Improved Aequorin for Monitoring Ca and Superoxide in Cells

光蛋白aequorin用于监测细胞内钙已有多年的历史，该蛋白的官能团coelenterazine及其类似物是测量超氧阴离子的敏感探针。本项目的主要目的是设计和制备最佳改进形式的青霉素和铜肠嗪，用于研究涉及游离钙和超氧阴离子的各种生物事件。Aequorin是1962年由PI在水母中发现的。通过分子内反应，它在电离钙存在的情况下发出蓝光，并将自身分解成apoaequorin、coelenteramide和二氧化碳。在有氧存在的条件下，与胶肠嗪孵育后可再生为原始的水芹素。在过去的几年里，大约有50种coelenterazine类似物被合成并掺入到apoaequorin中。许多产品，被称为半合成金黄素，显示出优异的性能，用于测量细胞钙。有的灵敏度非常高，有的灵敏度非常低，有的响应速度非常快，从而可以监测宽浓度范围（1nm - 1mm）内钙离子的快速瞬态变化。最近的一种趋势是在细胞中表达重组apoaequorin，然后通过添加coelenterazine原位重建apoaequorin。在这个过程中，选择coelenterazine类似物对于成功地重组aequorin至关重要。超氧自由基在生物系统中产生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。这主要是由于没有足够灵敏的超氧化物探针。在过去的两年中，在PI的实验室中制备和测试了20多种coelenterazine类似物。其中一些对超氧化物的反应有明显的改善，这些类似物的结构-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)通过扩展coelenterazine在咪唑吡嗪酮环8位的偶联体系，制备能发出红光（550-650 nm）的半合成青蒿素。该产品将用于监测彩色和不透明细胞和组织中的钙，也可用于某些双波长技术。(2)确定合适的coelenterazine类似物，用于重组apoaequorin在细胞中表达。这一信息将对许多实验室正在进行的工作作出重大贡献。(3)基于现有信息，设计和制备新的、改进的超氧化物探针；这种探针的灵敏度将比MCLA高至少100倍，MCLA是目前可用的最灵敏的探针，背景发光非常低。(4) PI实验室将继续为细胞生物学家提供各种青霉素制剂和铜肠嗪类似物，以协助他们的研究。还将提供制备好的超敏超氧化物探针。外部环境效应（如光、温度和化学物质）的感知影响细胞和生物体，使其在生理和发育方面产生大量明显的变化。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的快速变化或通量的机制将信号传递到细胞下游。PI在水母中发现了一种用于生物发光的蛋白质，即含有胶肠嗪的apoaequorin，这种蛋白质对钙非常敏感。apoaequarin基因可以在细胞中表达，为钙提供了一种内在的视觉探针，添加了coelenterazine。在研究细胞对环境和发育变化的反应时，能够可视化细胞内钙响应信号的变化和通量是非常有用的。有了这个奖项，Shimomura博士将继续改进黄酮的使用，生产新的异肠嗪类似物，这种类似物将发出红光而不是蓝光，并且对伴随许多应激反应的氧化爆发敏感。实现这些目标将为科学界提供极有价值的工具，以探索动物、植物和真菌细胞生理变化的多种反应。＊＊＊

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. ***