MOLECULAR REGULATION OF FXR ACTIVITY

FXR 活性的分子调控

• 批准号: 7730715
• 负责人: Jongsook Kim Kemper
• 金额: $ 31.7万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7730715
• 关键词: Acetylation; Acetylesterase; Bile Acids; Biological; Cholesterol; Chromatin; Cultured Cells; DNA Binding; Deacetylase; Deacetylation; Development; Diabetes Mellitus; Disease; Disease model; Down-Regulation; EP300 gene; Fatty Acids; Fatty Liver; Fc Receptor; Fibroblast Growth Factor; Gene Targeting; Genes; Glucose; Goals; Growth; Health; Homeostasis; In Vitro; Individual; Intestines; Leptin; Lipids; Lipoproteins; Liver; Liver Regeneration; Lysine; Mass Spectrum Analysis; Mediating; Metabolic; Metabolic Diseases; Metabolism; Molecular; Mus; Mutation; Nuclear; Obesity; Orphan; Pathology; Physiological; Physiology; Play; Post-Translational Protein Processing; Proteins; Receptor Down-Regulation; Recruitment Activity; Regulation; Role; SRE-1 binding protein; Site; Small Intestines; Stress; TP53 gene; Testing; Therapeutic Agents; Transactivation; Transcription Regulatory Protein; blood glucose regulation; cofactor; design; functional outcomes; glucose metabolism; histone modification; hypercholesterolemia; improved; in vivo; insight; lipoprotein triglyceride; mouse model; mutant; novel therapeutics; receptor; receptor function; small heterodimer partner protein; tumorigenesis

The overall goal of this project is to understand molecular mechanisms by which the nuclear bile acid receptor, farnesoid X receptor (FXR), regulates metabolic homeostasis in normal and diseased states. The specific goal of this application is to elucidate the role of post-translational acetylation and deacetylation of FXR mediated by p300 and SIRT1 in FXR function in normal physiology and in pathological conditions. FXR plays a central role in cholesterol/bile acid, fatty acid, lipoprotein, and glucose metabolism by regulating expression of numerous its target genes. Although such important biological roles of FXR are now known, how FXR activity is regulated remains relatively unknown. Transcriptional cofactors, such as p300 acetylase and SIRT1 deacetylase, modulate receptor activity, not only by histone modification of their target gene chromatin, but also by post-translational modification of the receptor itself. In preliminary studies, we found that FXR is acetylated and deacetylated by p300 and SIRT1, respectively. P300 and SIRT1 antagonize each other’s activity in the regulation of FXR transactivation. Down-regulation of p300 altered expression of FXR target genes, such that beneficial changes in lipid and glucose metabolic profiles would be expected. FXR acetylation was dynamically regulated under normal physiological states but surprisingly, FXR acetylation levels were substantially elevated in ob/ob mouse liver. Specific lysine residues in FXR were identified as acetylation sites by mass spectrometry and mutation analyses. Interestingly, mutation of these individual sites had different effects on FXR transactivation activity. These intriguing results led us to hypothesize first, that acetylation profoundly modulates FXR activity and is dynamically regulated by p300 and SIRT1 under normal physiological conditions but is highly elevated under metabolic disease and stress conditions and second, that FXR acetylation at different sites may have distinct functional outcomes in normal and disease states by regulating different FXR target genes. To test our hypothesis, we will: 1) determine whether p300 and SIRT1 are critical in vivo cofactors of FXR by down-regulation of these cofactors in cultured cells and in vivo ; 2) identify acetylated sites in FXR and determine the effects of mutations of these sites on FXR function in vitro, in cultured cells, and in vivo; and 3) determine whether FXR acetylation is dysregulated in pathophysiological conditions and the role of acetylation at specific sites in the disease pathology. Our studies will provide substantial insight into the molecular mechanism of FXR action in vivo and information that may be important for the development of novel therapeutic agents for metabolic disorders, such as fatty liver (liver steatosis), hypercholesterolemia, and diabetes. The bile acid receptor FXR has important biological roles in cholesterol and bile acid homeostasis, triglyceride and lipoprotein metabolism, and glucose regulation in the liver, but how the activity of FXR is regulated remains largely unknown. Our studies to define how FXR acetylation controls FXR activity in health and disease states will provide important information about the mechanisms regulating levels of cholesterol, triglycerides, lipoproteins, and glucose which are abnormal in diseases such as hypercholesterolemia, obesity, and diabetes. The studies may also facilitate the design of therapeutic agents for treating these metabolic disorders.

该项目的总体目标是了解核胆汁酸受体法尼醇 X 受体 (FXR) 在正常和患病状态下调节代谢稳态的分子机制。本申请的具体目标是阐明 p300 和 SIRT1 介导的 FXR 翻译后乙酰化和脱乙酰化在正常生理和病理条件下 FXR 功能中的作用。 FXR 播放 通过调节众多靶基因的表达，在胆固醇/胆汁酸、脂肪酸、脂蛋白和葡萄糖代谢中发挥核心作用。尽管 FXR 如此重要的生物学作用现已为人所知，但 FXR 活性如何调节仍然相对未知。转录辅因子，例如 p300 乙酰化酶和 SIRT1 脱乙酰酶，不仅通过其靶基因染色质的组蛋白修饰来调节受体活性，而且还通过 也可以通过受体本身的翻译后修饰。在初步研究中，我们发现FXR分别被p300和SIRT1乙酰化和去乙酰化。 P300 和 SIRT1 在 FXR 反式激活调节中相互拮抗。 p300 的下调改变了 FXR 靶基因的表达，从而预期脂质和葡萄糖代谢谱会发生有益的变化。 FXR 乙酰化在正常生理状态下受到动态调节，但令人惊讶的是，ob/ob 小鼠肝脏中 FXR 乙酰化水平显着升高。通过质谱和突变分析将 FXR 中的特定赖氨酸残基鉴定为乙酰化位点。有趣的是，这些单独位点的突变对 FXR 反式激活活性有不同的影响。这些有趣的结果让我们首先假设 乙酰化深刻调节 FXR 活性，并在正常生理条件下受 p300 和 SIRT1 动态调节，但在代谢疾病和应激条件下高度升高；其次，不同位点的 FXR 乙酰化可能通过调节不同的 FXR 靶基因在正常和疾病状态下产生不同的功能结果。为了检验我们的假设，我们将： 1) 通过在培养细胞和体内下调这些辅助因子来确定 p300 和 SIRT1 是否是 FXR 的关键体内辅助因子； 2) 鉴定 FXR 中的乙酰化位点，并确定这些位点的突变对体外、培养细胞和体内 FXR 功能的影响； 3) 确定 FXR 乙酰化在病理生理条件下是否失调以及乙酰化在疾病病理学中特定位点的作用。我们的研究将提供 对 FXR 体内作用的分子机制的深入了解，以及对于开发代谢性疾病（如脂肪肝（肝脂肪变性）、高胆固醇血症和糖尿病）的新型治疗剂可能很重要的信息。 胆汁酸受体FXR在胆固醇和胆汁酸稳态、甘油三酯和脂蛋白代谢以及肝脏葡萄糖调节中具有重要的生物学作用，但FXR的活性如何调节仍然很大程度上未知。我们对 FXR 乙酰化如何在健康和疾病状态下控制 FXR 活性的研究将提供有关调节胆固醇、甘油三酯、脂蛋白和葡萄糖水平的机制的重要信息，这些水平在高胆固醇血症、肥胖和糖尿病等疾病中是异常的。这些研究还可能有助于设计治疗这些代谢性疾病的治疗剂。