激光烧蚀电感耦合等离子体质谱。它提供了通过非变性聚丙烯酰胺凝胶电泳在蛋白质组学背景中定位分离的锌蛋白的机会。结合传感器荧光和蛋白质定位的灵敏分析，传感器-Zn-蛋白质可以定位并进行质谱分析以进行鉴定。这种方法的变体将用于开始回答由基于Zn 2+传感器的显微镜提出的问题：“什么是成像？“公共卫生相关性：锌是一种必需的营养素，在正常的胎儿发育，生长，免疫中枢神经系统功能和癌细胞增殖等方面起着关键作用。锌荧光传感器越来越多地用作显微探针，以研究锌如何参与这些过程。由于对这种传感器如何成像细胞内锌或它们成像的内容知之甚少，因此本提案的目的是了解常用锌传感器的显微荧光成像的化学基础。

DESCRIPTION (provided by applicant): There may be as many as 2800 Zn-Proteins in the proteome, more than 10 times the number of non-heme iron proteins or copper proteins. Besides its key functions in enzyme catalysis and protein folding and stability, Zn2+ plays major roles in normal development and growth, in cancer, in immune response, in neuro-synaptic function etc. In many of these activities, Zn2+ trafficking seems to be involved. In order to observe the cellular distribution of "free" or "accessible" Zn2+ and its perturbation by physiological and pathological stimuli, increasing attention has been given to the use of Sensors that undergo changes in their fluorescent properties in the presence of Zn2+. The most commonly used Sensors, TSQ (N-(6-methoxy-8-quinolyl)-p-toluensulfonamide) and its close relative, Zinquin, reveal a highly asymmetric distribution of intracellular "chelatable" Zn2+ and fluorescence enhancement in response to agents such as nitric oxide donors. It is generally thought that TSQ and Zinquin bind Zn2+ from pools of "free" or modestly bound metal ion to form fluorescent Zn(TSQ)2 or Zn(Zinquin)2. To the contrary, preliminary results are fully consistent with the hypothesis that these Sensors become fluorescent by forming Sensor-Zn-Protein ternary complexes. In addition, model studies with TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine), a cell permeant Zn2+ chelator used to quench Zn-Sensor fluorescence, and nitric oxide, an agent that increases intracellular Zinquin fluorescence, suggest that some of their effects may involve Sensor-Zn-Protein adduct chemistry as well. These findings raise the question with respect to TSQ, Zinquin, and other Zn2+ Sensors, "What is being imaged?" The overall objective of the proposal is to address this question with complementary in vivo and in vitro methods that are both needed to resolve this question. The specific aims are: 1. To establish a set of basic properties that characterizes intracellular imaging with TSQ and Zinquin. 2. To isolate and identify individual proteins to which TSQ is bound, putatively, TSQ-Zn-Protein adducts. 3. To define the cellular and molecular characteristics of the reaction of TPEN with TSQ and Zinquin-treated cells. 4. To investigate the generality of the findings of Specific Aims 1 and 2 in other cell types and conditions. 5. To conduct model studies with TSQ, Zinquin, and other Sensors and a selection of Zn-Proteins. 6. To test the hypothesis that Sensor-Zn-Protein adducts play a significant role in cellular Zn2+ imaging by other Sensors. The major new tool that will be employed in this study is laser ablation-inductively coupled plasma mass spectrometry. It provides the opportunity to locate Zn-Proteins separated within a proteomic background by native polyacrylamide gel electrophoresis. Together with sensitive analysis of Sensor fluorescence and protein location, Sensor-Zn-Proteins can be located and subjected to mass spectral analysis for identification. Variants of this methodology will be used to begin to answer the question raised by Zn2+ Sensor based microscopy: "What is being imaged?" PUBLIC HEALTH RELEVANCE: Zinc is an essential nutrient that plays key roles in normal fetal development, growth, immune central nervous system function, and cancer cell proliferation, among others. Zinc fluorescent Sensors are increasingly used as microscopic probes to study how zinc participates in these processes. Because relatively little is known about how such Sensors image intracellular zinc or what they image, the objective of this proposal is to understand the chemistry underlying microscopic fluorescent imaging by commonly used zinc Sensors.