Molecular Mechanisms of Phytochrome Signaling

说明(申请人提供)：光敏色素是一种胆蛋白光敏传感器，能协调基因表达和植物的生长发育。当光线有限时，它们优化了光收集，当光线太强时，它们将光损害降至最低。光敏色素在真细菌和真菌中的广泛存在表明，这些感受器对于异养生物适应昼夜光环境也是重要的。我们试图定义光信号是如何被光敏色素感知并传递到目标分子的。我们的研究提出了这样一种假说：在原核(蓝藻)和真核(植物)光敏色素模型中，碧林光异构化诱导光感受器内的反离子开关，从而改变依赖于ATP的蛋白质-蛋白质相互作用和磷酸转移酶的活性。这些研究将结合使用计算方法，即量子计算、同源建模和分子动力学模拟、线性生色团类似物的化学酶合成、用于突变和分离具有不同发色团的光感受器的生物化学和分子生物学、荧光、量热和单分子分析来探索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。配套实验利用我们发现的一个荧光的，成分激活的等位基因，使用荧光显微镜技术和抑制突变的组合在体内解剖光敏色素信号，在拟南芥遗传模型中。光敏色素是重要的调控靶标，不仅可以最大限度地减少远红浓荫和反射光对作物产量的损失，还可以设计针对病原微生物和有益微生物的药物。了解植物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，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导致大量人类死亡的原因。虽然哺乳动物缺乏这一家族的光感受器，但光敏色素研究已经为癌症和糖尿病重要的细胞信号传递的常见机制提供了宝贵的见解。

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.