Structural Genomics of Orphan Nuclear Receptors

描述（由申请人提供）：该项目的长期目标是确定所有孤儿核受体配体结合域（lbd）的结构，将其结构与这些受体的生物学功能联系起来，并探索结构信息，以发现针对这些受体的药物。核受体（NRs）构成了一个dna结合转录因子的大家族，它调节了参与广泛生理的基因的表达。与其他转录因子不同的是，NRs的活性受直接结合NRs的小分子配体调控，以诱导活性或抑制性构象。小分子调控使nr成为治疗药物最重要和最成功的靶点之一。所有核受体至少包含两个高度保守结构域中的一个：位于中心的dna结合结构域（DBD）和c端LBD。LBD是NR的关键结构域和功能域。除了配体结合外，LBD还包含二聚化基序和一个保守的表面，该表面介导配体调节的共激活因子和共抑制因子的募集，以进行转录调节。因此，LBD已成为激烈的结构研究的焦点和药物发现的直接目标。所有“经典”内分泌核受体和所有“收养孤儿受体”（受体确定后再确定配体的受体）的晶体结构已经解决。这些结构揭示了一个保守的夹心折叠，其中包含一个配体结合袋和一个介导配体调节核受体功能的c端激活螺旋（AF-2）。重要的是，这些结构有助于揭示配体调控和核受体配体发现的关键机制，正如本资助的第一期对SF-1和COUP-TFII所证明的那样。目前，人类核受体只有三个亚家族（SHP、GCNF和TLX/PNR）的LBD结构有待解决。SHP、GCNF、TLX和PNR分别是胆固醇稳态、神经发生、眼睛发育和干细胞再生的重要调节因子，但对它们的结构和配体调控知之甚少。结构信息的缺乏已经成为理解这些受体介导的生物学和信号通路的关键障碍。基于对其他孤儿核受体的配体鉴定和核受体的共同结构特征，我们假设剩余的孤儿核受体（SHP， GCNF, TLX/PNR）也受到配体调控。在这次更新的应用中，计划使用x射线晶体学结合生化和功能分析来验证这一假设。通过为这些受体的配体调节提供一个全面的结构和分子框架，以及为针对这些受体的药物发现提供知识基础，实现我们的特定目标将克服上述与这些受体相关的科学进步的关键障碍。

DESCRIPTION (provided by applicant): The long-term objective of this project is to determine the structures of all orphan nuclear receptor ligand binding domains (LBDs), to correlate their structures with the biological functions of these receptors, and to explore the structural information for discovery of drugs that target these receptors. Nuclear receptors (NRs) constitute a large family of DNA-binding transcription factors that modulate the expression of genes involved in a broad spectrum of physiology. In contrast to other transcription factors, the activity of NRs is regulated by small-molecule ligands that directly bind NRs to induce active or repressive conformations. Their regulation by small molecules has made NRs one of the most important and successful targets for therapeutic drugs. All nuclear receptors contain at least one of two highly conserved domains: the centrally located DNA-binding domain (DBD) and the C-terminal LBD. The LBD is the key structural and functional domain of a NR. In addition to ligand binding, the LBD contains dimerization motifs and a conserved surface that mediates ligand-regulated recruitment of coactivators and co-repressors for transcriptional regulation. The LBD has thus been the focus of intense structural study and the direct target of pharmaceutical discovery. Crystal structures of all "classical" endocrine nuclear receptors and all "adopted orphan receptors" (receptors for which ligands were identified after the receptor was identified) have been solved. These structures reveal a conserved sandwich fold that harbors a ligand binding pocket and a C-terminal activation helix (AF-2) that mediates the ligand-regulated function of nuclear receptors. Importantly, these structures have been instrumental in revealing key mechanisms of ligand regulation and ligand discovery for nuclear receptors, as demonstrated for SF-1 and COUP-TFII in the first period of this grant. Currently, there are only three subfamilies (SHP, GCNF, and TLX/PNR) of human nuclear receptors for which the LBD structure remains to be solved. SHP, GCNF, TLX, and PNR are essential regulators in cholesterol homeostasis, neurogenesis, eye development, and stem cell regeneration, respectively, but little is known about their structures and ligand regulation. The lack of structural information has become a critical barrier for understanding the biology and signaling pathways mediated by these receptors. Based on ligand identification for other orphan nuclear receptors and the common structural features of nuclear receptors, we hypothesize that the remaining orphan nuclear receptors (SHP, GCNF, TLX/PNR) are also ligand-regulated. In this renewed application, the plan is to use X-ray crystallography in combination with biochemical and functional assays to test this hypothesis. Achieving our specific aims will overcome the above critical barrier to scientific advancements related to these receptors by providing a comprehensive structural and molecular framework for ligand regulation of these receptors, as well as a knowledgeable foundation for drug discovery focused on targeting these receptors. PUBLIC HEALTH RELEVANCE: SHP, TLX/PNR, and GCNF are orphan nuclear receptors that play crucial roles in the metabolism of lipids and bile acid, or maintenance and development of neural and embryonic stem cells. However, little is known about their structures and aspects of ligand regulation. The lack of structural information for these receptors has become critical barriers to progress of these receptors. Crystal structures of these receptors and their functional correlation under their respective biological context will not only establish whether these orphan nuclear receptors are ligand-regulated receptors, but will also serve as a rational template for drug design targeting of these receptors for cancers, metabolic diseases, and stem cell therapy for neurodegenerative diseases.