Competitive Supplement for 5R01 DK 58829

5R01 DK 58829 的竞争性补充品

• 批准号: 6570812
• 负责人: RAJ KUMAR
• 金额: $ 9.82万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-15 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6570812
• 关键词: Baculoviridae; X ray crystallography; binding sites; circular dichroism; corticosteroid receptors; fluorescence spectrometry; intermolecular interaction; nuclear magnetic resonance spectroscopy; protein folding; protein structure function; recombinant proteins; site directed mutagenesis; surface plasmon resonance; tertiary amine

DESCRIPTION (provided by applicant): We wish to investigate the functional consequences of mutations in the AF1/taul I/enh2 transcription transactivation domain of the human glucocorticoid receptor (GR). We hypothesize that this domain is naturally unfolded. Under DK58829 we are studying aspects of AF1 folding in vitro and are developing mutants that define intra- and extra-AF1 amino acids or regions important for AF1 to gain structure. Exposure to the osmolyte trimethylamine oxide or binding of the GR's DNA-binding domain impart structure to AF1, and when thus structured, it binds several nuclear proteins, including the TATA box binding protein and CBP. The review of the proposal funded as DK58829 criticized the lack of in vivo cellular studies to complement the in vitro work. This request for supplemental funding is in response to that criticism. Under this supplemental funding, we will pursue the Specific Aim of testing in cells the functional consequences of GR mutations that may alter the folding of AF1. We will transfect two types of cells - lymphoid and epithelioid - in which we will compare the transcription-regulating efficacy of mutants affecting AF1 function in both holo GR constructs and those lacking a ligand binding domain. Simple and more complex promoters controlling a reporter gene will be compared in responsiveness. In the lymphoid cells regulation of both induced and repressed endogenous genes will be evaluated, and the bioendpoint of lymphoid apoptosis will be followed. Each mutant will also be tested for folding in vitro, thus the experiments will directly test the correlation between AF1 folding and its functions. With this supplement, our results will provide a comprehensive evaluation of AF1 function, with relevance to both basic biochemical mechanisms and the practical use of hormones and other ligands in medical applications.

描述（由申请人提供）：我们希望研究人类糖皮质激素受体（GR）的AF1/TAUL I/ENH2转录反式反式激活结构域中突变的功能后果。我们假设这个领域是自然展开的。在DK58829下，我们正在研究体外AF1折叠的各个方面，并正在开发突变体，这些突变体定义了对AF1至关重要的AF1内和额外AF1氨基酸或区域，以获得结构。暴露于渗透剂三甲胺氧化物或GR的DNA结合结构域的结合赋予AF1的结构，并在结构结构时结合了几种核蛋白，包括TATA盒结合蛋白和CBP。对DK58829资助的提案的审查批评了缺乏体内细胞研究来补充体外工作。这种补充资金的要求是​​对这种批评的回应。在这种补充资金下，我们将追求在细胞中测试可能会改变AF1折叠的GR突变的功能后果的具体目的。我们将转染两种类型的细胞 - 淋巴样和上皮样细胞 - 在其中我们将比较影响Holo GR构建体中影响AF1功能的突变体和缺乏配体结合域的转录调节功效。控制记者基因的简单且更复杂的启动子将在响应中进行比较。将评估诱导和抑制性内源基因的淋巴样细胞中，将评估淋巴样细胞凋亡的生物端点。每个突变体还将在体外进行折叠测试，因此实验将直接测试AF1折叠及其功能之间的相关性。通过这种补充，我们的结果将对AF1功能进行全面评估，与基本的生化机制以及在医疗应用中的基本生化机制和其他配体的实际使用相关。