Improved Aequorin for Monitoring Ca and Superoxide in Cells

9722982下村光蛋白Aequorin多年来一直被用于监测细胞内钙离子，而该蛋白的官能团Coelenterazine及其类似物是测量超氧阴离子的灵敏探针。该项目的主要目标是设计和制备用于研究涉及游离钙和超氧阴离子的各种生物事件的最佳改进形式的木犀草素和土豆蔻嗪。Aequorin是在1962年由PI在水母中发现的。在电离钙存在的情况下，它通过分子内反应发出蓝光，将自己分解为载脂蛋白、腔肠酰胺和二氧化碳。在氧气存在的情况下，辅酶环素可以通过与乙酸乙酯孵育而再生为原辅酶环素。在过去的几年里，合成了大约50种腔肠静类似物并将其与罗布麻黄碱进行了结合。许多产品，被称为半合成的Aequorin，在测量细胞钙方面表现出了极好的性能。有些灵敏度很高，有些灵敏度很低，有些响应非常快，因此可以在很大的浓度范围内(1 NM-1 mm)监测钙离子的快速、瞬时变化。最近的一个趋势是在细胞中表达重组apoequorin，然后通过添加coelenterazine来原位重组aequorin。在这一过程中，腔肠静类似物的选择是成功重组水飞蓟素的关键。超氧阴离子自由基的产生在生物系统中的重要性早已被认识到，但测量它的技术远远落后于测量钙的技术。这在很大程度上是由于无法获得对此目的足够敏感的超氧化物探针。在过去的两年里，在PI的实验室里制备了20多种Coelenterazine类似物并进行了测试。其中一些化合物对超氧化物的响应明显增强，其结构-灵敏度关系为进一步改进超氧化物探针提供了有用的信息。未来三年的研究计划包括：(1)通过在咪唑并吡嗪酮环的8位扩展腔肠净水杨素的共轭体系，制备半合成的红光(550-650 nm)的水杨酸乙酯。这些产品将用于监测有色和不透明细胞和组织中的钙，以及某些双波长技术。(2)在细胞中表达的重组夹竹桃素的合适的腔肠静类似物的鉴定。这些信息应对许多实验室正在进行的工作作出重大贡献。(3)将根据现有信息设计和制备新的、改进的超氧化物探头；这些探头的灵敏度将至少比目前可用的最灵敏的探头MCLA高100倍，背景发光非常低。(4)PI的实验室将继续向细胞生物学家提供各种马钱子素制剂和腔肠净类似物，以帮助他们进行研究。当制备好超灵敏的超氧化物探针时，还将提供。对外部环境效应的感知，如光、温度和化学物质，影响细胞和有机体在生理和发育过程中施加大量明显的变化。对环境变化的最初感知是由特定的蛋白质检测到的，这些蛋白质通过通常涉及细胞内钙离子快速变化或流动的机制将信号传递到细胞下游。PI发现了一种蛋白质，Apoaequorin和coelentazine，用于水母对钙高度敏感的生物发光。载脂蛋白的基因可以在细胞中表达，以提供钙离子的内在视觉探针，并加入腔肠静。在研究细胞对环境和发育变化的反应中，能够直观地看到细胞内钙的变化和钙的流动是非常有用的。有了这一奖项，下村博士将继续改进aequorin的使用，生产出新的coelenterazine类似物，这种类似物将发出红色而不是蓝色的光，并对伴随着许多应激反应的氧化猝发敏感。实现这些目标将为科学界提供极其宝贵的工具，以探索动物、植物和真菌细胞的生理学变化的许多类型的反应。***

9722982 Shimomura The photoprotein aequorin has been used in monitoring intracellular calcium for many years, and the functional group of this protein, coelenterazine, and its analogues are sensitive probes for measuring superoxide anion. The main objective of this project is to design and prepare optimally improved forms of aequorin and coelenterazine for studying various biological events that involve free calcium and superoxide anion. Aequorin was discovered in a jellyfish by the PI in 1962. It emits blue light in the presence of ionized calcium by an intramolecular reaction, decomposing itself into apoaequorin, coelenteramide and carbon dioxide. Apoaequorin can be regenerated into the original aequorin by incubation with coelenterazine in the presence of oxygen. In the past several years, about 50 kinds of coelenterazine analogue were synthesized and incorporated into apoaequorin. Many of the products, which are called semisynthetic aequorins, showed excellent properties for measuring cellular calcium. Some had a very high sensitivity, others had a very low sensitivity, and some had a very fast response, thus making possible the monitoring of rapid, transient changes of calcium ions in a wide concentration range (1 nM - 1 mM). A recent trend is to express recombinant apoaequorin in cells, which is followed by the in situ reconstitution of aequorin by the addition of a coelenterazine. In this procedure, the choice of coelenterazine analogue is critical for the success of the reconstitution of an aequorin. The significance of superoxide radical generation in biological systems has long been recognized, but the technique of measuring it lags far behind that of measuring calcium. This is largely due to the unavailability of the superoxide probes that are sufficiently sensitive for the purpose. In the past two years, more than 20 kinds of coelenterazine analogues were prepared and tested at the PI's lab. Some of them showed significantly improved responses to superoxide, and the structure- sensitivity relationships of the analogues provided useful information for further improvement of superoxide probes. The research plan for the next three years includes: (1) Preparation of the semisynthetic aequorins that emit red light (550-650 nm), by extending the conjugation system of coelenterazine at the position 8 of its imidazopyrazinone ring. The products will be useful in monitoring calcium in colored and opaque cells and tissues, and also in certain two-wavelength techniques. (2) Identification of suitable coelenterazine analogues for the reconstitution of the recombinant apoaequorin that is expressed in cells. This information should significantly contribute to the work in progress at many laboratories. (3) New, improved superoxide probes will be designed and prepared based on the information now available; the probes will have at least 100 times higher sensitivity than MCLA, the most sensitive probe currently available, with a very low background luminescence. (4) The PI's laboratory will continue to supply various aequorin preparations and coelenterazine analogues to cell biologists to aid their studies. The supersensitive superoxide-probes, when prepared, will also be provided. The perception of extrinsic environmental effects such as light, temperature and chemicals influences cells and organisms to exert a multitude of overt changes in physiology and development. The initial perception of environmental changes is detected by specific proteins that transmit the signal downstream in the cell through mechanisms that often involve rapid changes or fluxes of intracellular calcium. The PI discovered a protein, apoaequorin with coelenterazine, is used for bioluminescence in jelly fish is highly sensitive to calcium. The gene for apoaequarin can be expressed in cells to provide an intrinsic visual probe for calcium with the addition of coelenterazine. To be able to visualize the changes a nd fluxes of calcium within cells in response to signals is extremely useful in the study of cellular reactions to environmental and developmental changes. With this award, Dr. Shimomura will continue to refine the use of aequorin by producing new analogs of coelenterazine that will emit light in the red instead of blue and to be sensitive to oxidative burst that accompanies many stress reactions. Attaining these goals will provide the scientific community with extremely valuable tools to explore many types of reactions to changes in the physiology of animal, plant and fungal cells. ***