Competitive Supplement for 5R01 DK 58829

描述(申请人提供)：我们希望研究人糖皮质激素受体(GR)的AF1/TOUL I/EN2转录反式激活结构域突变的功能后果。我们假设这个领域是自然展开的。在DK58829的指导下，我们正在研究AF1的体外折叠，并正在开发定义AF1内和AF1外氨基酸或区域的突变体，这些氨基酸或区域对AF1获得结构至关重要。暴露于渗透分子三甲胺氧化物或GR的DNA结合域的结合赋予AF1结构，当这样的结构时，它与几个核蛋白结合，包括TATA盒结合蛋白和CBP。对由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的问题。这种补充资金的请求是对这种批评的回应。在这笔补充资金下，我们将追求在细胞中测试GR突变可能改变AF1折叠的功能后果的具体目标。我们将转染两种类型的细胞-淋巴样细胞和上皮样细胞-在这两种细胞中，我们将比较影响AF1功能的突变体在完整GR结构和缺乏配体结合域的情况下的转录调节效率。控制报告基因的简单启动子和更复杂的启动子的响应性将进行比较。在淋巴细胞中，将评估诱导和抑制的内源性基因的调节，并追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AF1折叠与其功能之间的相关性。通过这一补充，我们的结果将提供AF1功能的全面评估，与基本的生化机制和激素和其他配体在医疗应用中的实际应用相关。

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.