Fluorescent Cellular Zinc Sensors. What Are They Imaging?

描述（由申请人提供）：蛋白质组中可能存在多达2800个zn - protein，是非血红素铁蛋白或铜蛋白数量的10倍以上。Zn2+除了在酶催化、蛋白质折叠和稳定性方面发挥关键作用外，还在正常发育和生长、癌症、免疫反应、神经突触功能等方面发挥重要作用。在许多此类活动中，似乎都涉及到Zn2+的贩运。为了观察“游离”或“可接近”的Zn2+的细胞分布及其在生理和病理刺激下的扰动，越来越多的人关注使用在Zn2+存在下发生荧光特性变化的传感器。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”Zn2+的高度不对称分布和对一氧化氮供体等药物的荧光增强反应。一般认为TSQ和Zinquin结合游离或适度结合金属离子池中的Zn2+形成荧光Zn(TSQ)2或Zn(Zinquin)2。相反，初步结果完全符合这些传感器通过形成传感器-锌-蛋白三元配合物而产生荧光的假设。此外，用TPEN （N，N，N‘，N’-tetrakis(2-pyridylmethyl)-乙二胺）（一种用于淬灭Zn-Sensor荧光的细胞渗透Zn2+螯合剂）和一氧化氮（一种增强细胞内Zinquin荧光的试剂）进行的模型研究表明，它们的一些作用也可能涉及传感器- zn - protein加合物的化学反应。这些发现提出了关于TSQ、Zinquin和其他Zn2+传感器的问题，“被成像的是什么？”该提案的总体目标是通过补充体内和体外方法来解决这个问题，这两个方法都需要解决这个问题。具体目标是：1。建立TSQ和Zinquin在细胞内成像的一组基本性质。为了分离和鉴定TSQ结合的单个蛋白质，推测TSQ- zn - protein加合物。3. 明确TPEN与TSQ和zinins处理的细胞反应的细胞和分子特征。4. 目的：探讨特异性Aims 1和2在其他细胞类型和条件下的普遍性。5. 用TSQ、Zinquin和其他传感器和精选的锌蛋白进行模型研究。6. 为了验证传感器-锌蛋白加合物在其他传感器对细胞Zn2+成像中起重要作用的假设。本研究将采用的主要新工具是激光烧蚀-电感耦合等离子体质谱法。它提供了通过天然聚丙烯酰胺凝胶电泳在蛋白质组学背景中分离的锌蛋白的定位机会。结合Sensor荧光和蛋白定位的灵敏分析，Sensor- zn - proteins可以定位并进行质谱分析进行鉴定。该方法的变体将用于开始回答基于Zn2+传感器的显微镜提出的问题：“正在成像的是什么？”公共卫生相关性：锌是一种必需营养素，在正常胎儿发育、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被用作微观探针来研究锌如何参与这些过程。由于对这些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微观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.