Molecular Mechanisms of Phytochrome Signaling

描述（申请人提供）：光敏色素是一种胆蛋白光传感器，可协调植物的基因表达和生长发育。当光线有限时，它们可以优化光捕获，当光线太强时，它们可以最大限度地减少光损伤。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。我们试图定义光信号是如何被光敏色素感知并转导到目标分子的。我们的调查解决的假设，胆色素光异构化诱导的“反开关”的感光细胞内改变ATP依赖的蛋白质-蛋白质相互作用和磷酸转移酶活性的原核生物（蓝藻）和真核生物（植物）光敏色素模型。这些研究将使用计算方法的组合，即量子计算、同源建模和分子动力学模拟、线性发色团类似物的化学-酶促合成、用于诱变和分离具有各种发色团的光感受器的生物化学和分子生物学、荧光、量热法和单分子测定以探测ATP和光调制的蛋白质-蛋白质相互作用，以及体外野生型和光敏色素突变体的光谱分析。伴随实验利用我们的发现的荧光，组成型激活的等位基因在体内解剖的光敏色素信号的荧光显微镜技术和抑制基因突变的遗传模型拟南芥的组合。光敏色素是重要的调控靶标，不仅用于最小化远红富集阴影和反射光对植物作物产量的损失，而且用于设计靶向病原微生物和有益微生物的药物。了解植物光敏色素信号传导的分子机制对发展中国家特别重要，因为发展中国家的作物产量不足和伴随营养不良的机会性疾病是造成重大人类死亡的原因。虽然哺乳动物缺乏这种光传感器家族，但光敏色素研究已经为癌症和糖尿病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.