Molecular Mechanisms of Phytochrome Signaling

描述（由申请人提供）：光敏色素是协调基因表达和植物生长发育的胆蛋白光传感器。当光线有限时，它们可以优化光收集，当光线太强时，它们可以最小化光损伤。光敏色素在真细菌和真菌中的广泛存在表明，这些传感器对于异养生物适应昼夜光环境也很重要。我们试图定义光敏色素如何感知光信号并将其转导到目标分子。我们的研究提出了一个假设，即在原核（蓝藻）和真核（植物）光敏色素模型中，十亿蛋白光异构化诱导了光感受器内的“反离子开关”，从而改变了atp依赖性蛋白质-蛋白质相互作用和磷酸转移酶活性。这些研究将结合计算方法，即量子计算、同源性建模和分子动力学模拟、线性发色团类似物的化学酶合成、生物化学和分子生物学的诱变和分离具有各种发色团的光感受器、荧光、量热法和单分子测定来探测ATP和光调制的蛋白质-蛋白质相互作用。并对野生型和光敏色素突变体进行了体外光谱分析。同伴实验利用我们在遗传模型拟南芥中发现的荧光、组成型激活等位基因，结合荧光显微镜技术和抑制突变，用于体内解剖光敏色素信号。光敏色素是重要的调控靶点，不仅可以最大限度地减少植物作物在远红色富集阴影和反射光下的产量损失，而且还可以用于设计针对致病和有益微生物的药物。了解植物中光敏色素信号的分子机制对发展中国家具有特别重要的意义，因为在发展中国家，作物产量不足和伴随营养不良的机会性疾病是造成大量人类死亡的原因。虽然哺乳动物缺乏这种光传感器家族，但光敏色素的研究已经为了解对癌症和糖尿病很重要的细胞信号传导的共同机制提供了有价值的见解。

DESCRIPTION (provided by applicant): Phytochromes are biliprotein photosensors that coordinate gene expression, and growth and development of plants. They optimize light harvesting when light is limiting, and minimize light damage when light is too intense. The widespread occurrence of phytochromes in eubacteria and fungi demonstrates that these sensors are also important for adapation by heterotrophic organisms to the circadian light environment. We seek to define how light signals are perceived by phytochromes and transduced to target molecules. Our investigations address the hypothesis that bilin photoisomerization induces a 'counterion-switch' within the photoreceptor which alters ATP-dependent protein-protein interactions and phosphotransferase activities for both prokaryotic (cyanobacterial) and eukaryotic (plant) phytochrome models. These studies will use a combination of computational approaches, i.e. quantum calculations, homology modeling and molecular dynamics simulations, chemi-enzymatic synthesis of linear chromophore analogs, biochemistry and molecular biology for mutagenesis and isolation of photoreceptors with various chromophores, fluorescence, calorimetry and single-molecule assays to probe ATP- and light-modulated protein-protein interactions, together with spectroscopic analysis of wild type and phytochrome mutants in vitro. Companion experiments exploit our discovery of a fluorescent, constitutively activated allele for in vivo dissection of phytochrome signaling using a combination of fluorescence microscopy techniques and suppressor mutagenesis in the genetic model Arabidopsis thaliana. Phytochromes are important regulatory targets, not only for minimizing plant crop yield losses to far-red enriched shade and reflected light, but also for design of drugs that target both pathogenic and beneficial microorganisms. An understanding of the molecular mechanisms of phytochrome signaling in plants is of particular significance to the developing world where inadequate crop yields and opportunistic diseases accompanying malnutrition are responsible for significant human mortality. While mammals lack this family of light sensors, phytochrome studies have already provided valuable insight into common mechanisms of cell signaling important to cancer and diabetes.