Imaging Nuclear Receptor LRH-1 in Functional Transcriptional Assemblies

描述（由申请人提供）：细胞转录程序需要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的蛋白质家族是核受体。核受体是多结构域蛋白，目前还没有完整结构的原子分辨率图像。我们的建议侧重于核受体LRH-1（肝脏受体同源物1，也称为胰腺受体同源物1），一个在肝脏，肠道和胰腺中发现的关键转录因子。我们的最终目标是成像全长LRH-1受体与伴侣共调节蛋白结合时的DNA。我们将使用一种先进的方法来制备和组装LRH-1的多个结构域与伴侣蛋白和特定的DNA片段，以便转录组装能够很好地进行x射线晶体学分析。我们计划使用一种稳定的转录伙伴蛋白，β -连环蛋白，它将允许生化制备以前难以处理的分子，并介导它们在功能复合物中的相互作用。我们将使用系统的方法来鉴定LRH-1的稳定组装及其特征的转录调节因子。代表LRH-1已知反应元件的特定DNA片段将包括在我们的组装评估中。我们开发的LRH-1构建的调控复合物成像方法将直接适用于其他核受体及其转录组装体。我们的目标是迈出重要的一步，学习如何确定功能性核受体的结构，并了解其组装和转录调节机制的原子水平细节。目前三分之一的药物靶向核受体的调控位点，因为它们在所有生物过程中的RNA转录中起主要作用。核受体在DNA上构建的转录机器的新结构信息将极大地扩展我们对转录及其调控的理解，以及这些转录过程控制的生理和病理生理途径。我们的建议侧重于核受体LRH-1及其协同调节因子β -连环蛋白构建的转录组装。我们之所以选择LRH-1作为主要靶点，是因为该受体在人类发育、代谢和许多病理生理过程中起着关键作用。正如我们在提案中所描述的那样，胰腺中LRH-1的失调与成熟型糖尿病有关。功能性LRH-1/ β -连环蛋白缺乏可导致炎症性肠病、克罗恩病和溃疡性结肠炎。相反，LRH-1/ β -连环蛋白的异常、上调活性与恶性肿瘤的触发和增殖有关，包括胃肠道癌症。因此，我们期望功能性LRH-1/ β -连环蛋白组装的原子分辨率结构将推动药物发现过程，并使靶向干预治疗的新型有效疗法的开发成为可能。公共卫生相关性：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起主要作用。我们专注于核受体LRH-1的成像，这是一种发现于肝脏、肠道和胰腺的关键转录因子，当它与DNA结合时，它的转录调控伙伴。我们期望这种受体形成的功能调控组合的成像不仅将极大地扩展我们对转录及其调控的理解，而且还将使许多疾病的靶向治疗，包括慢性肠道炎症、胃肠道和胰腺癌的有效治疗的发展成为可能。

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.