Fluorescent Cellular Zinc Sensors. What Are They Imaging?

描述(申请人提供)：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的10倍以上。除了在酶催化、蛋白质折叠和稳定等方面发挥重要作用外，锌在正常发育和生长、癌症、免疫反应、神经突触功能等方面也发挥着重要作用。为了观察“游离的”或“可接近的”锌离子在细胞内的分布及其受到生理和病理刺激的干扰，人们越来越关注在锌离子存在下发生荧光性质变化的传感器的使用。最常用的传感器，TSQ(N-(6-methoxy-8-quinolyl)-p-toluensulfonamide)和它的近亲，Zquiin，揭示了细胞内高度不对称的可络合锌的分布和对一氧化氮供体等试剂的荧光增强。一般认为，TSQ和Zquiin通过结合“游离”或适度结合的金属离子与锌离子结合形成荧光的锌(TSQ)_2或锌(锌)_2。相反，初步的结果与传感器-锌-蛋白质三元络合物的形成而成为荧光的假设完全一致。此外，对TPEN(N，N，N‘，N’-tetrakis(2-pyridylmethyl)-ethylenediamine)，，一种用于猝灭锌传感器荧光的细胞螯合剂)和一氧化氮(一种增强细胞内锌奎宁荧光的试剂)的模型研究表明，它们的一些作用可能也涉及传感器-锌蛋白加合物化学。这些发现提出了一个关于TSQ、Zquiin和其他锌传感器的问题，“正在成像的是什么？”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，这两种方法都是解决这个问题所需的。其具体目的是：1.建立一套表征TSQ和Zquin细胞内成像的基本性质。2.分离和鉴定TSQ结合的单个蛋白，推测为TSQ-锌蛋白加合物。3.明确TPEN与TSQ和Zquin处理细胞反应的细胞和分子特征。4.探讨特异靶蛋白1和2在其他细胞类型和条件下的发现的共性。5.用TSQ、Zquin等传感器和精选的锌蛋白进行模型研究。6.通过其他传感器验证传感器-锌蛋白加合物在细胞锌成像中的重要作用这一假设。在这项研究中将使用的主要新工具是激光消融-电感耦合等离子体质谱。它提供了通过天然聚丙烯酰胺凝胶电泳法在蛋白质组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，可以定位传感器锌蛋白质并进行质谱分析以进行鉴定。这种方法的变种将被用来开始回答由基于锌传感器的显微镜提出的问题：“成像的是什么？”与公众健康相关：锌是一种基本的营养物质，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发挥着关键作用。锌荧光传感器越来越多地被用作研究锌如何参与这些过程的微观探针。由于对这类传感器如何成像细胞内锌或它们成像什么的了解相对较少，因此这项提议的目的是了解常用锌传感器显微荧光成像背后的化学原理。

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.