Imaging Nuclear Receptor LRH-1 in Functional Transcriptional Assemblies

描述(申请人提供)：细胞转录程序要求许多不同功能的蛋白质聚集在染色质上，以响应来自细胞核外的信号。启动这些转录过程的最大蛋白质家族是核受体。核受体是多结构域蛋白质，没有其完整结构的原子分辨率图像。我们的建议集中在核受体LRH-1(肝脏受体同源1，也称为胰腺受体同源1)，一个关键的转录因子，发现在肝脏，肠道和胰腺。我们的最终目标是在与DNA结合时，将全长LRH-1受体与伙伴共调节蛋白成像。我们将使用一种先进的方法来制备和组装LRH-1的多个结构域和伙伴蛋白质和特定的DNA片段，以便转录组装足够有序，以便通过X射线结晶学进行分析。我们计划使用一种稳定的转录伙伴蛋白，β-连环蛋白，它将允许对以前难以处理的分子进行生化准备，并在功能复合体中调节它们的相互作用。我们将使用系统的方法来确定LRH-1及其特有的转录调控因子的稳定组装。代表LRH-1已知反应元件的特定DNA片段将包括在我们组装的评估中。我们开发的用于对LRH-1构建的调节复合体进行成像的方法将直接适用于其他核受体及其转录组件。我们的目标是在学习如何确定功能性核受体的结构方面迈出重要的一步，并了解关于它们组装和转录调控机制的原子水平的细节。目前有三分之一的药物针对核受体的调节位点，因为它们在所有生物过程中的RNA转录中发挥主要作用。关于DNA上核受体构建的转录机器的新的结构信息将极大地扩展我们对转录及其调控以及由这些转录过程控制的生理和病理生理途径的理解。我们的建议集中在核受体LRH-1及其辅助调节因子β-连环蛋白构建的转录组件上。我们选择LRH-1作为主要靶点，是因为LRH-1受体在人类发育、代谢和许多病理生理过程中发挥着关键作用。正如我们在提案中所描述的，胰腺中LRH-1的解除调控与成熟期糖尿病有关。功能性LRH-1/β-连环蛋白缺乏会导致炎症性肠病、克罗恩病和溃疡性结肠炎。相反，LRH-1/β-catenin伙伴关系的异常上调活性与癌症的触发和增殖有关，包括胃肠癌。因此，我们期望功能性LRH-1/β-连环蛋白组装的原子分辨结构将推动药物发现进程，并使靶向干预治疗的新的有效疗法的开发成为可能。与公共卫生相关：我们正在研究核受体--启动基因转录变化的蛋白质分子，因此在所有生物过程中发挥主要作用。我们集中精力对核受体LRH-1进行成像的挑战性目标，LRH-1是一种在肝脏、肠道和胰腺中发现的关键转录因子，当与DNA结合时，它与其转录调控伙伴结合。我们预计，对该受体形成的功能调控组件的成像不仅将极大地扩大我们对转录及其调控的理解，还将使许多疾病的靶向治疗方法得以开发，包括慢性肠炎以及胃肠道和胰腺癌。

DESCRIPTION (provided by applicant): Cellular transcription programs require that many proteins of different function assemble on chromatin in response to signals that originate outside the nucleus. The largest family of proteins initiating these transcriptional processes is that of the nuclear receptors. The nuclear receptors are multidomain proteins, and there are no atomic resolution images of their complete structures. Our proposal focuses on the nuclear receptor LRH-1 (Liver Receptor Homologue 1, termed also Pancreas Receptor Homologue 1), a critical transcription factor found in liver, intestines and pancreas. Our ultimate goal is to image the full length LRH-1 receptor with partner co-regulatory proteins when bound to DNA. We will use an advanced methodology to prepare and assemble the multiple domains of LRH-1 with partner proteins and specific DNA fragments so that the transcriptional assembly would be ordered well enough for analysis by X-ray crystallography. We plan to employ a stabilizing transcriptional partner protein, beta-catenin, that will permit biochemical preparation of previously intractable molecules and mediate their interactions in the functional complexes. We will use a systematic approach to identify stable assemblies of LRH-1 with its characterized transcriptional regulators. Specific DNA fragments representing known response elements of LRH-1 will be included in evaluation of our assemblies. Methods that we develop for imaging the regulatory complexes built by LRH-1 will be directly applicable to other nuclear receptors and their transcriptional assemblies. Our goal is to take a major step in learning how to determine structures for functional nuclear receptors and learn atomic level details about the mechanisms of their assembly and regulation of transcription. One third of current pharmaceuticals target regulatory sites of the nuclear receptors because of their primary roles in RNA transcription in all biological processes. Novel structural information about the transcriptional machines built by nuclear receptors on DNA would vastly expand our understanding of transcription and its regulation as well as physiological and pathophysiological pathways controlled by these transcriptional processes. Our proposal focuses on the transcriptional assemblies built by nuclear receptor LRH-1 and its co-regulator beta-catenin. We chose LRH-1 as a primary target because of the receptor's critical roles in human developmental, metabolic and numerous pathophysiological processes. As we describe in our proposal, deregulation of LRH-1 in pancreas is linked to maturity-onset diabetes. Functional LRH-1/beta-catenin deficiencies lead to inflammatory bowel disease, Crohn's disease and ulcerative colitis. In contrast, the aberrant, up-regulated activity of the LRH-1/ beta-catenin partnership is implicated in triggering and proliferation of cancerous tumors, including gastrointestinal cancers. Therefore, we expect that the atomic resolution architecture of the functional LRH-1/beta-catenin assembly will propel drug discovery process and enable the development of novel potent therapeutics for targeted intervention therapies. PUBLIC HEALTH RELEVANCE: We are studying nuclear receptors - protein molecules that initiate changes in gene transcription and therefore play primary roles in all biological processes. We concentrate our efforts on challenging goal of imaging the nuclear receptor LRH-1, a critical transcription factor found in liver, intestines and pancreas, with its transcriptional regulatory partners when bound on DNA. We expect that imaging of functional regulatory assemblies formed by this receptor will not only vastly expand our understanding of transcription and its regulation but also enable the development of potent therapeutics for targeted treatments of numerous diseases, including chronic intestinal inflammation and gastrointestinal and pancreatic cancers.