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）：我们希望研究人糖皮质激素受体（GR）的AF 1/taul I/enh 2转录反式激活结构域突变的功能后果。我们假设这个域是自然展开的。在DK 58829下，我们正在研究AF 1体外折叠的各个方面，并正在开发定义AF 1内和外氨基酸或对AF 1获得结构重要的区域的突变体。暴露于渗透剂氧化三甲胺或GR的DNA结合结构域的结合赋予AF 1结构，并且当如此结构化时，它结合几种核蛋白，包括TATA盒结合蛋白和CBP。作为DK 58829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。这一补充资金的要求是对这一批评的回应。在这笔补充资金下，我们将追求在细胞中测试可能改变AF 1折叠的GR突变的功能后果的具体目标。我们将选择两种类型的细胞-淋巴和上皮样细胞-在其中，我们将比较影响AF 1功能的突变体在两个holo GR结构和那些缺乏配体结合域的转录调节功效。控制报告基因的简单和更复杂的启动子将在响应性方面进行比较。在淋巴细胞中，将评价诱导和抑制的内源性基因的调节，并将跟踪淋巴细胞凋亡的生物终点。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AF 1折叠与其功能之间的相关性。有了这个补充，我们的研究结果将提供一个全面的评估AF 1功能，相关的基本生化机制和激素和其他配体在医学应用中的实际使用。