Arabidopsis 2010: Systematic Discovery of Ligands for Arabidopsis START proteins

智力上的优点。植物生长部分是由称为激素的小分子信号控制的。这些激素通过与受体蛋白结合而起作用，受体蛋白反过来又起到协调基因转录和细胞生长等反应的作用。例如，激素脱落酸（阿坝）是一种小的亲脂性化合物，由植物在逆境中合成。一旦合成，它与受体蛋白结合，受体蛋白反过来抑制生长并激活适应压力环境条件所需的转录反应。已经确定了许多受体对，并且已知它们控制植物生长的许多方面。因此，激素信号及其受体蛋白是现代植物生物学研究的中心。有了完整的基因组序列，很明显高等植物的基因组编码的受体蛋白比已知的激素多数百种;这一观察和其他观察表明，存在的激素比我们目前所知的还要多。鉴于植物激素的重要性已被充分证明，发现激素的新方法和工具可能会对我们对植物生物学的理解产生变革性的影响。原则上，鉴定激素的一种方法是凭借它们与受体蛋白的结合来纯化它们。为了发展、探索和实施这一想法，该项目将执行一个简单而通用的策略来识别与受体蛋白结合的化合物。该项目的重点是START蛋白，它形成了一个已知或疑似激素受体的大家族。拟南芥基因组编码89个START蛋白，其中一些在发育、非生物或生物胁迫生物学中起重要作用。尽管它们的重要性，大多数START蛋白的内源性配体是未知的。在这个项目中，我们利用最新的蛋白质配体鉴定技术的进展，系统地表征拟南芥START蛋白的配体。使用最近开发的小分子下拉方法，我们将使用START蛋白作为试剂，指导其内源性配体的鉴定和随后的纯化。一旦分离，这些配体的结构将使用高灵敏度微探针2D-NMR和其他方法的组合来确定。我们的建议基于多种方法的综合基础，并为新一轮的蛋白质靶向配体发现奠定了坚实的基础，这将有助于发现新的激素。更广泛的影响这个跨学科项目将为学生和博士后提供一个国际上独特的植物化学生物学和遗传学的培训环境。此外，通过UCR？的REU计划和其他途径，本科生，包括代表性不足的少数民族将在代谢物分析，纯化，分离和一般植物生物学的方法进行培训。PI维护一个网站，提供有关植物化学基因组学和化学生物学的信息和技术建议。该网站是PI与世界各地数十个实验室共享的实验室化学资源信息的门户。小分子唐斯和代谢物纯化方案将通过PI网站公开提供和传播。该项目将系统地定义整个蛋白质家族的蛋白质-代谢物相互作用，这在任何生物体中都是前所未有的，并将创造一个令人兴奋的跨学科培训环境，使科学家准备好迎接与采用新方法解决重要生物学问题相关的挑战。此外，新激素和生长调节剂的发现预计将对农业产生长期效益。

Intellectual merit. Plant growth is controlled in part by small molecule signals called hormones. These hormones work by binding to receptor proteins, which in turn function to coordinate responses like gene transcription and cellular growth. For example, the hormone abscisic acid (ABA) is a small lipophilic compound that is synthesized by plants in times of stress. Once synthesized, it binds to a receptor protein, which in turn inhibits growth and activates the transcriptional responses needed for acclimation to stressful environmental conditions. Numerous hormone-receptor pairs have been defined and are known to control many aspects of plant growth. Thus, hormone signals and their receptor proteins are a central preoccupation of modern plant biological studies. With complete genome sequences in hand, it is apparent that the genomes of higher plants encode hundreds more receptor proteins than known hormones; this and other observations imply that more hormones exist than we currently know about. Given the well-documented importance of plant hormones, new methods and tools for discovering hormones may have a transformative influence on our understanding of plant biology.In principle, one way to identify hormones is to purify them by virtue of their association with their receptor proteins. To develop, explore and implement this idea, this project will execute a simple and general strategy for identifying compounds that bind to receptor proteins. The project focuses on START proteins, which form a large family of known or suspected hormone receptors. The Arabidopsis genome encodes 89 START proteins, several of which play important roles in developmental, abiotic or biotic stress biology. In spite of their importance, the endogenous ligands for most START proteins are not known. In this project, we leverage recent technological advances in protein-ligand identification to systematically characterize the ligands of Arabidopsis START proteins. Using recently developed small-molecule pull down methods, we will use START proteins as reagents that guide the identification and subsequent purification of their endogenous ligands. Once isolated, the structure of these ligands will be determined using a combination of high-sensitivity microprobe 2D-NMR and other methods. Our proposal rests on an integrative base of diverse approaches and builds a robust foundation for a new wave of protein-targeted ligand discovery, which will enable the discovery of new hormones.Broader impacts This interdisciplinary project will provide students and post-docs an internationally unique training environment for plant chemical biology and genetics. In addition, through UCR?s REU program and other avenues, undergraduates, including underrepresented minorities will be trained in methods for metabolite analysis, purification, isolation and general plant biology. The PI maintains a website that provides information and technical advice about plant chemical genomics and chemical biology. This site is gateway to information about the laboratory's chemical resources that the PI has shared with dozens of laboratories across the world. Protocols for small molecule pull downs and metabolite purification will be made publicly available and disseminated through the PI's website. This project, which will systematically define protein-metabolite interactions for an entire protein family, is unprecedented in any organism and will create an exciting, interdisciplinary training environment that produces scientists ready to embrace the challenges associated with adopting new approaches to important biological questions. The discovery of new hormones and growth regulators is additionally expected to be of long-term benefit to agriculture.